INHIBITION OF PRIMARY COLONIZERS BY - USP …digilib.library.usp.ac.fj/gsdl/collect/usplibr1/index/...INHIBITION OF PRIMARY COLONIZERS BY MARINE SURFACE-ASSOCIATED BACTERIA By Vipra

INHIBITION OF PRIMARY COLONIZERS BY

MARINE SURFACE-ASSOCIATED

BACTERIA

By

Vipra Nandani KUMAR

A Thesis Submitted in Partial Fulfilment of the

Requirements for the Degree of

Master in Science in Biology

School of Biological, Chemical and Environmental Sciences

Faculty of Science and Technology

The University of the South Pacific

2009

i

Abstract

Surfaces immersed in seawater rapidly accumulate a complex biofouling community,

of which bacteria and diatoms are among the first colonisers. However marine

organisms have evolved several defence mechanisms and it has been suggested that

green algae of the genus Ulva rely on microbial defence. The antibacterial properties

of epiphytic bacteria are well established, but relatively little is known about their

anti-diatom properties. In this study the hypothesis that surface-associated bacteria

from tropical Ulva species have anti-fouling characteristics that may have a role in

preventing surface fouling on the algae was investigated. Bacterial isolates from the

surface of Ulva growing in tropical waters were obtained and tested for antibacterial

and anti-diatom properties. It was found that 60% of the isolates expressed some

inhibitory action against the remaining bacteria isolated in the study and 80%

inhibited growth of the diatom Cylindrotheca fusiformis. Most effective bacteria were

members of the Pseudoalteromonas genus. Also showing inhibitory properties were

members of the genus Bacillus, Vibrio and Shewanella. Since Pseudoalteromonas

spp. and the Roseobacter clade are model surface-associated bacteria, both groups

were screened for anti-diatom property. Results showed that anti-diatom activity was

present in 100% and 44% of tested Pseudoalteromonas and Roseobacter strains

respectively. In order to better comprehend the anti-diatom property of marine

surface-associated bacteria, a transposon mutant library of Pseudoalteromonas

tunicata was generated and screened for mutants lacking in anti-diatom activity.

Genetic analysis of transposon insertion sites into the P. tunicata genome was then

used to identify loci linked with anti-diatom activity. Genes identified in this way

include a cation/multidrug efflux pump, a beta-hexosaminidase protein, a RTX toxin-

like gene and a member of the HemeO protein family. A hypothetical model for the

regulation of anti-diatom activity in P. tunicata was suggested and this will form the

basis of future studies that aim to identify the mechanism of anti-diatom activity in

bacteria, especially in P. tunicata. Additionally, the presence of epiphytic bacteria

engaged in antifouling activities on the surface of tropical Ulva sp. emphasizes the

prevalence of microbial-mediated defence systems which can be manipulated to find

solutions to current biofouling-associated problems.

ii

Acknowledgement

I extend my appreciation to the Faculty of Science and Technology of the University

of the South Pacific for awarding me with a Graduate Assistantship and funding this

research. Sincere thanks are also conveyed to the Centre for Marine Bio-Innovation,

University of New South Wales for collaborating in this research. To my supervisors,

Dr. Dhana Rao of University of the South Pacific, Dr. Suhelen Egan and Prof. Staffan

Kjelleberg of University of New South Wales, I am greatly honoured to have worked

under you. Your constant guidance and companionship has brought out the best in this

project. Please accept my utmost gratitude.

I owe much thanks to Ani, Francesco, Mel, Cathy and Flavia for going over various

procedures with me. Thanks to Torsten for expert advice with phylogenetics. Special

thanks go out to Richard and Preeti for their assistance with editing and proof-reading.

I am grateful also to the Division of Biology for assisting in having my isolates sent

over. To Debra, Anne, Jeyran, Neil, Nico and the 304 team, thanks for making

everyday at CMB so much fun. A hearty thanks to all relatives and friends for your

continuous love and support. Heartfelt thanks also to friends and elders of Rooty Hill

Sai Centre for having made me a part of their ‘Sai family’.

I offer my highest salute to my devoted and loving parents for ensuring the best of

everything for me. Thank you for your constant encouragement and endless support.

And to my most treasured brother and sister-in-law, I do not know how to thank you

enough. Thank you for looking after me and ensuring warm meals and a cosy bed. I

am deeply indebted to your immense love and understanding.

Finally, thank you to my most beloved Swami for being my inspiration and guide. I

humbly dedicate this thesis to you, dear Lord.

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Table of Contents

List of Acronyms

ATP adenosine triphosphate

BLAST Basic Local Alignment

Search Tool

bp base pairs

Da Dalton

DM non anti-diatom mutant

DNA deoxyribonucleic acid

dNTP deoxynucleotide

triphosphate

EDTA ethylene diamine

tetraacetic acid

EPS extracellular polymeric

substances

g grams

g gravitational force

hr hours

IMG Intergrated Microbial

Genomes

kb kilobases, 1000bp

kDa kilodaltons, 1000 Da

Km kanamycin

l litres

LB Luria Broth

m milli (10-3)

M molar

min minutes

mm millimetres

mol moles

n nano (10-9)

NCBI National Centre for

Biotechnology

Information

ORF open reading frame

p pico (10-12)

PCR polymerase chain

reaction

rRNA ribosomal ribonucleic

acid

SDS sodium dodecyl sulfate

sec seconds

Sm streptomycin

SmR streptomycin resistant

sp. species

TBE tris-boric acid-EDTA

buffer

TBT tributyl tin

v/v volume to volume

w/v weight to volume

� lambda

μ micro (10-6)

°C degrees Celsius

ii

List of Tables

Table 2.1: Antibacterial activity expressed by bacteria isolated from Ulva…………22

Table 2.2: Inhibition of C. fusiformis as expressed by bacteria isolated from Ulva…23

Table 2.3: 16S rRNA gene identification of bacteria isolated from Ulva……………24

Table 3.1: Restriction enzymes used for panhandle PCR……………………………38

List of Figures

Figure 1.1: The stages of colonisation of surfaces immersed in seawater………….....2

Figure 1.2: Stages of biofilm development…………………………………….….…..4

Figure 1.3: Schematic diagram of diatom structure and frustule terminology…….....12

Figure 3.1: Diatom growth inhibition by Pseudoalteromonas spp………………......40

Figure 3.2: Diatom growth inhibition by members of the Roseobacter clade..……...41

Figure 3.3: Anti-diatom activity of wild type P. tunicata and non-antidiatom

mutants (DM1, DM2, DM3 and DM4)………………………………….…...42

Figure 3.4: Growth curve of wild type and mutant strains of P. tunicata………........43

Figure 3.5: Agarose gel showing the results from a typical panhandle-PCR ….…….44

Figure 3.6: Genomic location of PTD2_12754, homologous to AcrB/AcrD/AcrF

family protein………………………………………………………………...45

Figure 3.7: Genomic location of PTD2_01386 and PTD2_01391, homologous to

beta-hexosaminidase and RTX toxin respectively…………………………. ..45

Figure 3.8: Genomic location of PTD2_02946, a HemeO protein family..………….46

Figure 4.1: Hypothetical model for the regulation of anti-diatom activity

in P. tunicata………………………………………………………………….57

Figure 8.1: Phylogenetic relationship of isolate U3 to bacteria on ARB

Project Database…………………………………………………...…………63


Project Database………………………….…………………………...…...…64

Figure 8.3: Phylogenetic relationship of isolate U7 and U11 to bacteria on

ARB Project Database………………………………………………………..65


Project Database……………………………………………..…………….…66

iii

Figure 8.5: Phylogenetic relationship of isolate U13 and U14 to bacteria on

ARB Project Database………………………………...………………….…..67


Project Database…………………………………………………...…………68

Figure 9.1: P. tunicata genome sequence section showing the point of insertion

of Tn10 in DM1……………………………………………………..………..69


of Tn10 in DM2…………………………………………………….…….…..71


of Tn10 in DM3………………………………………………………………75

List of Appendices

Appendix I: Media and buffers 78

Appendix II:Primers 81

Appendix III: Phylogenetic relationship of algal isolates to bacteria on ARB Project

database 82

Appendix IV: Transposon insertion sites in the P. tunicata genome 88

iv

Abstract i

Chapter 1: Introduction 1

1.1 Biofouling in the marine environment 1

1.1.1 The process and its importance 1

1.1.2 The role of biofilms 2

1.1.3 Current biofouling control strategies and its impact on the marine ecosystem 6

1.1.4 Natural defence mechanisms against biofouling 6

1.2 Diatoms as marine surface colonisers 11

1.2.1 Diatom biology 11

1.2.2 Diatom adhesion, motility and extracellular polymeric substances 13

1.3 Aims of this study 14

Chapter 2: Inhibitory activity of epiphytic bacteria isolated from Ulva 16

2.1 Introduction 16

2.2 Materials and methods 17

2.2.1 Isolation of epiphytic bacteria from Ulva 17

2.2.2 Bioassay against growth of bacteria 18

2.2.3 Bioassay against growth of diatoms 18

2.2.4 Identification of isolates with inhibitory properties 19

2.2.4.1 DNA extraction 19

2.2.4.2 Agarose gel electrophoresis 19

2.2.4.3 PCR amplification of 16S rRNA gene 20

2.2.4.4 DNA sequencing and sequence analysis 20

2.3 Results 21

2.3.1 Epiphytic bacteria isolated from Ulva 21

2.3.2 Production of extracellular antibacterial compounds 21

2.3.3 Growth inhibition of diatoms 22

2.3.4 Characterization of active bacterial strains 23

2.4 Discussion 27

2.4.1 Antibacterial activity of bacterial isolates 27

2.4.2 Anti-diatom activity of bacterial isolates 27

v

2.4.3 Identification of active bacterial strains 28

2.5 Conclusion 32

Chapter 3: Anti-diatom property of Pseudoalteromonas and Roseobacter

strains 33

3.1 Introduction 33

3.2 Materials and methods 34

3.2.1 Screening Pseudoalteromonas and Roseobacter strains for anti-diatom activity

34

3.2.2 Analysis of anti-diatom strategy of P. tunicata 35

3.2.2.1 Transposon mutagenesis 35

3.2.2.2 Screening for P. tunicata mutants lacking anti-diatom property 36

3.2.2.3 Growth rates of mutants 36

3.2.2.4 Genomic DNA extraction of non anti-diatom mutants 37

3.2.2.5 Generation of adaptor ligated DNA for panhandle PCR 37

3.2.2.6 Panhandle PCR 38

3.2.2.7 Sequencing 39

3.3 Results 39

3.3.1 Growth inhibition of diatoms 39

3.3.2 Mutants lacking in anti-diatom activity 42

3.3.3 Growth curve of wild type and mutants of P. tunicata 43

3.3.4 Panhandle PCR and DNA sequencing 43

3.3.5 Genotype characterization of the non anti-diatom mutants 44

3.3.5.1 DNA regions flanking the transposon insertion site in DM1 44



3.4 Discussion 46

3.4.1 Anti-diatom activity of Psedoalteromonas spp. and Roseobacter clade 46

3.4.2 Analysis of transposon insertion sites within the P. tunicata genome 48

3.5 Conclusion 53

vi

Chapter 4: General discussion 54

4.1 Antifouling properties of surface-associated bacteria 54

4.2 Modelling anti-diatom mechanism in P. tunicata 55

4.3 Future directions and implications 58

References 59

Chapter 1: Introduction

Inhibition of primary colonizers by marine surface-associated bacteria 1


1.1 Biofouling in the marine environment

1.1.1 The process and its importance

Biofouling is the undesirable accumulation of microorganisms, plants and animals on

surfaces immersed in water. It is a dynamic process involving a sequence of

colonization events that lead to the formation of a mature fouling community.

Dobretsov et al., (2006) viewed the colonization of a substratum in aquatic systems as

a three-step process (Figure 1.1). The process involves (i) adsorption of dissolved

organic molecules to a newly submerged or otherwise uncolonised surface, (ii)

colonization of the surface by bacteria and microscopic eukaryotes (e.g., diatoms,

fungi, and other heterotrophic eukaryotes) and (iii) settlement and subsequent growth

of invertebrate larvae and algal spores.

Biofouling impacts on humans in a number of ways, perhaps most important are the

potential economic effects. Any industry that is reliant upon or linked to aquatic

environments must deal with the effects of biofouling (e.g., offshore oil and gas

sectors, fishing and aquaculture industries and the transport industry). Perhaps the

most common biofouling sites are ships hulls. A heavily fouled vessel suffers

increased drag and decreased manoeuvrability due to the roughness of the hull. This

has major economic implications, potentially resulting in a significant increase in fuel

costs. Biofouling may also lead eventually to corrosion of the hull that may reduce the

lifespan for the vessel. Other surfaces, in particular those exposed directly to water

(e.g., heat exchangers, ballast tanks, and propellers), may also be subject to biofouling

(Brizzolara, 2002). In addition, equipment used in fishing and fish farming (e.g., mesh

cages and trawls) are also likely to harbour fouling organisms.

Living surfaces in the marine environment are also prone to biofouling. Macroalgae

are particularly susceptible since they are sessile and often restricted to the photic

zone where conditions for fouling are optimal (de Nys et al., 1995).



Molecular fouling Micro-fouling

Settlement of bacteria and diatoms

Adsortpion of conditioning film

Macro-fouling

Settlement of macroalgae and invertebrate larvae

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Besides providing a large surface area for colonization, algae may also provide a

source of nutrients and shelter for epibionts. Biofouling on marine organisms can

have detrimental effects on the host such as loss of photosynthetic area, reduced

viability, and organism death (Holmstrom and Kjelleberg, 2000). Biofouling may

impair the ability of the host to exchange gases and nutrients. Toxins, digestive

enzymes and waste produced by the biofouling community may also cause physical

damage to the host (Felgenhaur et al., 1989).

1.1.2 The role of biofilms

Each of the three stages in the formation of a biofouling community is an important

pre-requisite for the establishment of subsequent layers. In particular the microfouling

stage (the second stage in Figure 1.1), which involves the formation of a microbial

biofilm, initiates colonization for higher organisms. Studies have found that the

bacterial component of marine biofilms are important for induction of larval

settlement in several groups including echinoderms (Johnson et al., 1991), cnidarians

(Negri et al., 2001), polychaetes (Unabia and Hadfield, 1999), gastropods (Rodriguez

et al., 1995) and crustaceans (Neal and Yule, 1994). Hence an understanding of

biofilm formation is essential for developing an overall understanding of the



biofouling process. Although biofilms are composed of both bacteria and diatoms, our

current general understanding of biofilm formation has been gained from bacterial

biofilms. Biofilms are multicellular associations, consisting of closely spaced cells

embedded in an extracellular matrix. The process of bacterial biofilm formation

involves cell attachment, microcolony formation, biofilm maturation and cell

dispersal (Figure 1.2).

The first stage in biofilm formation involves surface attachment. Upon encountering a

suitable surface, planktonic cells adhere to the conditioning film and establish a weak

interaction with the surface. This initial phase is referred to as “reversible attachment”

(Busscher et al., 1992). Adhesion is influenced by many factors including the

physico-chemical properties of the cell surface (e.g., cell surface hydrophobicity;

Bruinsma, 2001), genetic determinants of the cell (e.g., the expression of cell surface

components and matrix material; Caiazza and O’Toole, 2004) and hydrophobicity and

charge of the substratum (Harkes et al., 1992; Mueller et al., 1992). Commitment, the

next phase, which involves “irreversible attachment”, is a crucial step in biofilm

formation since these initial colonizers form the foundation of the mature biofilm.

After this transition, cells cannot be removed from the surface by simple washing

procedures (Oliveira, 1992).

Following adhesion to a surface, bacterial cells aggregate, forming the basic structural

unit of a biofilm, referred to as a microcolony (Davey and O’Toole, 2000).

Microcolonies may form by one of three mechanisms - surface translocation, cell

recruitment and clonal growth. The first two mechanisms involve recruitment of new

cells to a microcolony. During surface translocation cells attached to the surface

utilize swarming or twitching motility to join existing microcolonies. In contrast cell

recruitment involves planktonic cells (Tolker-Nielsen et al., 2000) or cell flocs

(Stoodley et al., 2001) attaching directly to cell aggregates from the bulk fluid. In

clonal growth increase in microcolony size results from division of existing resident

bacteria. The relative contribution of each mechanism varies depending on the

organism involved, the surface being colonized and the environmental conditions

(Stoodley et al., 2002).



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During biofilm maturation the microcolonies reach their maximum dimensions. Cells

within a given microcolony are non-motile and are usually segregated into a number

of distinct cell clusters (Sauer et al., 2002). A microcolony is usually a simple conical

structure or mushroom-shaped. Microcolony structure is dependant upon the presence

of an extracellular cell-to-cell interconnecting matrix consisting mainly of

exopolymers (e.g. polysaccharides, proteins and DNA). In addition, outer membrane

proteins and cell appendages such as fimbriae, pili, and flagella may also form part of

the biofilm matrix (Pamp et al., 2007). The interconnecting matrix is interspersed

with highly permeable water-channels. These act as a “circulatory system,” delivering

nutrients and removing metabolic waste from the microcolony (Lawrence et al., 1991;

Costerton et al., 1994). Importantly biofilm growth form protects the biofilm-forming

organisms against the negative effects of antimicrobial agents and predation by

protozoans (Stewart, 2002; Matz and Kjelleberg, 2005). Additionally, biofilms

facilitate horizontal gene transfer and intracellular communication (Hausner and

Wuertz, 1999; Parsek and Greenberg, 2000) as well as promoting increased genetic

diversity of the bacterial populations (Boles et al., 2004). Such characteristics

improve the survival of bacterial communities in harsh environmental conditions.

For some bacteria, cell-cell communication is essential for the establishment of an

ordered biofilm community. Bacteria achieve this using secreted signalling molecules

called autoinducers in a process called “quorum sensing” (Nealson and Hastings,

1979). This enables the population to collectively regulate gene expression and,

therefore, behave as a group. Quorum sensing is known to control bioluminescence,



secretion of virulence factors, sporulation, and conjugation. Thus, quorum sensing is a

mechanism that allows bacteria to function much like a multi-cellular organism

(Hammer and Bassler, 2007).

As a biofilm matures dispersal of cell aggregates from the main body occurs. The

dispersal process may be either passive or active. Passive dispersal is a direct

consequence of the immediate environmental conditions and usually occurs by

erosion, sloughing, abrasion or predator grazing (Bryers, 1988). Cell aggregates are

released as a result of physical disruption of the biofilm. On the other hand active

dispersal mechanisms are used when environmental conditions become unfavourable.

In this case release of cells or cell aggregates is initiated and regulated by the biofilm.

For example, Pseudomonas spp., Escherichia coli, and Acinetobacter spp. biofilms

will release cells in response to nutrient starvation (Delaquis et al., 1989; Sawyer and

Hermanowicz, 2000; Jackson et al., 2002). Several studies have shown programmed

cell death is responsible for active dispersal. In Pseudoalteromonas tunicata the

antibacterial protein, AlpP, mediates cell death. This acts as a lysine oxidase resulting

in the production of hydrogen peroxidase and cell death within the microcolony (Mai-

Prochnow et al., 2004, 2006, 2008). Death of specific regions of the microcolony

results in the release of isolated cell aggregates. Phaeobacter gallaeciensis biofilms,

formerly Roseobacter gallaeciensis, also displays cell death within microcolonies

(Martens et al., 2006).

Cells dispersing from biofilms often exhibit phenotypic and genotypic variation, a

feature that is thought to enhance survival in the face of changing environmental

conditions and competitive regimes (Boles et al., 2004; Webb et al., 2004; Ho, 2008).

For example, dispersed cells of Ph. gallaeciensis express varying levels of

antimicrobial activity against the competitive colonizer, P. tunicata. This is expected

to lead to differential colonisation ability (Ho, 2008). In Pseudomonas aeruginosa

biofilms, small colony variants exhibit enhanced attachment and accelerated biofilm

development relative to the wild type strain (Webb et al., 2004).



1.1.3 Current biofouling control strategies and its impact on the marine ecosystem

To minimize the impact of biofouling on artificial structures in the marine

environment (e.g. ship’s hulls) many are protected with antifouling coatings. These

are paint-based and contain a biocide or toxin, often tributyl tin (TBT) or copper

based compounds (Thomas, 2001; Yebra et al., 2004). The biocide is slowly released

into the environment poisoning organisms that adhere to the surface.

However, since the biocides are non-specific they may also have harmful effects on

non-fouling organisms (Evans, 1999; Yebra et al., 2004). In the case of TBT, organo-

tin moieties are released as the coating degrades. These have a range of sublethal

effects on non-target species. For example, low concentrations of TBT may cause

defective shell growth in the oyster Crassostrea gigas and development of male

characteristics in female dog whelk Nucella lapillus (Evans, 1995). Perhaps most

alarming for the present study, studies indicate that the highest levels of TBT-

contamination have been recorded in Fiji, the most contaminated site recording a TBT

concentration of 360μgg-1 (Maata and Koshy, 2001).

Due to the non-specificity of TBT and other tin containing biocides in antifouling

paints, the International Maritime Organization and Marine Environmental Protection

Committee (MEPC) have banned their usage (Champ, 1999). This has prompted a

search for alternate antifoulants that are non-toxic and “environment-friendly.” An

understanding of the natural defence mechanisms of marine organisms against

biofouling is the first step towards “safer” antifoulants. It is hoped that these natural

defence strategies and the associated bioactive compounds may be manipulated to

develop novel antifouling technologies that are less harmful to the marine

environment.

1.1.4 Natural defence mechanisms against biofouling

Marine algae are also prone to biofouling. Seaweeds employ a number of physical

defence systems to prevent fouling. These include shedding of outer cell layers (Keats

et al., 1997), mucilaginous coverings on blades (Filion-Myklebust and Norton, 1981;

Moss, 1982) and continuous erosion of the distal ends of blades (Mann, 1973; Ott,



1980). Water turbulence and abrasion may also limit fouling (Sieburth and Tootle,

1981).

In addition, marine algae may produce inhibitory chemicals to prevent fouling and

grazing (Dworjanyn, et al., 1999). A well studied example is the red alga Delisea

pulchra, which produces an array of structurally related secondary metabolites known

as halogenated furanones (Kazlausks et al., 1977; de Nys et al., 1993). These

compounds interfere with bacterial colonization and prevent settling of invertebrate

larvae and the spores of common fouling algae (Kjelleberg et al., 1997; Maximilien et

al., 1998). Scanning electron microscopy of the alga reveals a significantly higher

abundance of epibacteria near the holdfast than closer to the blade apices (Steinberg et

al., 1997). This corresponds to a gradient in the concentration of halogenated

furanones - which are highest close to apices. Studies indicate that furanones may

control bacterial colonization by specifically interfering with acylated homoserine

lactone (AHL)-mediated gene expression at the level of the LuxR protein (Manefield

et al., 1999).

Although toxin mediated mechanisms are effective, they are energy expensive.

Generally, defence costs are the sum of (1) the energy and nutrients consumed for

defence production (and, therefore, lost to growth), (2) the energy necessary for

sequestering the toxins away from active cell processes, (3) the interference of the

defence with photosynthesis and (4) the loss of productivity from the tissue given it

would have photosynthesized if it were not co-opted for defence (Coley, 1986).

Hence, large amounts of energy are invested in toxin-mediated defence systems, and

this is costly for smaller and simpler algal forms.

Organisms that lack chemical or physical defences are thought to rely on secondary

metabolites produced by bacterial symbionts to provide defence against surface-

colonizing organisms (Armstrong et al., 2001; Berland et al., 1972; Thomas and

Allsopp, 1983). For example, symbiotic interactions have been found in the marine

crustaceans Palaemon macrodactylus and Homarus americanus where symbiotic

bacteria defend embryos from fungal infection (Gil-Turness and Fenical, 1992). More

generally, Holmstrom et al., (1996) investigated the frequency with which bacterial

strains isolated from living and inanimate surfaces displayed inhibitory activity



against fouling organisms. Results showed that 10% of isolates from rock surfaces

inhibited the settlement of invertebrate larvae compared with 30% of isolates from

marine animals and 74% from algal surfaces. These data suggest that many of the

bacteria that form epiphytic communities on living surfaces are able to regulate

fouling by other organisms (Egan et al., 2000).

Additionally, results from behavioral assays demonstrate that secondary metabolites

may be produced to ensure colonization by preferred epibionts (Wahl et al., 1994;

Bryan 1996; Engel et al., 2002). It is interesting to note that secondary metabolites

may control the density of surface associated microbes, allowing growth of a

community of preferred microbes rather than maintaining an axenic surface (Engel et

al., 2002). For example, in D. pulchra halogenated furanones affect bacterial

colonization differently. Attachment is inhibited in strains associated with surface-

fouling while growth and swarming is inhibited in the preferred strains (Maximilien et

al., 1998). The results explain why fouling strains are absent from the alga’s surface

and preferred bacterial strains have limited surface distribution.

The ecology of marine algal surfaces is known to be highly complex. As space and

nutrients are limited, colonization by bacteria often requires them to compete with one

another (Egan et al., 2008). Bacterial strains known to be associated with algal

surfaces include members of the Flavobacterium group of Bacterioidetes, members of

the Roseobacter clade (Rao et al., 2005) as well as various Pseudoalteromonas and

Alteromonas spp. (Holmstrom and Kjelleberg. 1999). Using a culture-independent

method, Longford et al. (2007) compared bacterial diversity on the red maccroalga D.

pulchra with that of U. australis. Approximately 79 species from 7 phyla were

isolated from D. pulchra while an estimated 36 species from only 4 phyla were

isolated from Ulva. Alpha-, Delta- and Gammaproteobacteria were all well

represented with Planctomycetes and Bacteroidetes common on both algae. However,

there were very few species common to both algae (Longford et al., 2007).

One of the major arguments for preserving biodiversity is the potential for discovery

of new bioactive compounds. For this reason, the genus Pseudoalteromonas has

received a lot of attention in the last two decades. The focus reflects the bacterium’s

frequent association with eukaryotic hosts in the marine environment. Studies of such



associations are useful for understanding the mechanisms underlying microbe-host

interactions. Also, many pigmented species of Pseudoalteromonas produce

biologically active metabolites (Egan et al., 2002b). Species of Pseudoalteromonas

display antibacterial, bacteriolytic, agarolytic and algicidal properties, as well as

various other pharmaceutically-relevant activities. Several Pseudoalteromonas strains

prevent the settlement and colonisation of marine surfaces by common fouling

organisms (Holmstrom and Kjelleberg, 1999; Bowman, 2007).

Within the genus, Pseudoalteromonas tunicata is thought to exhibit the broadest

range of inhibitory activities (Holmstrom et al., 2002). This species produces a

diverse range of biologically active compounds, many of which target marine fouling

organisms (Holmstrom et al., 1998). To date a range of antifouling compounds have

been isolated from P. tunicata. The antifungal compound is a yellow, tambjamine-like

alkaloid (YP1), the biosynthetic pathway of which is encoded by a cluster of 19 genes

(tamA to tamS; Franks et al., 2005; Burke et al., 2007). Moreover, the autolytic

antibacterial protein (AlpP) produces hydrogen peroxide which causes cell death,

mediates differentiation, dispersal and phenotypic variation during the dispersal event

(James et al., 1996; Mai-Prochnow et al., 2004, 2008). Other bioactive compounds

include a polar, heat-stable anti-larval molecule (Holmstrom et al., 1992), a heat-

sensitive anti-algal peptide (Egan et al., 2001) and an uncharacterized anti-diatom

compound. With such a wide range of antifouling characteristics present in P.

tunicata, the antifouling potential of the remainder of the genus is worth investigating.

More knowledge of the biologically active chemicals produced by

Pseudoalteromonas would also be potentially pharmacologically beneficial (Bowman,

2007).

The Roseobacter clade is another group of marine bacteria that due to its worldwide

distribution, abundance and physiological diversity is well studied (Brinkhoff et al.,

2008). The group has been isolated from both coastal and open waters, a variety of

micro- and macro-algae, microbial mats, sediments, polar sea ice, and marine

invertebrates (Buchan et al., 2005; Wagner-Dobler and Biebl, 2006). Members of the

group often form symbioses with higher organisms (Bruhn et al., 2007). For example,

the symbiotic association between Silicibacter sp. strain TM1040 (a member of the

Roseobacter clade) and the dinoflagellate Pfiesteria piscicida involves bacterial



chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP

demethylation, and ultimately a biofilm on the surface of the dinoflagellate host

(Alavi et al., 2001; Miller and Belas, 2004; Miller et al., 2004). Biofilm formation

coincides with the production of an antibiotic, a sulfur-containing compound,

tropodithietic acid (TDA). Since the genes critical for TDA biosynthesis are located

on plasmids in both Silicibacter sp. strain TM1040 and Phaeobacter sp. strain 27-4, it

is suggested that both members of the Roseobacter clade may use a common pathway

for TDA biosynthesis that involves plasmid-encoded proteins (Geng et al., 2008).

This suggests that investigating other members in the Roseobacter clade for bioactive

properties is important.

Phaeobacter gallaeciensis (a member of the Roseobacter clade) is a commonly

studied temperate, biofilm-forming strain. Together with Pseudoalteromonas

tunicata, Ph. gallaeciensis benefits its algal host by producing compounds that inhibit

common fouling organisms. Studies have shown Ph. gallaeciensis to be more

competitive than P. tunicata during biofilm formation and having the capacity to

invade and disperse pre-established biofilms (Rao et al., 2006). Hence, given strong

competitive characteristics, participation in symbiotic interactions with eukaryotic

hosts and wide distribution, members of the Roseobacter clade are also strong

candidates for future antifouling solutions.

While it is acknowledged that the technology available to assess and exploit microbial

diversity is limited, there is a need for studies that will enhance current understanding

of microbial associations (Egan et al., 2008). It is recognized that many of the marine

invertebrates (e.g. sponges, bryozoans and tunicates) that are sources of secondary

metabolites also contain endo- and epibiotic microorganisms. Indeed, some

invertebrate-derived natural products are structurally related to the bacterial

metabolites (Sudek et al., 2007). With the complexity of associations in marine

organisms, it is difficult to determine the biosynthetic source of many marine natural

products (Konig et al., 2006). However, it is now recognised that many of these

metabolites may well be of microbial origin (Sudek et al., 2007). The marine

bryozoan Bugula neritina synthesizes bryostatins, complex polyketides that render the

B. neritina larvae unpalatable to predators (Sharp et al., 2007). A recent study has

shown that bryostatin, isolated from B. neritina, is actually produced by the



uncultured symbiotic bacterium “Candidatus Endobugula sertula" (Sudek et al.,

2007). The finding highlights the important role of biotechnological advancement in

the discovery and exploitation of microbial defence mechanisms. The need was

recently re-emphasized when for the first time a strain of the Roseobacter clade-

affiliated (RCA) cluster was successfully isolated and propagated (Mayali et al.,

2008). This was accomplished through the application of novel techniques with algal

cultures. Previous efforts to culture the RCA cluster as well as many other bacteria

abundant in the marine environment, using traditional culture methods have not been

successful. This recent finding stresses the need for developing novel molecular

approaches to study uncultivated microbial diversity which could potentially lead to

the discovery of new compounds (Egan et al., 2008) and improved biofouling-control

techniques.

1.2 Diatoms as marine surface colonisers

Like bacteria, diatoms are also an important constituent of microfouling communities

on marine surfaces. However, most studies have focused on the bacterial component.

In the current search for improved-control strategies, it is essential to also develop an

understanding of the role that diatoms play. This will be critical for finding more

effective means of tackling the issue of biofouling.

1.2.1 Diatom biology

Diatoms are among the earliest eukaryotic colonizers of submerged surfaces and are

among the most conspicuous components of natural biofilms (Evans 1988). Diatoms

belong to the Bacillariophyceae with over 250 genera and perhaps as many as 100,000

species (Norton et al., 1996; Van Den Hoek et al., 1997). Characteristically, the cell

walls are highly patterned with pores and ridges. They are unique among the algae

due to the presence of silica-based cell walls. Most diatoms contain silicon

transporters (SITs) for transferring Si(OH)4 across lipid bilayer membranes

(Hildebrand et al., 1997). Diatoms take up silicon predominantly as silicic acid that is

then polymerized and deposited into the cell wall as silica (Del Amo and Brzezinski,

1999). Silicic acid is co-transported with sodium in marine diatoms with zinc also

suggested to play a role in silicic acid uptake (Sullivan, 1977; Rueter and Morel,



1981). The diatom of interest in this research is Cylindrotheca fusiformis. This

autotrophic, marine, pennate diatom is being used as a model organism for studying

transport, deposition and patterning of silica in diatom cell walls. Biosilica from all

diatom species investigated so far has shown to be a composite material containing

proteins (mainly the silaffins) and long-chain polyamines as organic components.

These organic constituents have been recognised as important players in silica

biomineralisation. Several recent reviews have described the structure and properties

of these organic molecules (mainly from C. fusiformis) as well as possible function in

silica formation and patterning (Pohnert, 2002; Foo et al., 2004; Sumper et al., 2004;

Sumper and Brunner, 2006).

The wall is constructed of two sections or thecae, with the smaller hypotheca fitting

within a larger epitheca much like a Petri dish (Figure 1.3). In diatoms one of the most

important cell wall proteins is pleuralin, which is involved in the cell cycle-dependent

frustule development. To maintain the integrity of the frustule, coupling between

biogenesis of new frustule components and cell cycle is required. The molecular

mechanism by which this coupling occurs is unknown. Interestingly, although the

thecae are morphologically similar, immunolocalisation with anti-pleuralin antibodies

demonstrates that their protein composition is clearly different (Kroger and

Wetherbee, 2000). It is hypothesized that pleuralins are involved in hypotheca-

epitheca differentiation, a crucial process that ensures proper frustule development.

(Redrawn from Hasle and Syvertsen, 1997)

Centric Diatoms Pennate Diatoms

Diagrammatic section showing frustule terminology

epivalve

valve view girdle viewvalve view girdle view

epetheca

hypotheca

hypovalve

��



Generally, two types of diatoms are recognised, pennate or bilaterally symmetrical

diatoms and centric or radially symmetrical diatoms. The former are mostly

planktonic and the latter predominately benthic, associated with sediments or attached

to rocks or macroalgae (Falciatore and Bowler, 2002; Leblanc et al., 1999). Diatoms

can be unicellular or colonial and either autotrophic or heterotrophic (Gilabert, 2007).

The brown colour of diatoms is due to the presence of the carotenoid pigment

fucoxanthin, which is located together with chlorophyll a and c in their plastids

(Round and Crawford, 1990). Reproduction in diatoms is mainly asexual, the

daughter cells each receiving one half of the parental cell wall and constructing a new

frustule half within it (Raven et al., 1999). Benthic diatoms are able to glide along

surfaces; mucilage is secreted into furrow (known as a raphe) which allows movement

(Falciatore and Bowler, 2002). In contrast, most planktonic diatoms are non-motile

and rely on mixing of the water column to remain suspended (Stoermer et al., 2004).

1.2.2 Diatom adhesion, motility and extracellular polymeric substances

Diatoms are abundant in benthic habitats where they adhere to surfaces using copious

quantities of mucilage. Depending on the nature of the surface, initial contact may or

may not result in bonding by the diatom (Wetherbee et al., 1998). Adhesion in

diatoms is Ca2+ dependent and the process requires metabolic energy, protein and

glycoprotein synthesis (Cooksey and Wigglesworth-Cooksey, 1995). Thus diatom

bonding requires an active commitment involving the activation of specific adhesion

mechanisms. In most benthic diatoms, cell-substratum adhesion occurs at the raphe,

resulting in cell reorientation and a unique form of cell motility called “gliding”

(Edgar and Pickett-Heaps, 1984). The mucilage secreted into the raphe links the cell

cytoplasm to the substratum. This provides for 'gliding' motility via an actin-myosin

system located adjacent to each raphe (Edgar and Pickett-Heaps, 1984; Poulsen et al.,

1999). Cell-substratum adhesion at the raphe is a requirement for diatom gliding

(Edgar and Pickett-Heaps, 1984; Wetherbee et al., 1998). As diatoms glide, the

secreted mucilaginous strands are detached and left behind as diatom “trails” that

eventually accumulate as a component of biofilms (Edgar and Pickett-Heaps, 1984;

Higgins et al., 2000; Wetherbee et al., 1998). In addition, the trails remain adhesive

and may aid in the accumulation of other biofouling agents (Lind et al., 1997).



The mucilage or extracellular polymeric substance (EPS) is produced by benthic

diatoms both as part of the motility system and as a response to environmental

conditions. The EPS is composed of polysaccharides, proteins, and glycoproteins

(Chiovitti et al., 2003). Typically carbohydrates are the dominant component of EPS,

but the constituent sugars are often complex and highly diverse (Hoagland et al.,

1993). Characterization of EPS structure, serology, and lectin interactions, provides

for a broad classification of EPS materials. Several subtypes are recognized including

frustule EPS, outer capsular EPS, motility EPS, and matrix EPS (Wigglesworth-

Cooksey and Cooksey, 2005). Combined, diatoms and their insoluble EPS are

common features of biofouling communities.

1.3 Aims of this study

The green alga Ulva spp. does not produce secondary metabolites with recognised

roles in fouling prevention (Awad, 2000; Abd El-Baky et al., 2008). Ulva spp. is

found in temperate and tropical waters, including the shores of Fiji. While

antibacterial activities have been identified for epiphytic bacteria isolated from

temperate Ulva spp., the occurrence of anti-diatom properties has been less

intensively investigated. The latter is likely due to the inherent, technical challenges

of performing anti-diatom bioassays.

The major hypothesis addressed in this study is that surface-associated bacteria from

tropical Ulva spp. have characteristics that have a role in limiting surface fouling of

the algae. First the study aimed to isolate epiphytic bacteria from Fijian collection of

Ulva spp. growing in tropical Fiji waters and characterise their inhibition of bacteria

and diatoms. The second part concentrated on the prevalence of anti-diatom activity

across two ecologically significant bacterial groups, namely Pseudoalteromonas and

the Roseobacter clade. A further focus was the anti-diatom strategy of the model

epibiont, P. tuncata, with gene identity information used to propose a preliminary

model describing the mechanism.



To address the hypothesis the specific aims of this study were to:

1. Isolate and assess inhibitory activity of the epibionts against bacteria and

diatoms.

2. Identify the taxonomy of the epibionts with inhibitory properties and establish

a correlation with previous studies.

3. Screen Pseudoalteromonas and Roseobacter strains for anti-diatom activity.

4. Identify potential genes involved in the expression of the anti-diatom

compound produced by P. tunicata and suggest a hypothetical model to

describe the mechanism.

Chapter 2: Inhibitory activity of epiphytic bacteria isolated from Ulva


Chapter 2: Inhibitory activity of epiphytic bacteria isolated

from Ulva

2.1 Introduction

Marine macroalgae are prone to colonization by fouling organisms. To limit fouling,

some macroalgae produce secondary metabolites that mediate intra- and interspecific

interactions (Harborne, 2001; Rosenthal and Berenbaum, 1992). Although such

chemical defences are effective, they are expensive in terms of the energy required for

metabolite production (Thomas, 2001). Instead simpler algal forms are suggested to

have alternative mechanisms to prevent surface fouling.

Some bacteria produce inhibitory compounds that prevent surface fouling. Algae that

lack physical or chemical defence mechanisms are thought to form symbioses with

these bacteria (Holmström and Kjelleberg, 1999; Rao et al., 2005; Longford et al.,

2007). Various studies show that epiphytic bacteria on marine surfaces display

inhibitory activity against fouling organisms (Armstrong et al., 2001; Berland et al.,

1972; Thomas and Allsopp, 1983; Holmstrom et al., 1996). For example, Lemos et

al., (1985) isolated epibionts from five species of green and brown algae and found

that 38 of 224 isolates displayed antibacterial activity.

Bacteria of the Flavobacterium group, various Pseudoalteromonas and Alteromonas

spp. (Holmstrom and Kjelleberg. 1999) and members of the Roseobacter clade (Rao

et al., 2005) are commonly found on marine surfaces. Amongst these bacteria,

Pseudoalteromonas is unique. Fairly recently, the genus was established to contain

various species that produced biologically active molecules (Holmstrom and

Kjelleberg, 1999). In particular P. tunicata has the ability to influence the behaviour

of higher organisms (Holmstrom et al., 1998). Another ecologically important group

is the Roseobacter clade. The group includes the species Phaeobacter gallaeciensis,

frequently isolated from the surface of U. australis (Shiba, 1992), marine snow

particles (Gram et al., 2002) and dinoflgellates (Alavi et al., 2001; Lafay et al., 1995;

Miller and Belas, 2004). The bacterium demonstrates antibacterial activity (Brinkhoff

et al., 2004; Rao et al., 2005; Ruiz-Ponte et al., 1998). Advancement of current



biofouling-control strategies depends on the study of such inhibitory epiphytic

bacteria which may lead to the discovery of new bioactive compounds.

Much is already known about the antibacterial, anti-algal and anti-larval properties of

surface colonizing bacteria. However, anti-diatom capacity remains largely

unexplored. Diatoms are amongst the early colonizers of marine substrates and are

important components of the biofouling community. The lack of study largely reflects

lack of suitable culture techniques of diatoms in bioassay screens.

The green algae, Ulva spp. does not produce secondary metabolites with recognised

roles in fouling prevention (Awad, 2000; Abd El-Baky et al., 2008) but does play host

to antifoulant producing bacteria (Holmstrom et al., 1996; Lemos et al., 1985). Hence

the alga is a suitable model system for exploring the role of inhibitory bacteria. Ulva

is a cosmopolitan chlorophyte and found in temperate and tropical waters, including

the shores of Fiji. Compared to temperate climates, tropical conditions may support a

greater microbial diversity, but this remains unexplored. The study aimed to

investigate whether Ulva growing in Fiji waters had surface microflora similar to that

of temperate waters, which it relied upon for the prevention of surface fouling. The

approach was to test isolated bacteria for inhibitory activity against both bacteria and

diatoms. The epibionts with inhibitory properties were taxonomically classified and

correlated with previous studies.

2.2 Materials and methods

2.2.1 Isolation of epiphytic bacteria from Ulva

Samples of the green algae Ulva, were randomly collected from the intertidal zone of

Laucala Bay, Suva, Fiji (18°06'S, 175°30'E). Collections were made at low tide and

samples stored in sterile polyethylene bags for transport to the laboratory. The algal

samples were washed with sterile seawater to remove loosely attached bacteria. To

isolate bacteria that were tightly bound to the algal surface, samples were placed in

vials containing 10 ml of sterile seawater and vortexed for 5 min. Aliquots of the cell

suspensions were used to inoculate marine agar (Difco marine broth solidified with

1.5% agar) which were then incubated at 23°C for 48 hr. Morphologically distinct



bacterial colonies were selected and stored at -80°C in 30% glycerol (v/v). Isolates

were routinely grown and maintained on marine agar at 23°C.

2.2.2 Bioassay against growth of bacteria

Bacteria isolated from the surface of Ulva were tested for antibacterial activity

alongside three common laboratory strains; E. coli, Pseudomonas aeruginosa PAO1

and Bacillus strain CC6 (culture collection, Centre for Marine Bio-Innovation,

University of New South Wales, Australia). The antimicrobial assay was modified

from Rao et al., (2005). Broth cultures of algal isolates were grown for two days at

room temperature (23°C) and the supernatant isolated by centrifugation at 13 000 × g

for 5 min. Supernatant samples were assayed for inhibitory activity using the drop

assay. Briefly, 100 μl of 48 hr old target marine isolate and 24 hr old target laboratory

isolate were spread on marine agar plates, and the plates were dried at 30°C for 30-60

min. Drops containing 10 μl of the test isolate supernatant, as well as a control

(uninoculated marine broth) were placed on the agar surface and incubated at room

temperature (23°C) for two days to allow formation of inhibition zones. Assays were

conducted in triplicate.

2.2.3 Bioassay against growth of diatoms

The effect of the bacterial isolates on diatom growth was assessed using the pennate

diatom, Cylindrotheca fusiformis, (CSIRO microaglae culture collection, Hobart,

Australia). C. fusiformis was chosen as the target diatom as it grows rapidly on agar

plates (Chan et al., 1980). Diatoms were subcultured routinely by aseptically

transferring 5 ml of the inoculum to 100 ml of the diatom culture solution-f/2

(Guillard and Ryther, 1962; Appendix I) and incubating at 20°C. A photoperiod of 16

hr light: 8 hr dark was provided (as per supplier’s instructions).

To determine inhibitory activity, the diatom plating assay (Chan et al., 1980) was

used with some modifications. Briefly, 300 μl of exponential phase diatom culture

(with optical density of higher than 0.06, at 600nm) was spread evenly over the

surface of marine agar (Difco marine broth solidified with 1.5% agar) using a sterile



glass spreader and allowed to dry. The bacterial test culture was transferred to the

centre of inoculated plates by spotting with an inoculating loop. Plates were incubated

inverted at 20°C with a photoperiod of 16 hr light: 8 hr dark. Light was provided both

from above and below the plates. Growth was monitored over 4 days. Anti-diatom

activity was indicated by the presence of growth inhibition zones. Plates inoculated

with diatom cultures only, served as a control for diatom growth. Assays were

conducted in triplicate.

2.2.4 Identification of isolates with inhibitory properties

2.2.4.1 DNA extraction

DNA was extracted from bacterial isolates with inhibitory activity against bacteria

and diatoms. Extractions were conducted using the XS-buffer method (Tillett and

Neilan, 2000; Appendix I). A 2 ml aliquot of a 2 day old culture was pelleted by

centrifugation, the supernatant discarded, and cells subsequently resuspended in 1 ml

of XS-buffer. The suspension was incubated at 70°C for 60 min. After incubation,

tubes were vortexed for 10 sec and placed on ice for 30 min. Tubes were then

centrifuged at 21 000 × g for 10 min. The supernatant was transferred to a clean 2 ml

microcentrifuge tube, 1 volume of isopropanol was added and the solutions mixed.

The tubes were incubated at room temperature for 5 min and then centrifuged at

21000 × g for 10 min. The supernatant was decanted and the pellet washed with 70%

(v/v) ethanol. The pellet was then air dried before being resuspended in 50-100 μl of

sterile deionised water.

2.2.4.2 Agarose gel electrophoresis

The extracted DNA was examined by electrophoresis on 1% (w/v) agarose gel using

�-DNA digested with EcoRI/HindIII as a size marker and for concentration

estimation. Gels were run in 1 × TBE buffer (Appendix I) at 80 volts for 20-30 min,

stained with ethidium bromide, destained in TBE buffer, and then photographed using

the Gel-Doc Imaging system (BioRad).



2.2.4.3 PCR amplification of 16S rRNA gene

The 16S rRNA gene was amplified using the polymerase chain reaction (PCR).

Reaction volumes of 20 μl contained 1 × PCR Reaction Buffer (Invitrogen), 250 μM

of each deoxynucleotide triphosphate (dNTP), 25 pmol of each of F27 and R1492

primers (Appendix II), 2.5 mM MgCl2 (Invitrogen), 0.05 unit Platinum Taq DNA

Polymerase (Invitrogen) and 10 ng of extracted DNA template. Reaction mixture was

thermocycled as follows: 30 cycles of denaturation at 94°C for 30 sec, annealing at

50°C for 30 sec and extension at 72°C for 2 min. A final extension step was

performed at 72°C for 5 min and samples were held at 4°C.

PCR product concentration was estimated by agarose gel electrophoresis as described

in section 2.2.4.2. For successful amplifications, the PCR product was purified using

QIAquick PCR Purification Kit as per manufacturer’s instructions. Purified products

were examined using gel electrophoresis as described above (section 2.2.4.2.).

2.2.4.4 DNA sequencing and sequence analysis

The purified PCR product were sequenced unidirectionally using 25 pmol of either of

F27 or R1492 primers (Appendix II), 20 ng of DNA template, 5 × CSA sequencing

buffer (Applied Biosystems), 1 unit of BigDyeTM terminator cycle sequencing

reaction mix v.3.1 (Applied Biosystems) and sterile deionised water in a final volume

of 20 μl. Cycle sequencing was conducted using the following thermoprofile: 94°C

for 10 sec, 50°C for 5 sec and extension at 60°C for 4 min in 99 cycles. Extension

products were purified by ethanol precipitation. Specifically, 5 μl of 125 mM EDTA

and 60 μl of 100% ethanol were added to each reaction tube and vortexed briefly. The

extension products were left to precipitate for 30 min at room temperature. Tubes

were then centrifuged at 21 000 × g for 20 min and the supernatant aspirated. The

pellet was washed twice with 70% ethanol (v/v), the tubes briefly vortexed and

centrifuged at 4°C, at 21 000 × g for 10 min. Samples were dried in a speedvac for 15

min. Sequencing was performed on an ABI 3730 DNA sequencing system at the

Automated Sequencing Facility, UNSW. Sequences obtained were compared to

sequences available in the NCBI BLAST 2.0 database (Altschul et al., 1990).

Phylogenetic analysis was performed using the sequence data software, ARB (Ludwig



et al., 2004) and the Greengenes database (DeSantis et al., 2006). Specifically,

sequences were aligned to the Greengenes database in ARB using the integrated

aligner. The sequence alignments were manually checked and redefined, if necessary.

Placement of the sequences in the phylogenetic tree was determined by the maximum

parsimony algorithm implemented in ARB. Taxonomic assignment was based on

closely-related strains in the tree and the Hugenholtz taxonomy included in the

Greengenes database. In addition, taxonomic classification was also undertaken by the

Ribosomal Database Project II classifier based on a naïve Bayesian rRNA classifier

(Wang et al., 2007).

2.3 Results

2.3.1 Epiphytic bacteria isolated from Ulva

Temporal replicates of culturing efforts from Fijian Ulva spp. yielded different

bacterial morphotypes, of which 14 could be routinely sub-cultured. Using colony

morphology and Gram staining, redundant isolates were eliminated. A total of 10

distinct bacterial isolates were chosen and used for further experiments.

2.3.2 Production of extracellular antibacterial compounds

Bacteria isolated from Ulva along with laboratory strains were used as target strains in

assessing antibacterial activity of the Ulva epibionts. Table 2.1 summarizes the effect

of epiphytic bacteria on the growth of 13 bacterial strains. Of the 10 isolates, 60%

expressed some inhibitory action. The broadest ranges of antibacterial activity were

displayed by U15 and U11, which were effective against 50% and 33% of the target

strains, respectively. Isolates U8, U12, U13 and U14 failed to inhibit the growth of any

of the bacterial strains tested.



2.3.3 Growth inhibition of diatoms

The effect of the 10 isolated epiphytic bacteria on the growth of C. fusiformis is

summarized in Table 2.2. Eight of the ten isolates showed inhibitory activity against

C. fusiformis. Isolates U7, U11 and U15 were the most effective whereas isolates U1 and

U12 had no effect on C. fusiformis.

Table 2.1: Antibacterial activity expressed by bacteria isolated from Ulva

Target

Strain

Bacterial Isolate

U1

(mm)a

U3

(mm)

U4

(mm)

U7

(mm)

U8

(mm)

U11

(mm)

U12

(mm)

U13

(mm)

U14

(mm)

U15

(mm)

U1 * 1 0 0-1e 0 0-1e 0 0 0 0-1e

U3 0 * 0 0 0 0 0 0 0 0-1e

U4 0 0 * 0 0 0-1e 0 0 0 0

U7 0 0 0 * 0 0-1e 0 0 0 0-1e

U8 1 1-2 0-1e 0 * 0-1e 0 0 0 0-1e

U11 0 0 0 0 0 * 0 0 0 0

U12 0 0 0 0 0 0 * 0 0 0

U13 0 0 0 0 0 0 0 * 0 0

U14 0 0 0 0 0 0 0 0 * 0

U15 0 0 0 0 0 0 0 0 0 *

E. colib 0 0 0 0 0 0 0 0 0 0

PAO1c 0 0 0 0 0 0 0 0 0 1

CC6d 0 0 0 0 0 0 0 0 0 2

*Autoinhibitory activity was not tested. a The radius of growth inhibition measured in millimetres. b Escherichia coli c

Pseudomonas aeruginosa PAO1 d

Bacillus strain CC6

e Growth inhibition radii of a value greater than zero and less than one millimetre.



Table 2.2: Inhibition of C. fusiformis as expressed by bacteria isolated from Ulva

Bacterial Isolate Inhibition Zone Radii (mm)*

U1 0

U3 0-1a

U4 0-1a

U7 0-2b

U8 0-1a

U11 0-2b

U12 0

U13 0-1a

U14 0-1a

U15 0-2b

*The radius of growth inhibition measured in millimetres. a Growth inhibition radii of a value greater than zero and less than one millimetre. b Growth inhibition radii of a value greater than zero and less than two millimetres.

2.3.4 Characterization of active bacterial strains

On the basis of screening results, 8 active strains were chosen for species

identification. Table 2.3 summarises the identity of the isolates based on comparison

to NCBI, Ribosomal Project and ARB Project databases. Isolates were found to have

high identity (99-100% base identity) with those of previously sequenced marine

bacteria. Phylogenetic relations of the isolates to their closest groups are given in

Figures 8.1-8.6 (Appendix III).

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2.4 Discussion

2.4.1 Antibacterial activity of bacterial isolates

Antibacterial activity is known to be present in many surface-associated bacteria

(Holmström and Kjelleberg, 1999; Rao et al., 2005; Longford et al., 2007). Many

isolates in this study also displayed antibacterial activity (Table 2.1). Additionally,

U15 which inhibited the largest number of isolates was pigmented. This correlates

with a previous study where pigmentation was linked to the production of antifouling

compounds. The study by Egan et al., (2002b) observed that colour mutants of P.

tunicata differed in antifouling characteristics. Moreover, loss of antifouling activities

and pigmentation was the result of disruption to genes with sequence similarities to

transcriptional regulators, ToxR from Vibrio cholerae and CadC from Escherichia

coli (Egan et al., 2002a).

During colonization, epiphytic bacteria compete for space and nutrients. Having

antimicrobial properties confers selective advantages during colonization. It also

provides protection to the host by reducing colonization of fouling organisms.

Numerous studies show that bacteria produce active compounds against other

microorganisms as well as against higher organisms (Egan et al., 2000; Kjelleberg et

al., 1997). This characteristic may contribute to the overall microbial diversity on an

algal surface since colonization and settlement of other organisms becomes highly

regulated. Generally, a multitude of factors such as chemical-mediated interactions,

communication, space and nutrient limitation and competition may shape the

composition and properties of a surface community (Egan et al., 2008).

2.4.2 Anti-diatom activity of bacterial isolates

Anti-diatom activity was observed to occur widely amongst the bacterial isolates

(Table 2.2). The presence of growth inhibition zones on assay plates may indicate the

production of inhibitory substances by these isolates. A few studies have investigated

anti-diatom activity in marine bacteria. Silva-Aciares and Riquelme (2007) recently

studied the effect of bacterial biofilms of Alteromonas sp. strain Ni1-LEM on the

settlement of 8 marine benthic diatoms. Comparison was made against Halomonas



marina (ATCC 25374) and Pseudoalteromonas tunicata, reference strains with

proven antifouling properties. The highest antifouling activity was found for the

Alteromonas strain. Similarly, a study of diatom settlement responses to crude

extracts of several sponge species linked laboratory results with field evidence. In the

laboratory, 6 out of 7 sponge extracts inhibited growth and caused mortality of the

pennate diatom Nitzschia paleacea at tissue-level concentration. For field

experiments, sponge metabolites immobilized in a gel matrix were exposed to natural

microbial communities. After 7 days of exposure, 6 extracts suppressed the

recruitment of diatoms (Dobretsov et al., 2005). These results indicate that anti-

diatom activity is a common feature of marine surface-associated organisms.

However, lack of suitable assay techniques has limited diatom-related studies.

The ecological significance of anti-diatom activity may be that bacteria are able to

symbiotically exist with the host by preventing settlement and growth of diatoms in

exchange for space and nutrients. Additionally, bacteria with the ability to inhibit

diatom growth may be at an advantage during colonization with more effective

isolates being more competitive than other surface colonizers.

2.4.3 Identification of active bacterial strains

Most of the isolates characterised in this study share sequence similarity to surface-

associated marine bacteria previously isolated from temperate habitats. The majority

of the isolates in the present study fall within the Proteobacteria (Table 2.3), a finding

consistent with the global distribution of this group (Britschgi and Giovannoni, 1991;

Schmidt et al., 1991; Field et al., 1997). As noted by Longford et al., (2007)

comprehensive studies have led to the recognition of bacterial distribution in

planktonic communties. In contrast, the study of living surface-associated biofilms is

still in its infancy. Hence there is insufficient data to make similar large-scale

comparisons of epibionts.

Several studies have looked at the diversity of bacteria on Ulva (Longford et al.,

2007; Skovhus et al., 2007; Egan et al., 2000). Using a culture-independent method,

Longford et al., (2007) found bacteria from an estimated 36 species and 4 phyla

present on the surface of U. australis. These included Alpha-, Delta- and



Gammaproteobacteria along with Planctomycetes and Bacteroidetes. Moreover,

bacteria of the genus Pseudoaltermonas has also been isolated from the surface of

Ulva (Skovhus et al., 2007; Egan et al., 2000). In addition, a study showed that

bacterial cells on the surface of U. australis consisted of approximately 70%

Alphaproteobacteria and 13% Cytophaga-Flavobacteria (Tujula, 2006). In this study,

except for one isolate, all were identified as Gammaproteobacteria.

It is speculated that expertise in culture-independent studies may be useful for

demonstrating a greater microbial diversity on tropical Ulva spp. that has potential for

more antifouling activities. Since marine bacteria have low culturability and are slow

growing, cell recovery may be increased by increasing incubation period. In addition,

using alternative media (e.g. a polysaccharide containing marine medium with a

variety of substrates such as sea salts) could lead to the isolation of a wider range of

bacteria.

Two inhibitory isolates (U4 and U8) had highest sequence similarity with Shewanella

sp. (Table 2.3; Figures 8.2 and 8.4, Appendix III). Isolate U4 showed 100% similarity

to Shewanella oneidensis SCH0402, Genbank accession AY881235. This bacterium

was originally isolated from temperate South Korean waters. The strain was found to

be most active against a range of target bacteria and to have stronger repellent activity

than tributyltin oxide (Bhattarai et al., 2006). Two antifouling compounds have been

isolated from Shewanella oneidensis SCH0402, identified as 2-hydroxymyristic acid

and cis-9-oleic acid (Bhattarai et al., 2007). These may or may not be the same

compounds responsible for antifouling activity observed in this study. Isolate U8 also

showed high sequence similarity (99%) to Shewanella sp. However, based on

phylogenetic analysis on the ARB database, U8 appeared to be a different species to

U4 (Figures 8.2 and 8.4, Appendix III).

Also identified in this study were 3 isolates (U7, U11, and U15) belonging to the genus

Pseudoalteromonas (Table 2.3; Figures 8.3 and 8.6, Appendix III). Bacteria of the

genus Pseudoalteromonas are often present on the surface of Ulva spp. (Egan et al.,

2000; Skovhus et al., 2007). Pseudoalteromonas sp. influence biofilm formation in

various marine niches; are involved in predator-like interactions within the microbial

loop; influence settlement, germination and metamorphosis of various invertebrate



and algal species; and are adopted by marine flora and fauna as defence agents

(Bowman, 2007).

Pigmented species of Pseudoalteromonas produce an array of both low and high

molecular weight compounds with anti-fouling activities. The compounds formed

include toxic proteins, polyanionic exopolymers, substituted phenolic and pyrolle-

containing alkaloids, cyclic peptides and a range of bromine-substituted compounds

(Bowman, 2007). In the current study, isolate U15, that had 100% sequence similarity

with Pseudoalteromonas rubra, was also red-pigmented and had the strongest

antibacterial and antidiatom activities (Tables 2.1 and 2.2). The bright red pigment of

P. rubra, is a low molecular weight substance, identified as cycloprodigiosin HCl

(Gerber and Gauthier, 1979; Kawauchi et al., 1997). This compound is of

pharmaceutical importance as an immuno-proliferation suppressesor (Magae et al.,

1996), for displaying anti-malarial activity (Kim et al., 1999) and for inducing

apoptosis in several cancer cell lines (Campàs et al., 2003; Perez-Tomas et al., 2003).

Substituted phenylalkenoic acids, referred to as rubrenoic acids, purified from P.

rubra show bronchodilatatoric activity. P. rubra also forms a high molecular weight

substance, which is possibly a glycoprotein or polysaccharide known to expresses

antibacterial activity. Growth inhibition by the antibiotic is due to the induction of

oxidative stress in target cells through increased O2 uptake and an accumulation of

hydrogen peroxide (Gauthier, 1976a; b). Currently there are no reports of anti-diatom

activity in P. rubra and either of the known bioactive molecules or a new molecule

may be responsible for the antidiatom characteristic of the bacterium. Isolates U7 and

U11 had respectively 99% and 100% sequence similarity to Pseudoalteromonas sp.

Moreover, phylogenetic analysis showed that the two isolates are possibly closely

related (Figure 8.3; Appendix III).

Two of the bacterial isolates (U13 and U14) were identified as members of the genus

Vibrio (Table 2.3; Figure 8.5, Appendix III). Phylogenetic analysis showed that the

two isolates may be closely related (Figure 8.5; Appendix III). Isolate U13 had closest

sequence similarity (99%) to Vibrio sp. A356, Genbank accession DQ005876. This

bacterium was originally isolated from the surface of coralline algae. The isolate

induced larval settlement in the common Australian sea urchin Heliocidaris

erythrogramma (Hugget et al., 2006). In another study, a Vibrio sp. isolated from



Ulva reticulata, growing in Hong Kong waters inhibited settlement and

metamorphosis of the polychaete Hydroides elegans larvae (Dobretsov and Qian,

2002). Such findings suggest that Vibrio spp. live in close association with algal

surfaces and may either play an inhibitive or inductive role. The results of this study

correlate with findings of Dobretsov and Qian (2002), highlighting inhibitory

characteristics of Vibrio spp. Prior to the results presented here, antidiatom activity in

Vibrio spp. have not been reported.

Isolate U3 had activity against C. fusiformis and was identified as having closest

sequence similarity (99%) to Bacillus sp. (Table 2.3; Figure 8.1, Appendix III). The

general understanding of marine bacterial diversity gained from planktonic and

epiphytic communities suggest that the majority of isolates are Gram-negative

(Farmer and Hickman-Brenner, 1992). Also, recent culture-independent studies of

bacterial diversity on the surface of Ulva spp. have not reported the presence of

Bacillus sp. (Tujula, 2006; Longford et al., 2007). However, there is now an increased

awareness of the presence of true marine Gram-positive bacteria, with a large number

being isolated from marine sediments. Recently, Gontang et al., (2007) isolated 1,624

diverse Gram-positive bacteria spanning 22 families with 66% belonging to the class

Actinobacteria and the remaining 34% being members of the class Bacilli. Gram-

positive bacteria of the genus Bacillus have been reported in another study where B.

firmus and B. mojavensis were isolated from the marine environment (Gontang et al.,

2007; Ivanova et al., 1999; Ortega-Morales et al., 2008). Furthermore, members of

the genus are known to produce bioactive lipopeptides that are responsible for the

antibacterial activity observed (Ortega-Morales et al., 2008). This study has

highlighted anti-diatom characteristics within Bacillus, which has not been reported

previously. The mode of inhibition remains to be explored.



2.5 Conclusion

This chapter highlights the presence of surface associated bacteria on Ulva that may

be responsible for the algae remaining unfouled in an environment prone to

biofouling. Both antibacterial and anti-diatom activities are common characteristics of

the epiphytic bacteria, with anti-diatom activity being a more widely prevalent

feature. The epiphytic anti-fouling bacterial groups identified include the genera

Shewanella, Vibrio, Bacillus and Pseudoalteromonas. The study further highlights

anti-diatom activity in Bacillus and Vibrio, groups where such action has not

previously been recognised. The close association between Pseudoalteromonas and

algal surfaces has gained a lot of attention recently. This has mainly been due to its

significant antifouling properties and success as surface colonizers. This study also

suggests a close affiliation between Pseudoalteromonas and Ulva since the strains

isolated from the algae displayed both antibacterial and anti-diatom properties.

The microfloral similarity on Ulva isolated from temperate and tropical regions is

highlighted. It is evident that Ulva supports growth of many epiphytic bacteria that

have potential as antifoulants. The abundance of Ulva, combined with warm tropical

conditions may support high bacterial diversity, leading to the discovery of novel

antifoulants. Together with advances in studies of microbial mediated defence

systems, its application will be highly beneficial for the development of antifoulants.

Chapter 3: Anti-diatom properties of Pseudoalteromonas and Roseobacter strains


Chapter 3: Anti-diatom properties of Pseudoalteromonas

and Roseobacter strains

3.1 Introduction

Diatoms, like bacteria are primary colonizers of marine surfaces. Upon adhesion,

through their characteristic secretion of adhesive mucilage, diatoms play an important

role in early film formation. As the biofilm grows, adhesive exudates are released,

trapping additional particles and microorganisms, progressively leading to the

formation of a mature biofilm community (Silva-Aciares and Riquelme, 2007).

Once mature biofilms have formed on vessels, they are difficult to remove. The

development of silicone-fouling release coatings have decreased the adhesion strength

of attached organisms, which are removed as the vessel moves through water.

Although macroalgae and some hard foulers such as barnacles detach relatively

easily, diatom slimes, oysters and tubeworms are attached tenaciously and are not

easily removed, even at high speed (Callow and Callow, 2002). Due to the need for

more efficient diatom control measures researchers are now exploring natural

products for more effective alternatives (Silva-Aciares and Riquelme, 2007).

Living surfaces in the marine environment have developed efficient means of keeping

their surface free of diatoms. Dobretsov et al. (2005) assessed the settlement response

of diatoms to crude extracts of several sponge species from Hong Kong waters.

Experiments showed that 6 out of 7 sponge extracts inhibited growth and caused

mortality of the pennate diatom Nitzschia paleacea at tissue level concentration.

Similarly, a study explored the anti-diatom strategy of the blue mussel, Mytilus edulis�

(Bers et al., 2006). It was found that attachment of the benthic diatom Amphora

coffeaeformis was significantly reduced by dichloromethane extracts, whereas ethyl

acetate and diethyl ether fractions slowed diatom growth. These results provide

evidence that living surfaces in the marine environment may moderate surface

colonization of diatoms.



Pseudoalteromonas tunicata and Ph. gallaeciensis are regarded as model epiphytic

bacteria living in close association with marine algae. Both species are often isolated

from the surface of Ulva spp. and are known specifically to prevent the settlement of

common fouling organisms (Bowman, 2007; Brinkhoff et al., 2008). With the success

of P. tunicata and Ph. gallaeciensis as surface colonisers, it is predicted that other

members of these genera may also express antifouling activities. Additionally, anti-

diatom activity is a characteristic largely unexplored in Pseudoalteromonas and

Roseobacter spp. Such inhibitory activities may involve novel bioactive compounds.

The study aimed to explore and compare the prevalence of anti-diatom activity across

Pseudoalteromonas and Roseobacter spp. Since relatively little is known about the

defence strategies of epiphytic bacteria against diatoms, the study further aimed to

provide an insight into the mechanism. Information on gene identity and the

transposon Tn10 were used as tools to manipulate the genome of the model epibiont,

P. tunicata. Finally, a hypothetical model for the expression of anti-diatom activity in

P. tunicata was proposed.

3.2 Materials and methods

3.2.1 Screening Pseudoalteromonas and Roseobacter strains for anti-diatom

activity

Eight Pseudoalteromonas species strains and sixteen Roseobacter clade strain

members (Pseudoalteromonas aurantia, Pseudoalteromonas citrea,

Pseudoalteromonas piscicida, Pseudoalteromonas undina, Pseudoalteromonas ulvae,

Pseudoalteromonas haloplanktis, Pseudoalteromonas nigrifaciens,

Pseudoalteromonas tunicata, Rhodobacter sphaeroides 2.4.1, Rhodobacter

sphaeroides 17025, Rhodobacter sphaeroides 17029, Roseovarius nubinhibens ISM,

Sulfitobacter sp. EE-36, Roseovarius nubinhibens ISM, Sulfitobacter sp. NAS-14.1,

Sagittula stellata E-37, Silicibacter pomeroyi DSS-3, Dinoroseobacter shibae DFL

12, Maricaulis maris MCS10, Jannaschia sp. CCS1, Roseobacter sp. CCS2, Ruegeria

R11, Phaeobacter gallaciensis sp. 2.10 and Phaeobacter gallaciensis BS107) were

obtained from the culture collection at Centre for Marine Bio-innovation, University

of New South Wales, Australia. To screen for anti-diatom properties, the previously

described anti-diatom bioassay (section 2.2.3) was used.



3.2.2 Analysis of anti-diatom strategy of P. tunicata

Transposons are discrete DNA segments that can repeatedly insert at sites in a

genome and are therefore useful tools for genetic manipulation. In transposon

mutagenesis, random insertions occur within a specific gene and result in the loss of

function of that gene. One such system is a modified version of the transposon Tn10

known as mini-Tn10 (Herrero et al., 1990). This transposon carries a kanamycin-

resistance marker that allows for easy selection of mutants. In addition, the

transposase gene is outside of the mobile element, which allows for a stable insertion

because of the loss of the transposase gene during the transfer. A transposon mutant

library was created for P. tunicata. The library was screened for mutants lacking in

anti-diatom activity and genetic analysis of transposon insertion sites in mutants were

used to identify the affected genes.

3.2.2.1 Transposon mutagenesis

The transposon mutagenesis protocol established by James (1998) with modifications

was used to generate non anti-diatom mutants of P. tunicata. Overnight cultures of

both donor E. coli Sm 10 (containing pLOF mini-Tn 10 system) and the streptomycin

resistant recipient P. tunicata (SmR) were prepared. The E. coli strain was grown

with shaking at 37°C in LB10 medium (Appendix I) containing 85 μg/ml kanamycin

and 100 μg/ml ampicillin. The P. tunicata strain was grown with shaking at room

temperature in marine broth containing 200 μg/ml streptomycin.

The overnight cultures were washed twice to remove residual antibiotics. This was

done by spinning the cultures at 5400 × g for 2 min, then resuspending in fresh media

lacking antibiotics. After washing, the cells were resuspended in 1 volume of sterile

10 mM magnesium sulphate and gently mixed. To allow conjugation, a sterile 0.22

μm (2.5 cm diameter) membrane filter was placed onto LB15 agar plate (Appendix I)

containing 0.3 mM isopropyl-ß-D-thiogalactoside (IPTG). Donor and recipient

cultures were mixed on the membrane filter in a volume ratio of 1:3 (50 μl E. coli +

150 μl P. tunicata) and incubated for 4 hr at 30°C. Following incubation, cells were

removed from filters by gently rolling it up and placing into microcentifuge tubes

containing 1 ml marine broth and vortexed for 1 min. A 100 μl aliquot of the cell



suspension was plated onto marine agar (Difco marine broth solidified with 1.5%

agar) containing 85 μg/ml kanamycin and 200 μg/ml streptomycin to select for

recipient P. tunicata strains carrying the mini-Tn10 transposon. Plates were incubated

for 48 hr at 30°C. Colonies were transferred from agar plates to 96-well plates

containing 150 μl of marine broth containing 85 μg/ml kanamycin and 200 μg/ml

streptomycin. Plates were incubated with shaking at 30°C for 48 hr. A 65 μl aliquot of

glycerol was added to each well after the incubation period and plates stored at -80°C.

3.2.2.2 Screening for P. tunicata mutants lacking anti-diatom property

Screening for mutant phenotype was performed on 12 inch square agar plates. A 2 ml

aliquot of diatom culture was spread plated onto each plate. An ethanol sterilized 96-

pin replicator was used to transfer mutants from 96-well plates to the agar surface.

Plates were incubated inverted at 20°C in a photoperiod of 16 hr light: 8 hr dark.

Light was provided both from above and below the plates. Growth was monitored

over 5 days. Loss of anti-diatom activity was inferred if a mutant failed to produce an

inhibition zone. Plates inoculated with P. tunicata wild type served as controls.

Confirmatory tests were performed on mutant strains by re-testing for the loss of anti-

diatom activity as described above.

3.2.2.3 Growth rates of mutants

A comparison of the growth rates of transposon mutants and wild-type P. tunicata

was performed. Strains were grown in 500 ml conical flasks containing 200 ml of

growth media. The wild type P. tunicata was grown in marine broth whereas the non

anti-diatom transposon mutants, DM1, DM2, DM3 and DM4 were grown in marine

broth containing 85 μg/ml kanamycin and 200 μg/ml streptomycin. A 2 ml aliquot of

an overnight culture was inoculated into an appropriate flask and incubated shaking at

23°C. Growth was monitored by absorbance readings at 610 nm over a 24 hr period.

The experiment was carried out in duplicate and mean absorbance values were used to

plot the growth curve of tested strains.



3.2.2.4 Genomic DNA extraction of non anti-diatom mutants

Genomic DNA was extracted from mutant cultures using the XS-buffer method

(Tillett and Neilan, 2000), outlined in section 2.2.4.1. The extracted DNA was

visualized by electrophoresis on 1% (w/v) agarose gel using �-DNA digested with

EcoRI/HindIII molecular weight marker for size and concentration estimation.

3.2.2.5 Generation of adaptor ligated DNA for panhandle PCR

A suppression PCR (Siebert et al., 1995) method termed pan-handle PCR can be used

to walk from a known region into an unknown region in genomic DNA. The DNA

sequence of the mini-Tn10 transposon is known. Hence, it is possible to use the

panhandle-PCR method to obtain sequence information for the genes disrupted by the

transposon. Due to the presence of inverted terminal repeats in adaptor molecules,

PCR amplification of fragments with adaptor sequence at both ends will result in the

ends of individual DNA strands forming “panhandle” structures following every

denaturation step. Since these structures are more stable than the primer-template

hybrid, exponential amplification is suppressed (Siebert et al., 1995). In contrast, PCR

products formed by gene-specific primer and adaptor primer combinations cannot

form panhandle structures, allowing PCR amplification to continue.

Genomic DNA extracted from mutant cultures was used for restriction digestion and

ligation of adaptor molecules in a one step process. Each reaction mixture contained 1

μg of genomic DNA, 10 pmol/μl adaptor 1 (Appendix II), 10 pmol/μl adaptor 2

(Appendix II), 20 mM ATP, 2.5 units of T4 ligase, 0.5 units of blunt-end restriction

enzyme (various, see Table 3.1), 1 × respective restriction enzyme buffer and

deionised water to give a final reaction volume of 20 μl. Reactions were incubated at

20°C for 16 hr, after which the reaction was deactivated by heating to 68°C for 10

min.

The DNA was then precipitated using ethanol. Briefly, a 1/10th volume of 3 M

sodium acetate (pH 5.2) was added and mixed well. Exactly 2.5 volumes of ice-cold

absolute ethanol was then added and again mixed well. Tubes were chilled at -20°C

for 60 min. After incubation, DNA was pelleted by centrifugation at 21 000 × g and



4°C for 15 min. The supernatant was discarded and the pellet was washed in 70 %

(v/v) ethanol to remove salt. The tubes were inverted to dry the pellet, following

which DNA was resuspended in 50 μl of sterile deionised water. This solution served

as the template DNA for the PCR reactions described below.

Table 3.1: Restriction enzymes used for panhandle PCR

Restriction enzyme1 Recognition sequence

DraI TTT↓AAA

EcoRV GAT↓ATC

HincII GT(T,C) ↓(A,G)AC

HpaI GTT↓AAC

PvuII CAG↓CTG

RsaI GT↓AC

ScaI ACT↓ACT

SspI AAT↓ATT

XmnI GAANN↓NNTTC

1All enzymes were purchased either from BioLabs, Promega or Roche

3.2.2.6 Panhandle PCR

PCR was performed in 20 μl reaction volumes using 1 μl of DNA solution (as

described above), 10 mM dNTPs, 10 pmol of adaptor primer 1 (Appendix II), 10

pmol of sequence specific primer (Tn10C or Tn10D, Appendix II), 1 × Taq buffer, 2.5

mM MgCl2 and 0.05 unit of Taq polymerase added after a hot start (95°C for 2 min).

The cycle parameters were denaturion at 95°C for 30 sec and annealing/extension at

65°C for 7 min for 25 cycles. A final extension was performed at 65°C for 1 min and

samples were held at 4°C.

PCR product concentration was estimated by agarose gel electrophoresis as described

in section 2.2.4.2. Single band products were purified using QIAquick PCR



Purification Kit as per the manufacturer’s instructions. When more than one band was

present, the band of interest was excised and DNA extracted from the gel slice using

Invitrogen Quick Gel Extraction Kit following the manufacturer’s instructions. The

purified product was examined by visualizing on agarose gel once again.

3.2.2.7 Sequencing

The purified PCR products were sequenced independently using the corresponding

transposon-specific and adaptor primers. Reaction mixtures contained 15-20 pmol of

either of Tn10C, Tn10D, AP1 or AP2 primers (Appendix II), 50-100 ng of DNA

template, 5 × CSA sequencing buffer, 1 unit of BigDyeTM terminator cycle

sequencing reaction mix v.3.1 (Applied Biosystems) and sterile deionised water in a

final volume of 20 μl. Cycle sequencing was conducted using the following

thermoprofile: 94°C for 10 sec, 50°C for 5 sec and extension at 60°C for 4 min in 99

cycles. Extension products were purified by ethanol precipitation as described in

section 2.2.4.4. Separation of sequencing products was performed on an ABI 3730

DNA sequencing system at the Automated Sequencing Facility, UNSW. Sequences

obtained were compared to protein sequences available in the NCBI BLAST 2.0

(Altschul et al. 1990) and IMG 2.41 (Markowitz et al., 2008) databases in order to

identify genes disrupted by the transposon.

3.3 Results

3.3.1 Growth inhibition of diatoms

All Pseudoalteromonas strains tested showed some degree of diatom growth

inhibition (Figure 3.1). Pseudoalteromonas aurantia, P. undina and P. haloplanktis

were the most effective while P. citrea and P. ulvae were the least inhibitory.

Additionally, replicated assay plates showed varying sizes of growth inhibiton zones

produced by P. nigrifaciens. The activity of Roseobacter strains against growth of C.

fusiformis is illustrated in Figure 3.2. Approximately 44% of the Roseobacter clade

species tested displayed anti-diatom activity. Jannaschia sp. CCS1 and Phaeobacter

gallaeciensis 2.10 were the most effective, with replicated plates showing variation in



the level of activity. Nine strains of the Roseobacter clade did not inhibit diatom

growth.

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3.3.2 Mutants lacking in anti-diatom activity

Using mini-Tn10 and the suicide vector pLOF for delivery (Way et al., 1984),

transposon mutants lacking the ability to inhibit diatom growth were successfully

generated in P. tunicata. The transposon bank consisted of approximately 1000

transconjugants. After screening it was found that 4% of the transconjugants had lost

activity. Four of the mutants (designated DM1, DM2, DM3 and DM4) were randomly

selected for re-testing. Of these, 3 were used for further genotypic analysis. Figure 3.3

illustrates the presence of a zone of growth inhibition when wild type P. tunicata was

tested against C. fusiformis and its absence when mutants of P. tunicata were used.

D2 Wild Type DM1 DM2

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To ensure that the loss of anti-diatom activity was not due to a mutation in an

essential pathway that would lead to decreased cell activity and growth, an overnight

growth experiment was performed. The wild type P. tunicata and the transposon

generated anti-diatom mutants showed no significant difference in general growth

pattern or rate. Cells entered logarithmic growth phase after approximately 2 hr and

reached stationary phase after approximately 22 hr. A graph of optical density versus

time is shown in Figure 3.4.

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Using panhandle PCR, the sites within the P. tunicata genome that had been

disrupted by insertion of the mini-Tn10 transposon were amplified (Figure 3.5).

Amplified regions were further used for sequencing to determine the transposon

insertion sites.



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3.3.5 Genotype characterization of the non anti-diatom mutants

The genomic DNA regions flanking the transposon insertion sites in each of the three

mutants were analysed by sequencing. Sequencing of the 3 mutants (DM1, DM2 and

DM3) indicated different transposon insertion sites. Descriptions of each of the

insertion sites are as follows:

3.3.5.1 DNA regions flanking the transposon insertion site in DM1

Sequencing results of the analysis of DM1 showed that the transposon had inserted

into a gene, IMG locus tag PTD2_12754, with homology to a cation/multidrug efflux

pump, AcrB/AcrD/AcrF family protein. The insertion had occurred at position 227 of

the 3075 bp open reading frame (ORF) (Figure 9.1, Appendix IV). A schematic

representation of the position of the cation/multidrug efflux pump gene within the P.

tunicata genome is shown in Figure 3.6.



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(homology to a beta-hexosaminidase) and PTD2_01391 (homology to a RTX toxin

and related Ca2+ binding protein). The gene positions are schematically represented in

Figure 3.7. The sequence obtained indicated that the transposon inserted at the end of

PTD2_01386 (position 2248 in the 2280 bp ORF), extended into the intergenic region

and partially into locus PTD2_01391 (Figure 9.2, Appendix IV).

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Sequence analysis of DM3 showed that the transposon had inserted into a 702 bp

ORF (Figure 9.3, Appendix IV), IMG locus tag PTD2_02946, a protein with

homology to the HemeO protein family. PTD2_02946 is clustered with several other

ORFs including a long chain fatty acid (LCFA)-coA ligase and a short chain alcohol

dehydrogenase-like protein. A schematic representation of the position of

PTD2_02946 is provided in Figure 3.8.

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3.4 Discussion

3.4.1 Anti-diatom activity of Psedoalteromonas spp. and Roseobacter clade

Since all the Pseudoalteromonas strains tested inhibited diatom growth, it is obvious

that this charcteristic is prevalent within the genus. Pseudoalteromonas aurantia, P.

undina and P. haloplanktis were the most active against growth of C. fusiformis

(Figure 3.1). Previously, P. aurantia has been isolated from the surface of U.

australis and shown to produce unknown compounds that inhibit settlement of

fouling organisms (Gauthier and Breittmayer, 1979; Bowman, 2007). Additionally, P.

haloplanktis produces diketopiperazines, a probiotic beneficial to shellfish. Currently

there are no reports of bioactive compounds from P. undina (Bowman, 2007).



During a study conducted to observe the range of antifouling activities expressed by

Pseudoalteromonas spp., members of the genus produced a variety of bioactive

compounds (Holmstrom et al., 2002). Most strains inhibited bacterial and fungal

growth, algal spore germination and invertebrate larvae settlement. However, P.

nigrifaciens displayed negligible activity in most bioassays. Interestingly, in this

study P. nigrifaciens inhibited the growth of C. fusiformis. The different patterns of

diatom growth inhibition by Pseudoalteromonas strains suggest diversity within the

genus. The prevalence of anti-diatom activity may be an adaptation that allows

Pseudoalteromonas to colonize a wide range of habitats.

Strains of the Roseobacter clade tested for activity against the growth of C. fusiformis

also produced varying results. The greatest diatom growth inhibition was caused by

Jannaschia sp. CCS1, followed by Phaeobacter gallaeciensis 2.10 and

Dinoroseobacter shibae DFL 12 (Figure 3.2). Although anti-diatom activity by

members of the Roseobacter clade has not been reported previously, their

antibacterial activity has been investigated in several studies (Brinkhoff et al., 2004;

Bruhn et al., 2005; Ruiz-Ponte et al., 1998). The latter characteristic is an advantage

that may contribute to the dominance of the clade in alga-associated bacterial

communities. There may be different reasons for variation in bioactive compound

production. Certainly some strains may lack the ability to produce bioactive

compounds. For example, Ph. strain 27-4 only produces antibiotic when grown in

liquid nutrient medium under static conditions, which also facilitates rosette and

biofilm formation (Bruhn et al., 2006; Bruhn et al., 2005). Such results suggest that

culture conditions influence the production of antibacterial compounds.

Approximately half (56%) of the Roseobacter strains tested did not inhibit diatom

growth. It has been suggested that the differing physiological characteristics reflect

adaptation to the diverse ecological niches that Roseobacter occupies (Brinkhoff et

al., 2008). The lack of activity against diatoms may therefore indicate that anti-

diatom activity is not conferring a specific advantage. Members of the Roseobacter

clade are found in temperate as well as polar oceans (Brinkhoff, et al., 2008) and

dominate among marine alga-associated bacteria (Alavi et al., 2001; Buchan et al.,

2005; Gonzalez et al., 2000). It is reported that 1 in 10 bacterial cells is a member of

the Roseobacter group (Giovannoni and Rappe, 2000). Since the first description of



Roseobacter sp. in 1991, 38 affiliated and validated genera have been described

(Brinkhoff, et al., 2008). In comparison, the Pseudoalteromonas group is smaller with

as few as 35 species and 2 sub-species known to date

(http://www.bacterio.cict.fr/p/pseudoalteromonas.html). As the members of the

Roseobacter clade tested were only a small representation of the actual group,

comparison of the prevalence of anti-diatom activity to the the much smaller

Pseudoalteromonas group may be biased. Additionally, these results may suggest that

the defence mechanisms of the two groups of bacteria differ. In recent years,

members of Roseobacter have gained recognition for their antifouling properties

(Brinkhoff, et al., 2008; Rao et al., 2006). In particular, Ph. gallaeciensis is known to

be a more competitive biofilm-forming bacterium than P. tunicata (Rao et al., 2006).

With such strong anti-diatom properties and competitive biofilm-forming

characteristics, this species of Phaeobacter is worth further investigating for its

potential in preventing biofouling.

3.4.2 Analysis of transposon insertion sites within the P. tunicata genome

To study the anti-diatom strategy of P. tunicata, transposon insertion sites in non anti-

diatom mutants were analysed. In the first mutant, DM1, the transposon had inserted

into a gene homologous to the cation/multidrug efflux pump (Figure 3.6). The

cation/multidrug efflux pump (AcrB/AcrD/AcrF family protein) is an extremely

conserved gene across many phyla. The protein is synonymous to acriflavin (a

common tropical antiseptic) resistance protein in Pseudoalteromonas atlantica T6c

(66% identity). In Escherichia coli, the AcrB genes encode a multi-drug efflux

system, believed to protect the bacterium against hydrophobic inhibitors (Ma et al.,

1993). The system is energized by proton-motive force and shows the widest

substrate specificity amongst known multidrug pumps, including antibiotics,

disinfectants, dyes, detergents and solvents.

ABC transporters are generally involved in transporting ions, carbohydrates, amino

acids, antibiotics, polysaccharides and proteins (Saurin et al., 1999). ABC importers

in particular, are involved in transporting ferri-siderophore complexes across the

periplasmic space and cytoplasmic membrane back into the cells (Andrews et al.,

2003). Whether an ABC system imports or exports molecule depends upon the



presence or absence of a periplasmic binding protein associated to the coding

sequences for the ABC and transmembrane domains (Linton and Higgins, 1998).

The ABC-transporters of P. tunicata have been suggested to be involved in iron

transport (Evans et al., 2007). Analysis of the conserved domains of AcrB to

determine protein function suggests a wide range of roles. This includes defence and

transport mechanisms with a weak homology to SecD. The membrane protein SecD

is a preprotein translocase subunit which is involved in intracellular trafficking and

secretion. It may be speculated that the cation/multidrug efflux pump disrupted in

DM1 is involved either in the secretion of molecules or putative toxins responsible

for the anti-diatom property of P. tunicata.

In mutant DM2 two ORFs are affected by transposon insertion (Figure 3.7). The

transposon inserted at the end of a beta-hexosaminidase homologue that in

Pseudoalteromonas sp. strain S91 is involved in chitin degradation (Techkarnjanaruk

and Goodman, 1999). Chitin is an important component of diatom cells. A study

investigated the effects of two commercial chitin synthesis inhibitors, dimilin and

polyoxin D, on chitin fiber formation and cell sedimentation in the diatoms

Thalassiosira fluviatilis and Cyclotella cryptica (Morin et al., 1986). While dimilin

treated diatoms were indistinguishable from controls, the polyoxin D treated cells of

both diatom species completely lacked the characteristic chitin fibers. The polyoxin D

cultures were also characterized by significantly lower population densities, increased

sedimentation rates and strong tendency to clump in comparison to control and

dimilin treatments (Morin et al., 1986). One function of chitin is in the formation of

spines for diatom cell buoyancy. When the spines are removed by physical shearing

or digestion by chitinase, the otherwise intact diatom cells lose their buoyancy,

settling 1.7 times faster (Walsby and Xypolyta, 1977; Smucker, 1991). These results

highlight the importance of chitin fiber formation in diatoms and suggest the likely

impact of chitin-degrading enzymes (such as beta-hexosaminidase) produced by P.

tunicata on diatoms.

To test whether a disruption at the beta-hexosaminidase gene homologue is

responsible for loss of production of a chitin degrading enzyme further studies are

required. An experiment of the mutant’s growth on media with chitin as the sole



carbon source is suggested. Growth could be monitored by measuring optical density

over a period of 48 hr. A standard logarithmic (S-shaped) graph would indicate the

mutant’s ability to degrade chitin whereas decreased growth would indicate a

disruption that results in the loss of production of the chitin-degrading enzyme.

Located downstream of the transposon insertion site is a RTX toxin-like gene (Figure

3.7). RTX toxins are pore-forming, soluble, secreted proteins produced by a broad

range of pathogenic Gram-negative bacteria. In vitro, these most often exhibit

cytotoxic and hemolytic activities. They are particularly common in the

Pasteurellaceae which are disease causing in animals and humans (Joachim and Peter,

2002). The RTX gene cluster has also been identified in Vibrio cholerae, where it

produces proteins with haemolytic activity but also has roles in biofilm formation and

cell-cell adherence (Chatterjee et al., 2008; Lin et al., 1999). Although the function of

the RTX toxin-like genes in P. tunicata is still unknown, since it is a

Gammaproteobacteria like Vibrio, it seems likely that there would be some degree of

functional similarity between these two bacteria. This is supported by the presence of

both cadherin/sarcoglycan-homologous domains and a secretory signal peptide in the

P. tunicata (PTD2_01391) gene. These suggest a secreted protein with roles in

adhesion or cell wall stabilisation. Functional similarity of Vibrio and

Pseudoalteromonas proteins has been noted previously (Egan et al., 2002a).

The sequence obtained through Panhandle PCR and sequencing overlapped mostly in

gene PTD2_01391 (homology to RTX toxin). It is unclear if the RTX toxin gene was

disrupted which led to any change in anti-diatom activity. However, it may also be

that the intergenic region upstream of the RTX toxin gene contains regulatory

regions, the disruption of which affected the expression of RTX toxin (or other gene)

leading to a loss in anti-diatom property. Research has shown that the intergenic

region between the divergently transcribed niiA and niaD genes of Aspergillus

nidulans contains multiple NirA binding sites, which act bidirectionally (Punt et al.,

1995). Apparently, the insertion of an unrelated upstream activating sequence into the

intergenic region strongly affected the expression of both genes, irrespective of the

orientation in which the element was inserted. Also, located downstream of the RTX

toxin is PTD2_ 01396 (Figure 3.7), which is a hypothetical protein, the function of



which is not yet known. However, analysis of the conserved domains predicts the

protein to be a peptidase or a periplasmic protease.

Located upstream of PTD2_01386, are two Ton-B receptor proteins (PTD2_01376

and PTD2_01381) with roles in iron transport. Faraldo-Gomez and Sansom (2003)

also suggest TonB-dependent receptors to be responsible for the transport of large

extracellular molecules, such as vitamin B12 and iron carriers (siderophores) into the

bacterial cells. The TonB proteins in E. coli interact with outer membrane receptor

proteins that bind and take up specific substrates into the periplasmic space

(Chimento et al., 2003). In the absence of TonB, the receptors bind their substrates

but do not carry out active transport (Koebnik, 2005). The TonB complex senses

signals from outside the bacterial cell and transmits them via two membranes into the

cytoplasm, leading to transcriptional activation of target genes.

In P. tunicata, gene expression of proteins involved in iron acquisition and uptake,

including TonB are controlled by WmpR (a ToxR-like regulator, that also controls

expression of bioactive compounds, type IV pili and biofilm formation; Stelzer et al.,

2006). Recent work by Evans et al. (2007) links iron transport by TonB receptors to a

type-II secretion pathway. A disruption of the type-II secretion machinery in P.

tunicata (wmpD- mutant) results in the loss of pigment production and loss of

bioactive compounds against all target organisms. In addition, the upregulation of

TonB system biopolymer-transport proteins in the wmpD- mutant suggests a role in

transport and acquisition of iron. It has thus been suggested that the type-II secretion

pathway may be responsible for the transport of extracellular enzymes that obtain

precursor molecules for pigments and other bioactive compounds.

In mutant DM3 the transposon had inserted into PTD2_02946, a hypothetical protein

with homology to the HemeO protein family (Figure 3.8). Analysis of the conserved

domains demonstrates that the protein has heme-binding capacity. Acquisition of

heme from the environment is often for the purpose of obtaining iron (Wilks, 2002).

Neisseria meningitides and Pseudomonas aeruginosa are pathogens known to utilize

the host’s heme as an iron source. These pathogens depend on heme oxygenase for

the release of iron (Ratliff et al., 2001; Zhu et al., 2000a; b). In living organisms,

including diatoms, heme biosynthesis represents an essential metabolic pathway that



provides the precursors for cytochrome prosthetic groups, photosynthetic pigments,

and vitamin B-12 (Obornik and Green, 2005).

These results imply that a mutation in PTD2_02946, may prevent iron acquisition,

which by some means impairs anti-diatom activity. It may be that P. tunicata acquires

iron from diatom heme, an interaction that disrupts an essential metabolic pathway in

diatoms. However, this remains to be studied. At this stage we can only speculate that

iron is important either indirectly (such as in the form of a precursor of a regulatory

protein) or directly for virulence against diatoms.

Analysis of the different transposon insertion sites and corresponding loss in anti-

diatom activity can be used to propose a mechanism for anti-diatom activity in P.

tunicata. Iron is suggested to be essential for the expression of anti-diatom activity.

The HemeO homologue participates in the acquisition of iron from the environment

or directly from diatoms. Type II-secretion pathway regulates the secretion of TonB,

which would be needed to bind and transport the iron. Acquired iron is then involved

either directly or indirectly in the regulation and expression of the RTX toxin-like

gene. A multidrug efflux system is involved in pumping toxic proteins out of the

bacterial cell which lead to diatom growth inhibition. This is clearly a complex

system involving a number of steps, disruption of any one leading to loss of activity.

Further study is needed to confirm this general mechanism and fill in critical

remaining details.



3.5 Conclusion

Anti-diatom activity is a characteristic feature of many Pseudoalteromonas and

members of the Roseobacter clade. These current findings emphasize the success of

these groups as marine surface colonizers and the diversity of these groups. With the

search on for more effective means of controlling biofouling, the genus

Pseudoalteromonas and the Roseobacter clade need to be studied in much greater

detail as possible sources of antifoulants.

The ability of P. tunicata to inhibit diatom growth was studied in detail by generating

transposon mutants lacking in anti-diatom activity. Four mutants were successfully

generated, of which three were further analysed. Results suggest several possible

mechanisms of expression of anti-diatom activity. Genes observed to be important in

anti-diatom activity were predicted to have functions including a cation/multidrug

efflux pump, a beta-hexosaminidase protein, a RTX toxin and a heme binding

protein. The experiments conducted in this research will form the basis of future

studies that will identify the mechanism of anti-diatom activity in P. tunicata.

Chapter 4: General discussion



This thesis describes an investigation of the antifouling characteristics of marine

epiphytic bacteria, against primary surface colonizers of Ulva spp. from Fiji. As

initial colonizers, both bacteria and diatoms are crucial for the subsequent

development of a mature biofouling community. The presence of epiphytic bacteria,

which may have a role in regulating growth of bacteria and diatoms on the surface of

Ulva, along with their identification, is addressed in Chapter 2. Anti-diatom

properties of epiphytic bacteria, which remains an under-explored area in marine

ecology, is studied in Chapter 3. This final chapter outlines and discusses the major

findings presented in the thesis and suggests directions for future work.

4.1 Antifouling properties of surface-associated bacteria

Bacteria live symbiotically on algal hosts by limiting colonization of surface foulers

in exchange for space and nutrients. Specifically, epibionts regulate biofilm formation

which benefits the host by preventing the development of a mature biofouling

community. To cooperate in such symbiotic interactions, bacteria often need adaptive

responses such as the production of antifouling molecules. Such antifouling

properties confer a competitive advantage to bacteria competing for resources.

The first two aims of this thesis were to, isolate and identify epibionts of Fijian Ulva

with inhibitory properties. Both antibacterial and anti-diatom activities were found to

be common to these bacteria. Approximately 60% of the isolates inhibited the target

bacteria and 80% inhibited growth of the diatom, C. fusiformis. The level of growth

inhibition varied widely. The red pigmented, isolate U15 (deposited under GenBank

accession FJ235137) was the most effective. This observation is consistent with

previous studies that have correlated pigmentation with natural product formation in

Pseudoalteromonas spp. (Egan et al., 2002b). Other isolates with inhibitory

properties were identified as members of Shewanella, Pseudoalteromonas, Vibrio and

Bacillus. The results were correlated with previous findings of antifouling activity

within the respective genera. Interestingly, anti-diatom activity was demonstrated in

both Bacillus and Vibrio, groups where such activity has not previously been



recognized. It is speculated that many epibiotic bacteria have antifouling potential but

these remain unrecognized.

The third aim of the thesis was to screen Pseudoalteromonas and Roseobacter

isolates for anti-diatom activity. Results showed that of the tested strains, all the

Pseudoalteromonas spp. and 44% of the Roseobacter strains inhibited growth of C.

fusiformis. The research highlights the prevalence of anti-diatom activity in

Pseudoalteromonas. The lower occurance of anti-diatom activity in Roseobacter may

reflect the clade’s greater ecological diversity. It may be that anti-diatom activity does

not provide specific advantages in all cases.

The study re-emphasizes the need to preserve bacterial biodiversity especially

symbiotic forms. The technology available to assess bacterial symbioses with higher

organisms is limited (Egan et al., 2008). Advances in techniques will not only assist

in exploring existing microbial diversity for novel bioactive compounds but also

contribute significantly towards the exploitation of microbial defence mechanisms.

4.2 Modelling anti-diatom mechanism in P. tunicata

The marine epiphyte, P. tunicata is a model organism for studies of surface-

associations. The bacterium is believed to exhibit the broadest range of inhibitory

activities including antibacterial, anti-fungal, anti-algal and anti-larval characteristics

(Holmstrom et al., 2002). Although the antifouling capability of P. tunicata has been

highlighted, its anti-diatom capacity has not been explored specifically.

The final aim of the study was to identify genes involved in anti-diatom activity in P.

tunicata and suggest a model describing the mode of action. Pseudoalteromonas

tunicata mutants lacking in anti-diatom activity were generated by transposon

mutagenesis. These provided some insight into the organism’s anti-diatom strategies.

Three mutants were chosen for study and DNA sequence analysis revealed

transposon insertion at three different locations in the genome. These included a gene

homologous to a cation/multidrug efflux pump, a beta-hexosaminidase gene, RTX

toxin-like gene and a member of the HemeO protein family. Sequence analysis of

DM1 showed that the transposon had disrupted a gene homologous to the



cation/multidrug efflux pump protein, belonging to the AcrB/AcrD/AcrF family.

Analysis of the conserved domains of AcrB gene suggests a wide range of roles

including defence, transport mechanisms, intracellular trafficking and secretion. The

study suggests the gene may be involved in the secretion of toxin/s responsible for the

anti-diatom activity.

The two ORFs found affected by transposon insertion in mutant DM2 were

homologous to beta-hexosaminidase and a RTX toxin. Beta-hexosaminidase is a

chitin-degrading enzyme which suggests that chitin fiber formation in diatoms may

be targeted by P. tunicata. Located downstream of the beta-hexosaminidase gene is

an RTX toxin gene. The RTX gene cluster is present in a range of pathogenic Gram-

negative bacteria and exhibits cytotoxic and haemolytic activities (Joachim and Peter,

2002; Chatterjee et al., 2008; Lin et al., 1999). Conserved domain analysis of the

RTX toxin gene suggests involvement in adhesion and cell wall stabilisation or

calcium-binding capacity. The non-coding region between the genes may contain

regulatory elements, the disruption of which could affect the expression of the RTX

gene, leading to a loss in anti-diatom activity. Interestingly, located upstream of the

beta-hexasoaminidase gene are a pair of genes encoding TonB receptors, which are

involved in iron transport. Recent work by Evans et al., (2007) links iron transport by

TonB receptors to a type-II secretion pathway. Sequence analysis of this mutant

reveals a complex system, the expression of which may be regulated by iron.

In the last mutant analysed the transposon inserted into an ORF homologous to a

member of the HemeO protein family. Conserved domain analysis suggests a heme-

binding capacity. Heme is critical for iron acquisition (Wilks, 2002). In diatoms,

heme biosynthesis represents an essential metabolic pathway which provides the

precursors for cytochrome prosthetic groups, photosynthetic pigments, and vitamin

B-12 (Obornik and Green, 2005). As with results of DM2, analysis of this mutant

suggests an important role for iron in the anti-diatom strategy of P. tunicata. Iron may

be obtained from diatoms by the HemeO homologue.



Based on these results a preliminary model for the expression of anti-diatom activity

is proposed (Figure 4.1). Briefly, iron is suggested to be essential for the expression

of anti-diatom activity and is obtained by the HemeO homologue. The type II-

secretion pathway likely relates to the secretion of TonB, resulting in the binding and

transport of the acquired iron. Iron is also suggested to be involved either directly or

indirectly in the expression of the RTX toxin. Finally, the multidrug efflux system

likely pumps toxins out of the bacterial cell that inhibit diatom growth.

Extracellular environment

Cell cytoplasm

TonB TonB HemeO

Type II

Acquires iron

IronIron

Iron

Iron

Beta-hexosaminidase

Intergeneic region (controls expression of neighbouring genes)

RTX toxin

Possible precursorIron

ChitinaseMultidrug efflux pump

Putative toxin/s

Diatom heme

Relates to secretion of TonB

Binds & transports iron

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4.3 Future directions and implications

Ecologically, antibacterial and anti-diatom properties give epibionts a competitive

advantage over other surface colonizing microbes when competing for space and

nutrients. Additionally, bacteria with inhibitory characteristics are often able to form

symbioses with algae, providing a microbial-mediated defence system in return for

nutrition. This study has highlighted the possibility of finding novel bioactive

compounds from tropical epibionts. Future work might investigate the defence

strategies of algal epibionts against specific target organisms. With appropriate

modification, bacterial defence systems could be used as environment-friendly

controls of biofouling.

Furthermore, the results of this research may also form the basis of future studies that

explore the mechanisms of anti-diatom activity in surface-associated bacteria,

especially P. tunicata. An in-depth study would help further develop the proposed

model. The generation of knockouts in genes already identified as important may

provide further insight into the role of the respective gene in anti-diatom activity. An

improved understanding of the anti-diatom mechanisms will provide both

environmental and economic benefits, by leading to improved methods for the control

of tenacious biofilms.

References


References

1. Abd El-Baky, H. H., El Baz, F. K. and El-Baroty, G. S. (2008) Evaluation

of marine alga Ulva lactuca L. as a source of natural preservative ingredient.

EJEAF Che. 7:3353-3367.

2. Alavi, M., Miller, T., Erlandson, K., Schneider, R., and Belas, R.

(2001) Bacterial community associated with Pfiesteria-like dinoflagellate

cultures. Environ. Microbiol. 3:380-396.

3. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J.

(1990).

Basic local alignment search tool. J. Mol. Biol. 215:403-410.

4. Andrews, S. C., Robinson, A. K., and Rodriguez-Quinones, F. (2003)

Bacterial iron homeostasis. FEMS Microbiol. Rev. 27:215-237.

5. Armstrong, E., Yan, L., Boyd, K. G., Wright, P. C., and Burgess, J. G.

(2001) The symbiotic role of marine microbes on living surfaces.

Hydrobiologia. 461:37-40.

6. Awad, N. E. (2000) Biologically active steroid from the green alga Ulva

lactuca. Phytother. Res. 14: 641-643.

7. Berland, B. R., Bonin, D. J., and Maestrini, S. Y. (1972) Are some

bacteria toxic for marine algae? Mar. Biol. 12:189-193.

8. Bers, A., D'Souza, F., Klijnstra, J., Willemsen, P., and Wahl, M.

(2006) Chemical defence in mussels: antifouling effect of crude extracts

of the periostracum of the blue mussel Mytilus edulis. Biofouling.22:251-

259.

9. Bhattarai, H. D., Granti, V. S., Paudel, B., Lee, Y. K., Lee, H. K.,

Hong, Y. K., and Shin, H. W. (2007) Isolation of antifouling compounds

from the marine bacterium, Shewanela oneidensis SCH0402. World J.

Microbiol. Biotechnol. 23:243-249.

10. Bhattarai, H. D., Lee, Y. K., Cho, K. H., Lee, H. K., and Shin, H. W.

(2006) The study of antagonistic interactions among pelagic bacteria: a

promising way to coin environmental friendly antifouling compounds.

Hydrobiologia. 568:417-423.

References


11. Boles, B. R., Thoendel, M., and Singh, P. K. (2004) Self-generated

diversity produces “insurance-effects” in biofilm communities. Proc. Natl.

Acad. Sci. U.S.A. 101:16630-16635.

12. Bowman, J. P. (2007) Bioactive compound synthetic capacity and

ecological significance of marine bacterial genus Pseudoalteromonas.

Mar. Drugs. 5:220-241.

13. Brinkhoff, T., Bach, G., Heidorn, T., Liang, L. F., Schlingloff, A., and

Simon, M. (2004) Antibiotic production by a Roseobacter clade-affiliated

species from the German Wadden Sea and its antagonistic effects on

indigenous isolates. Appl. Environ. Microbiol. 70:2560-2565.

14. Brinkhoff, T., Giebel, H. and Simon, M. (2008) Diversity, ecology, and

genomics of the Roseobacter clade: a short overview. Arch. Microbiol.

189:531-539.

15. Britschgi, T. B., and Giovannoni, S. J. (1991) Phylogenetic analysis of a

natural marine bacterioplankton population by rRNA gene cloning and

sequencing. Appl. Environ. Microbiol. 57:1707–1713.

16. Brizzolara, R. A. (2002) Adsorption of alginic acid to titanium investigated

using x-ray photoelectron spectroscopy and atomic force microscopy. Surf.

Interface Anal. 33:351-360.

17. Bruhn, J. B., Haagensen, J. A. J., Bagge-Ravn, D., and Gram, L. (2006)

Culture conditions of Roseobacter strain 27-4 affect its attachment and

biofilm formation as quantified by real-time PCR. Appl. Environ. Microbiol.

72:3011-3015.

18. Bruhn, J. B., K. Nielsen, K. F., Hjelm, M., Hansen, M., Bresciani, J.,

Schulz, S., and L. Gram. (2005) Ecology, inhibitory activity, and

morphogenesis of a marine antagonistic bacterium belonging to the

Roseobacter clade. Appl. Environ. Microbiol. 71:7263-7270.

19. Bruhn, J.B., Gram, L., and Belas, R. (2007) Production of antibacterial

compounds and biofilm formation by Roseobacter species are influenced by

culture conditions. Appl. Environ. Microbiol. 73:442-450.

20. Bruinsma, G. M., Rustema-Abbing, M., Van Der Mei, H. C., and

Busscher, H. J. (2001) Efects of cell surface damage on surface properties

and adhesion of Pseudomonas aeruginosa. J. Microbiol. Methods. 45:95-101.

References


21. Bryan, P., Rittschof, D., and McClintock, J. B. (1996) Bioactivity of

echinoderm ethanolic bodywall extracts: an assessment of marine bacterial

attachment and macroinvertebrate larval settlement. J. Exp. Mar. Biol. Ecol.

196:79-96.

22. Bryers, J. D. (1988). Modeling biofilm accumulation. In: Bazin, M. and

Prosser, J. I. (eds.) Biofilms II: process analysis and application. Wiley-Liss.

New York. pp. 45-88.

23. Buchan, A., Gonzalez, J. M. and Moran, M. A. (2005) Overview of the

marine Roseobacter lineage. Appl. Environ. Microbiol. 71:5665-5677.

24. Burke, C., Thomas, T., Egan, S., and Kjelleberg, S. (2007) The use of

functional genomics for the identification of a gene cluster encoding for the

biosynthesis of an antifungal tambjamine in the marine bacterium

Pseudoalteromonas tunicata. Env. Microbiol. 9:814-818.

25. Busscher, H. J., Cowan, M. M., and Vandermei, H. C. (1992) On the

relative importance of specific and nonspecific approaches to oral microbial

adhesion. FEMS Microbiol. Rev. 88:199-209.

26. Caiazza, N. C., and O’Toole, G. A. (2004) SadB is required for the transition

from reversible to irreversible attachment during biofilm formation by

Pseudomonas aeruginosa PA14. J. Bacteriol. 186:4476-4485.

27. Callow, M. E., and Callow, J. A. (2002) Marine biofouling: a sticky

problem. Biologist. 49:1-5.

28. Campàs, C., Dalmau, M., Montaner, B., Barragán, M, Bellosillo, B,

Colomer, D, Pons, G, Pérez-Tomás, R., and Gil, J. (2003) Prodigiosin

induces apoptosis of B and T cells from B-cell chronic lymphocytic leukemia.

Leukemia. 17:746-750.

29. Champ, M. A. (1999) The need for the formation of an independent,

International Marine Coatings Board. Mar. Poll. Bull. 38:239-246.

30. Chan, A. T., Andersen, R. J., Blanc, M. J. Le, and Harrison, P. J. (1980)

Algal plating as a tool for investigating allelopathy among marine microalgae.

Mar. Biol. 59:7-13.

31. Characklis, W. G. (1990) Microbial fouling. John Wiley & Sons. New York.

32. Chatterjee, R., Nag, S. and Chaudhuri, K. (2008) Identification of a new

RTX-like gene cluster in Vibrio cholerae. FEMS Microbiol. Lett. 284:165-

171.

References


33. Chimento, D.P., Kadner, R.J., and Wiener, M.C. (2003) The Escherichia

coli outer membrane cobalamin transporter BtuB: structural analysis of

calcium and substrate binding, and identification of orthologous transporters

by sequence/structure conservation. J. Mol. Biol. 332:999-1014.

34. Chiovitti, A., Higgins, J. M., Harper, R. E., Wetherbee, R., and Bacic, A.

(2003) The complex polysaccharides of the raphid diatom Pinnularia viridis

(Bacillariophyceae). J. Phycol. 39:543-554.

35. Coley, P. D. (1986) Costs and benefits of defence by tannins in a neotropical

tree. Oecologia. 70: 238-241.

36. Cooksey, K. E., and Wigglesworth-Cooksey, B. (1995) Adhesion of bacteria

and diatoms to surfaces in the sea: a review. Aquat. Microb. Ecol. 9:87-96.

37. Costerton, J. W., Lewandowski, Z., DeBeer, D., Caldwell, D., Korber, D.,

and James, G. (1994) Biofilms, the customized microniche. J. Bacteriol.

176:2137-2142.

38. Davey, M. E., and O'Toole, G. A. (2000). Microbial biofilms: from ecology

to molecular genetics. Microbiol. Mol. Biol. Rev. 64:847-867.

39. de Nys, R., Steinberg, P. D., Willemsen, P., Dworjanyn, S. A., Gabelish,

C. L. and R. J. King. (1995) Broad spectrum effects of secondary

metabolites from the red alga Delisea pulchra in antifouling assays.

Biofouling. 8:259-271.

40. de Nys, R., Wright, A. D. Konig, G. M., Sticher, O. (1993) New

halogenated furanones from the marine alga Delisea pulchra (cf. fimbriata).

Tetrahedron. 49: 11213-11220.

41. Del Amo, Y. and Brzezinski, M. A. (1999) The chemical form of dissolved

Si taken up by marine diatoms. J. Phycol. 35:1162–1170.

42. Delaquis, P. J., Caldwell, D. E., Lawrence, J. R., and Mccurdy, A. R.

(1989) Detachment of Pseudomonas fluorescens from biofilms on glass

surfaces in response to nutrient stress. Microbiol. Ecol. 18:199-210.

43. DeSantis, T. Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E. L.,

Keller, K., Huber, T., Dalevi, D., Hu, P. and Andersen, G. L. (2006)

Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench

Compatible with ARB. Appl. Environ. Microbiol. 72:5069-5072.

References


44. Dobretsov, S., and Qian, P. Y. (2002) Effect of bacteria associated with the

green alga Ulva reticulata on marine micro- and macrofouling. Biofouling

18:217-228.

45. Dobretsov, S., Dahms, H., and Qian, P. (2005) Antibacterial and anti-

diatom activity of Hong Kong sponges. Aquat. Microb. Ecol. 38:191-201.

46. Dobretsov, S., Dahms, H., and Qian, P. (2006) Inhibition of biofouling by

marine microorganisms and their metabolites. Biofouling. 22:43-54.

47. Dworjanyn, S. A., de Nys, R., and Steinberg, P. D. (1999) Localization and

surface quantification of secondary metabolites in the red alga Delisea

pulchra. Mar. Biol. 133:727-736.

48. Edgar, L. A., and Pickett-Heaps, J. D. (1984) Ultrastructural localisation of

polysaccharides in the motile diatom Navicula cuspidate. Protoplasma.

130:10-12.

49. Egan, S., James, S. and Kjelleberg, S. (2002) Identification and

characterization of a putative transcriptional regulator controlling the

expression of fouling inhibitors in Pseudoalteromonas tunicata. Appl.

Environ. Microbiol. 68:372-378.

50. Egan, S., James, S., Holmstrom, C., and Kjelleberg, S. (2001) Inhibition of

algal spore germination by the marine bacterium Pseudoalteromonas tunicata.

FEMS Microbiol. Ecol. 35:67-73.

51. Egan, S., James, S., Holmstrom, C., and Kjelleberg, S. (2002) Correlation

between pigmentation and antifouling compounds produced by

Pseudoalteromonas tunicata. Env. Microbiol. 4:433-442.

52. Egan, S., Thomas, T., and Kjelleberg, S. (2008) Unlocking the diversity and

biotechnological potential of marine surface associated microbial

communities. Curr. Opin. Microbiol. 11:219-225.

53. Egan, S., Thomas, T., Holmstrom, C., and Kjelleberg, S. (2000)

Phylogenetic relationship and antifouling activity of bacterial epiphytes from

the marine alga Ulva lactuca. Environ Microbiol.2:343-347.

54. Engel, S., Jensen, P., and Fenical, W. (2002) Chemical ecology of marine

microbial defence. J. Chem. Ecol. 28:1971-1985.

55. Evans, F. F., Raftery, M. J., Egan, S., and Kjelleberg, S. (2007) Profiling

the secretome of the marine bacterium Pseudoalteromonas tunicata using

amine-specific isobaric tagging (iTRAQ). J. Proteome Res. 6:967-975.

References


56. Evans, L. V. (1988) Marine biofouling. In: Lembi, C. A. and Waaland, J. R.

(eds). Algae and Human Affairs. Cambridge University Press. Cambridge. pp.

433-453.

57. Evans, S. M. (1999) TBT or not TBT? That is the question. Biofouling.

14:117-129.

58. Evans, S. M., Leksono, T., and McKinnell, P. D. (1995) Tributyltin

pollution: A diminishing problem following legislation limiting the use of

TBT-based anti-fouling paints. Mar. Poll. Bull. 30:14-21.

59. Falciatore, A., and Bowler, C. (2002) Revealing the molecular secrets of

marine diatoms. Annu. Rev. Plant Biol. 53:109-130.

60. Faraldo-Gomez, J. D. and Sansom, M. S. P. (2003) Acquisition of

siderophores in Gram negative bacteria. Nat. Rev. Mol. Cell Biol. 4:105-116.

61. Farmer III, J.J. & Hickmann-Brenner, F. W. (1992) The genera Vibrio and

Photobacterium. In: Balows, A., Trupper, H. G., Dworkin, M., Harder, W.

and Schleifer, K. H. (eds.) The Prokaryotes 2nd edition, Vol. III, Springer-

Verlag. New York. pp. 2952-3011.

62. Felgenhauer, B. E., Watling, L.,Thistle, A. B. (1989) Functional

morphology of feeding and grooming in Crustacea. CRC Press. USA.

63. Field, K. G., Gordon, T., Wright, M., Rappé, M., Vergin, K., and

Giovannoni, S. J. (1997) Diversity and depth-specific distribution of Sar11

cluster rRNA genes from marine planktonic bacteria. Appl. Environ.

Microbiol. 63:63–70.

64. Filion-Myklebust, C., and Norton, T. A. (1981) Epidermis shedding on the

brown seaweed Ascophyllum nodosum (L.) Le Jolis, and its ecological

significance. Mar. Biol. Lett. 2:45-51.

65. Foo, C. W. P., Huang, J. D., and Kaplan, L. (2004) Lessons from seashells:

silica mineralization via protein templating. Trends Biotechnol. 22:577-585.

66. Franks, A., Haywood, P., Holmstrom, C., Egan, S., Kjelleberg, S., and

Kumar, N. (2005) Isolation and structure elucidation of a novel yellow

pigment from the marine bacterium Pseudoalteromonas tunicata. Molecules.

10:1286-1291.

67. Gauthier, M. J. (1976) Alteromonas rubra sp. nov., a new marine antibiotic-

producing bacterium. Int. J. Syst. Bacteriol. 26:459-466.

References


68. Gauthier, M. J. (1976) Modification of bacterial respiration by a

macromolecular polyanionic antibiotic produced by a marine Alteromonas.

Antimicrob. Agents Chemother. 76:361-366.

69. Gauthier, M. J., and Breittmayer, V. A. (1979) A new antibiotic-producing

bacterium from seawater: Alteromonas aurantia sp. nov. Int. J. Syst.

Bacteriol. 29:366-372.

70. Geng, H. F., Bruhn, J. B., Nielsen, K. F., Gram, L., Belas, R. (2008)

Genetic dissection of tropodithietic acid biosynthesis by marine roseobacters.

Appl. Environ. Microbiol. 74:1535-1545.

71. Gerber, N. N. and Gauthier, M. J. (1979) New prodigiosin-like pigment

from Alteromonas rubra. Appl. Env. Microbiol. 37:1176-1179.

72. Gilabert, J. (2007) Phytoplankton ecological processes for ecosystem

modelling: some basic concepts. NATO Security though Science Series.

2007:245-258.

73. Gil-Turness, M. S., and Fenical, W. (1992) Embryos of Homarus

americanus are protected by epibiotic bacteria. Biol. Bull. 182:105-108.

74. Giovannoni, S. J., and Rappé, M. S. (2000) The uncultured microbial

majority. In: Kirchman, D. L. (ed.) Microbial ecology of the oceans. John

Wiley & Sons, New York. pp. 47-84.

75. Gontang, E. A., Fenical, W., and Jensen, P. R. (2007) Phylogenetic

diversity of Gram-Positive bacteria cultured from marine sediments. Appl.

Environ. Microb. 73:3272–3282.

76. González, J. M., Simo, R., Massana, R., Covert, J. S., Casamayor, E. O.,

Pedros-Alio, C., and Moran, M. A. (2000) Bacterial community structure

associated with a dimethylsulfoniopropionate-producing North Atlantic algal

bloom. Appl. Environ. Microbiol. 66:4237-4246.

77. Gram, L., Grossart, H. P., Schlingloff, A., and Kiorboe, T. (2002) Possible

quorum sensing in marine bacteria: production of acylated homoserine

lactones by Roseobacter strains isolated from marine snow. Appl. Environ.

Microbiol. 68:4111-4116.

78. Guillard, R.R.L. (1975) Culture of phytoplankton for feeding marine

invertebrates. In: Smith, W.L. and Chanle,y M.H. (eds.) Culture of Marine

Invertebrate Animals. Plenum Press. New York. pp 26-60.

References


79. Guillard, R.R.L., and Ryther, J.H. (1962) Studies of marine planktonic

diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J.

Microbiol. 8:229-239.

80. Hammer, B. K. and Bassler, B. L. (2007) Regulatory small RNAs

circumvent the conventional quorum sensing pathway in pandemic Vibrio

cholerae. Proc. Natl. Acad. Sci. 104: 11145-11149.

81. Harborne, J. B. (2001) Twenty-five years of chemical ecology. Nat. Prod.

Rep. 18:361-379.

82. Harkes, G., Dankert, J., and Feijen, J. (1992) Bacterial migration along

solid-surfaces. Appl. Environ. Microbiol. 58:1500-1505.

83. Hasle, G.R. and Syvertsen, E.E. (1997) Marine diatoms. In: Tomas, C.R

(ed.) Identifying marine phytoplankton. Academic Press. pp 858.

84. Hausner, M., and Wuertz, S. (1999) High rates of conjugation in bacterial

biofilms as determined by quantitative in situ analysis. Appl. Environ.

Microbiol. 65:3710-3713.

85. Herrero, M., de Lorenzo, V., and Timmis, K. (1990) Transposon vectors

containing non-antibiotic resistance selection markers for cloning and stable

chromosomal insertion of foreign genes in Gram-negative bacteria. J.

Bacteriol. 172: 6557-6567.

86. Higgins, M. J., Crawford, S. A., Mulvaney, P. and Wetherbee, R. (2000)

The topography of soft, adhesive diatom “trails” as observed by atomic force

microscopy. Biofouling. 16:133-139.

87. Hildebrand, M., Volcani, B. E., Gassmann, W., and Schroeder, J. I.

(1997) A gene family of silicon transporters. Nature. 385:688–689.

88. Ho, W. K. (2008) Structure development and dispersal of biofilms formed by

the marine bacterium Roseobacter sp. 2.10. Honours Thesis, University of

New South Wales, Sydney, Australia.

89. Hoagland, K. D., Rowoski, J. R., Gretz, M. R., and Roener, S. C. (1993)

Diatom extracellular polymeric substances: function, fine structure, chemistry

and physiology. J. Phycol. 29:646-653.

90. Holmstrom, C., and Kjelleberg, S. (1999) Marine Pseudoalteromonas

species are associated with higher organisms and produce biologically active

extracellular agents. FEMS Microbiol. Ecol. 30:285-293.

References


91. Holmstrom, C., and S. Kjelleberg. (2000) Bacterial interactions with marine

fouling organisms, p. 101-117. In: L. V. Evans (ed.), Biofilms: recent

advances in their study and control. Overseas Publishing Associates (UK),

Amsterdam, The Netherlands.

92. Holmstrom, C., Egan, S., Franks, A., McCloy, and S., Kjelleberg, S.

(2002) Antifouling activities expressed by marine surface associated

Pseudoalteromonas species. FEMS Microbiol. Ecol. 41: 47-58.

93. Holmstrom, C., James, S., Egan, S., and Kjelleberg, S. (1996) Inhibition of

common fouling organisms by marine bacterial isolates with special reference

to the role of pigmented bacteria. Biofouling. 10:251-259.

94. Holmstrom, C., James, S., Neilan, B. A., White, D. C., and Kjelleberg, S.

(1998) Pseudoalteromonas tunicata sp. nov., a bacterium that produces

antifouling agents. Int. J. Syst. Bacteriol. 48:1205-1212.

95. Holmstrom, C., Rittschof, D., and Kjelleberg, S. (1992) Inhibition of

settlement of larvae of Balanus amphitrite and Ciona intestinalis by a surface-

colonizing marine bacterium D2. Appl. Environ. Microbiol. 58:2111-2115.

96. Huggett, M., Williamson, J., Nys, R., Kjelleberg, S., and Steinberg, P.

(2006) Larval settlement of the common Australian sea urchin Heliocidairs

erythrogramma in response to bacteria from the surface of coralline algae.

Oceologia. 149:604-619.

97. Ivanova, E P., Vysotskii, M. V., Svetashev, V. I.,, Nedashkovskaya O. I.,

Gorshkova, N. M., Mikhailov, V. V.,Yumoto, N., Shigeri, Y., Taguchi, T.,

and Yoshikawa, S. (1999) Characterization of Bacillus strains of marine

origin. Int. Microbiol. 2:267–271.

98. Jackson, D. W., Suzuki, K., Oakford, L., Simecka, J. W., Hart, M. E., and

Romeo, T. (2002) Biofilm formation and dispersal under the influence of the

global regulator CsrA of Eshcerichia coli. J. Bacteriol. 184:290-301.

99. James, S. (1998), Antifouling and antibacterial effects of Pseudoalteromonas

tunicata. PhD Thesis, University of New South Wales, Sydney, Australia.

100. James, S., Holmstrom, C., and Kjelleberg, S. (1996) Purification and

characterization of a novel antibacterial protein from the marine bacterium

D2. Appl. Environ. Microbiol. 62:2783-2788.

101. Joachim, F. and Peter, K. (2002) RTX toxins in Pasteurellaceae. Int. J.

Med. Microbiol. 292:149-158.

References


102. Johnson, C. R., Sutton, D.C., Olson, R. R., and Giddins, R. (1991)

Settlement of crown-of-thorns starfish: role of bacteria on surfaces of coralline

algae and hypothesis of deep water recruitment. Mar. Ecol. Prog. Ser.

71:143–162.

103. Kawauchi, K., Shibutani, K., Yagisawa, H., Kamata, H., Nakatsuji, S.,

Anzai, H., Yokoyama, Y., Ikegami, Y., Moriyama, Y., and Hirata, H.

(1997) A possible immunosuppressant, cycloprodigiosin hydrochloride,

obtained from Pseudoalteromonas denitrificans. Biochem. Biophys. Res.

Comm. 237:543-547.

104. Kazlauskas, R., Murphy, P. T., Quinn, R. J., Wells, R. J. (1977) A new

class of halogenated lactones from the red alga Delisea fimbriata

(Bonnemaisoniaceae). Tetrahedron. Lett. 1:37-40.

105. Keats, D. W., Knight, M. A., and Pueschel, C. M. (1997) Antifouling

effects of epithelial shedding in three crustose corralling algae (Rhodophyta,

Coralines) on a coral reef. J. Exp. Mar. Biol. Ecol. 213:281-293.

106. Kim, H. S., Hayashi, M., Shibata, Y., Wataya, Y., Mitamura, T., Horii,

T., Kawauchi, K., Hirata, H., Tsuboi, S., and Moriyama, Y. (1999)

Cycloprodigiosin hydrochloride obtained from Pseudoalteromonas

denitrificans is a potent antimalarial agent. Biol. Pharm. Bull. 22:532-534.

107. Kjelleberg, S., Steinberg, P., Givskov, M., Gram, L., Manefield, M., and

de Nys, R. (1997) Do marine natural products interfere with prokaryotic AHL

regulatory systems? Aquat. Microb. Ecol. 13:85-93.

108. Koebnik R. (2005) TonB-dependent trans-envelope signalling: the

exception or the rule? Trends Microbiol. 13:343-7.

109. Konig, G. M., Kehraus, S., Seibert, S. F., Abdel-Lateff, A., and Muller,

D. (2006) Natural products from marine organisms and their associated

microbes. Chembiochem. 7:229-238.

110. Kroger, N. and Wetherbee, R. (2000) Pleuralins are involved in theca

differentiation in the diatom Cylindrotheca fusiformis. Protist. 151:263-273.

111. Lafay, B., Ruimy, R., de Traubenberg, C., Breittmayer, V., Gauthier,

M., and Christen, R. (1995) Roseobacter algicola sp. nov., a new marine

bacterium isolated from the phycosphere of the toxin-producing dinoflagellate

Prorocentrum lima. Int. J. Syst. Bacteriol. 45:290-296.

References


112. Lawrence, J. R., Korber, D. R., Hoyle, B. D., Costerton, J. W., and

Caldwell, D. E. (1991) Optical sectioning of microbial biofilms. J. Bacteriol.

173:6558-6567.

113. Leblanc, C., Falciatore, A., and Bowler, C. (1999) Semi-quantitative RT-

PCR analysis of photoregulated gene expression in marine diatoms. Plant

Mol. Biol. 40: 1031– 1044.

114. Lemos, M. L., Toranzo, A. E., and Barja, J. L. (1985) Antibiotic activity

of epiphytic bacteria isolated from intertidal seaweeds. Microbial Ecol.

11:149-163.

115. Lin, W., Fullner, K. J., Clayton, R., Sexton, J. A., Rogers, M. B., Calia,

K. E., Calderwood, S. B., Fraser, C., and Mekalanos, J. J. (1999)

Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly

linked to the cholera toxin prophage. Proc. Natl. Acad. Sci. 96:1071–1076.

116. Lind, J. L., Heimann, K., Miller, E. A., vanVliet, C., Hoogenraad, N. J.,

and Wetherbee, R. (1997) Substratum adhesion and gliding in a diatom are

mediated by extracellular proteoglycans. Planta. 203:213-221.

117. Linton, K. J., and Higgins, C. F. (1998) The Escherichia coli ATP-binding

cassette (ABC) proteins. Mol. Microbiol. 28:5-13.

118. Longford, S., Tujula, N. A., Crocetti, G. R., Holmes, A. J., Holmstrom,

C., Kjelleberg, S., Steinberg, P. D., and Taylor, M. W. (2007) Comparisons

of diversity of bacterial communities associated with three sessile marine

eukaryotes. Aquat. Microb. Ecol. 48: 217–229.

119. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H.,

Yadhukumar, Buchner, A., Lai, T., Steppi, S., Jobb, G. et al. (2004) ARB:

a software environment for sequence data. Nucleic Acids Res. 32:1363-1371.

120. Ma, D., Cook, D.N., Alberti, M., Pon, N.G., Nikaido, H. and Hearst, J.E.

(1993) Molecular cloning and characterization of AcrA and AcrE genes of

Escherichia coli. J. Bacteriol. 175:6299-6313.

121. Maata, M. and Koshy, K. (2001) A study on tributyltin contamination of

marine sediments in the major ports of Fiji. South Pac J Nat Sci. 19:1-4

122. Magae, J., Miller, M. W., Nagai, K., and Shearer, G. M. (1996) Effect of

metacycloprodigiosin, an inhibitor of killer T cells on murine skill and heart

transplants. J. Antibiot. 48:86-90.

References


123. Mai-Prochnow, A., Evans, F., Dalisay-Saludes, D., Stelzer, S., Egan, S.,

James, S., Webb, J. S., and Kjelleberg, S. (2004) Biofilm development and

cell death in the marine bacterium Pseudoalteromonas tunicata. Appl.

Environ. Microbiol. 70:3232-3238.

124. Mai-Prochnow, A., Lucas-Elio, P., Egan, S., Thomas, T., Webb, J. S.,

Sanchez-Amat, A., and Kjelleberg, S. (2008) Hydrogen peroxide linked to

lysine oxidase activity facilitates biofilm differentiation and dispersal in

several Gram negative bacteria. J. Bacteriol. 190:5493-5501.

125. Mai-Prochnow, A., Webb, J. S., Ferrari, B. C. and Kjelleberg, S. (2006)

Ecological advantages of autolysis during the development and dispersal of

Pseudoalteromonas tunicata biofilms. Appl. Environ. Microbiol. 72:5414-

5420.

126. Manefield, M., de Nys, R., Kumar, N., Read, R., Givskov, M., Steinberg,

P. Kjelleberg, S. (1999) Evidence that halogenated furanones from Delisea

pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression

by displacing the AHL signal from its receptor protein. Microbiol. UK.

145:283-291.

127. Mann, K. H. (1973) Seaweeds: their productivity and strategy for growth.

Science. 182:975-981.

128. Markowitz V. M., Ivanova, N., Szeto, E., Palaniappan, K., Chu, K.,

Dalevi, D., Chen, I. M., Grechkin, Y., Dubchak, I., Anderson, I., et al.

(2008) IMG/M: a data management and analysis system for metagenomes,

Nucleic Acids Res. 36:534-538.

129. Martens, T., Heidorn, T., Pukall, R., Simon, M., Tindall, B. J., and

Brinkhoff, T. (2006) Roseobacter gallaeciensis Ruiz-Ponte et al. 1998 as

Phaeobacter gallaeciensis gen. nov., comb. nov., description of Phaeobacter

inhibens sp. nov., reclassification of Ruegeria algicola (Lafay et al. 1995)

Uchino et al. 1999 as Marinovum algicola gen. nov., comb. nov., and

emended descriptions of the genera Roseobacter, Ruegeria and Leisingera.

Int. J. Syst. Evol. Microbiol. 56:1293-1304.

130. Matz, C. and Kjelleberg, S. (2005) Off the hook - how bacteria survive

protozoan grazing. Trends. Microbiol. 13:302-307.

131. Maximilien, R., de Nys, R., Holmstr�m, C., Gram, L., Givskov, M.,

Crass, K. (1998) Chemical mediation of bacterial surface colonization by

References


secondary metabolites from the red alga Delisea pulchra. Aquat. Microb.

Ecol. 15:233-246.

132. Mayali, X., Franks, P. J. S. and Azam, F. (2008) Cultivation and

ecosystem role of a marine Roseobacter clade-affiliated cluster bacterium.

Appl. Environ. Microbiol. 74:2595-2603.

133. Miller, T. R., and Belas, R. (2004) Dimethylsulfoniopropionate (DMSP)

metabolism by Pfiesteria-associated Roseobacter spp. Appl. Environ.

Microbiol. 70:3383-3391.

134. Miller, T. R., Hnilicka, K., Dziedzic, A., Desplats, P., and Belas. R.

(2004) Chemotaxis of Silicibacter sp. TM1040 toward dinoflagellate

products. Appl. Environ. Microbiol. 70:4692-4701.

135. Morin, L. G., Smucker, R. A., and Herth, W. (1986) Effects of two chitin

synthesis inhibitors on Thalassiosira fluviatilis and Cyclotella cryptica. FEMS

Microbiol. Lett. 37:263-268.

136. Moss, B. L. (1982) The control of epiphytes by Halidrys siliquosa (L.)

Lynbg. (Phaeophyta, Crystoseiraceae). Phycologia 21:185-191.

137. Mueller, R. F., Chacklis, W. G., Jones, W. L., and Sears, J. T. (1992)

Characterization of initial events in bacterial surface colonization by two

Pseudomonas species using image analysis. Biotechnol. Bioeng. 39:1161-

1170.

138. Neal, A. L., and Yule, A. B. (1994) The interaction between Elminius

modestus Darwin cyprids and biofilms of Deleya marina Ncmb1877. J. Exp.

Mar. Biol. Ecol. 176:127–139.

139. Nealson, K. H. and Hastings, J. W. (1979) Bacterial bioluminescence: its

control and ecological significance. Microbiol. Rev. 43:496–518.

140. Negri, A. P., Webster, N.S., Hill, R.T., and Heyward, A. J. (2001)

Metamorphosis of broadcast spawning corals in response to bacteria isolated

from crustose algae. Mar. Ecol. Prog. Ser. 223:121–131.

141. Norton, T. A., Melkonian, M., and Andersen, R. A. (1996) Algal

biodiversity. Phycologia 35: 308– 226.

142. Obornik, M., and Green, B. R. (2005) Mosaic origin of the heme

biosynthesis pathway in photosynthetic eukaryotes. Mol. Biol. Evol. 22: 2343-

2353.

References


143. Oliveira, D. R. (1992) Physico-chemical aspects of adhesion. In: Melo, L.

F., Bott, T. R., Fletcher, M. and Capdeville, B. (eds.) Biofilms- Science and

Technology. Kluwer Academic Press. London.

144. Ortega-Morales, B. O., Chan-Bacab, M. J., Miranda-Tello, E., Fardeau,

M. L., Carrero, J. C., and Stein, T. (2008) Antifouling activity of sessile

bacilli derived from marine surfaces. J. Ind. Microbiol. Biotechnol. 35:9-15.

145. Ott, J. A. (1980) Growth and production in Posidonia oceanica (L.) Delile.

P. S. Z. N. I. Mar. Ecol. 1:47-64.

146. Pamp, S. J., Gjermansen, M. and Tolker-Neilsen T. (2007) The biofilm

matrix: a sticky framework. In: Kjelleberg, S. and Givskov, M. (eds.) The

biofilm mode of life: mechanisms and adaptations. Horizon Scientific Press.

UK.

147. Parsek, M. R., and Greenberg, E. P. (2000) Acyl-homoserine lactone

quorum sensing in Gram-negative bacteria: a signaling mechanism involved

in associations with higher organisms. Proc. Natl. Acad. Sci. U.S.A 97:8789-

8793.

148. Perez-Tomas, R., Montaner, B., Llagostera, R., and Soto-Cerrato, V.

(2003) The prodigiosins, proapoptotic drugs with anticancer properties.

Biochem. Pharmacol. 66:1447-1452.

149. Pohnert, G., (2002) Biomineralization in Diatoms Mediated through

Peptide- and Polyamine-Assisted Condensation of Silica. Angew. Chem. Int.

Ed. 41:3167-3169.

150. Poulsen, N. C., Spector, I., Spurck, T. P., Schultz, T. F. and Wetherbee,

R. (1999) Diatom gliding is the result of an actin-myosin motility system. Cell

Motil. Cytoskeleton 44:23-33.

151. Punt, P. J., Strauss, J., Smit, R., Kinghorn, J. R., Hondel, C. A. van den

and Scazzocchio, C. (1995) The intergenic region between the divergently

transcribed niiA and niaD genes of Aspergillus nidulans contains multiple

NirA binding sites which act bidirectionally. Mol. Cell Biol. 15: 5688–5699.

152. Rao, D., Webb, J. S., and Kjelleberg, S. (2005) Competitive interactions

in mixed-species biofilms containing the marine bacterium

Pseudoalteromonas tunicata. Appl. Environ. Microbiol. 71:1729-1736.

References


153. Rao, D., Webb, J. S., and Kjelleberg, S. (2006) Microbial colonization and

competition on the marine alga Ulva australis. Appl. Environ. Microbiol.

72:5547-5555.

154. Ratliff, M., Zhu, W., Stojilijkovic, I., and Wilks, A. (2001) Homologues

of neisserial heme oxygenase in gram-negative bacteria: degradation of heme

by the product of a pigA gene of Pseudomonas aeruginosa. J. Bacteriol.

183:6394–6403.

155. Raven, P. H., Evert, R. F. and Eichhorn, S. E. (1999) Biology of Plants.

W. H. Freeman and company worth publishers. New York.

156. Rodriguez, S. R., Riquelme, C., Campos, E. O., Chavez, P., Brandan, E.,

and Inestrosa, N. C. (1995) Behavioral responses of Concholepas

concholepas (Bruguiere, 1789) larvae to natural and artificial settlement cues

and microbial films. Biol. Bull. 189:272–279.

157. Rosenthal, G. A., and Berenbaum, M. R., (1992) Herbivores: their

interactions with secondary plant metabolites, 2nd ed., Volume II,

Evolutionary and Ecological Processes. Academic Press. California.

158. Round, F. E. and Crawford, R. M. (1990). The Diatoms. Biology and

Morphology of the Genera. Cambridge University Press. UK.

159. Rueter, J. G. Jr. and Morel, F. M. M. (1981) The interaction between zinc

deficiency and copper toxicity as it affects the silicic acid uptake mechanisms

in Thalassiosira pseudonana. Limnol. Oceanogr. 26:67–73.

160. Ruiz-Ponte, C., Cilia, V., Lambert, C., and Nicolas. J. L. (1998)

Roseobacter gallaeciensis sp. nov., a new marine bacterium isolated from

rearings and collectors of the scallop Pecten maximus. Int. J. Syst. Bacteriol.

48:537-542.

161. Sauer, K. A., Camper, K., Ehrlich, G. D., Costerton, J. W., and Davies,

D. G. (2002) Pseudomonas aeruginosa displays multiple phenotypes during

development as a biofilm. J. Bacteriol. 184:1140-1154.

162. Saurin, W., Hofnung, M., and Dassa, E. (1999) Getting in or out: early

segregation between importers and exporters in the evolution of ATP-binding

cassette (ABC) transporters. J. Mol. Evol. 48:22-41.

163. Sawyer, L. K. and Hermanowicz, S. W. (2000) Detachment of Aeromonas

hydrophila and Pseudomonas aeruginosa due to variations in nutrient supply.

Water Sci. Technol. 41:139-145.

References


164. Schmidt, T. M., DeLong, E. F., and Pace, N. R. (1991) Analysis of a

marine picoplankton community by 16S ribosomal RNA gene cloning and

sequencing. J. Bacteriol. 173:4371–4378.

165. Sharp, K. H., Davidson, S. K., and Haygood, M. G. (2007) Localization

of 'Candidatus Endobugula sertula' and the bryostatins throughout the life

cycle of the bryozoan Bugula neritina. ISME J. 1:693-702.

166. Shiba, T. (1992) The genus Roseobacter, 2nd ed. Springer-Verlag.

Germany.

167. Siebert, P. D., Chenchik, A., Kellogg, D. E., Lukyanov, K. A. and

Lukyanov, S. A. (1995) An improved PCR method for walking in uncloned

genomic DNA. Nucleic Acids Res. 23:1087-1088.

168. Sieburth, J. M., and Tootle, J. L. (1981) Seasonality of microbial fouling

on Ascophyllum nodosum (L.) Lejol, Fucus vesiculosus L., Polysiphonia

Ianosa (L.) Tandy and Chondrus crispus Stackh. J. Phycol. 19:404-416.

169. Silva-Aciares, F. and Riquelme, C. (2007) Inhibition of attachment of

some fouling diatoms and settlement of Ulva lactuca zoospores by film-

forming bacterium and their extracellular products isolated from biofouled

substrata in Northern Chile. J. Biotechnol. 11:1-11.

170. Skovhus, T. L., Holmstrom, C., Kjelleberg, S., and Dahloff, I. (2007)

Molecular investigation of the distribution, abundance and diversity of the

genus Pseudoalteromonas in marine samples. FEMS. Microbiol. Ecol.

61:348-361.

171. Smucker, R. A. (1991) Chitin primary production. Biochem. System.

Ecol.19:357-369.

172. Steinberg, P. D., Schneider, R., Kjelleberg, S. (1997) Chemical defence of

seaweeds against microbial colonization. Biodegradation. 8: 211-220.

173. Stelzer, S., Egan, S., Larsen, M. R., Bartlett, D. H., and Kjelleberg, S.

(2006) Unravelling the role of the ToxR-like transcriptional regulator WmpR

in the marine antifouling bacterium Pseudoalteromonas tunicata.

Microbiology. 152:1385-1394.

174. Stewart, P. S. (2002) Mechanisms of antibiotic resistance in bacterial

biofilms. Int. J. Med. Microbiol. 292:107-113.

175. Stoermer, E. F., Kociolek, J. P., Shoshani, J., and Frisch, C. (2004)

Diatoms from the Shelton Mastodon Site. J. Paleolimnol. 1:193-199.

References


176. Stoodley, P., Sauer, K., Davies, D. G., and Costerton, J. W. (2002)

Biofilms as complex differentiated communities. Ann. Rev. Microbiol.

56:187-209.

177. Stoodley, P., Wilson, S., Hall-Stoodley, L., Boyle, J. D., Lappin-Scott, H.

M., and Costerton, J. W. (2001) Growth and detachment of cell clusters

from mature mixed-species biofilms. Appl. Environ. Microbiol. 67:5608-

5613.

178. Sudek, S., Lopanik, N. B., Waggoner, L. E., Hildebrand, M., Anderson,

C., Liu, H. B., Patel, A., Sherman, D. H., and Haygood, M. G. (2007)

Identification of the putative bryostatin polyketide synthase gene cluster from

“Candidatus endobugula sertula”, the uncultivated microbial symbiont of the

marine bryozoan Bugula neritina. J. Nat. Prod. 70:67-74.

179. Sullivan, C. W. (1977) Diatom mineralization of silicic acid. II. Regulation

of Si(OH)4 transport rates during the cell cycle of Navicula pelliculosa. J.

Phycol. 13:86–91.

180. Sumper, M., and Brunner, E. (2006) Learning from diatoms: nature’s

tools for the production of nanostructured silica. Adv. Funct. Mater. 16:17-26.

181. Sumper, M., Kroger, N., and Mater. J. (2004) Silica formation in

diatoms: the function of long-chain polyamines and silaffins, J. Mater. Chem.

14:2059-2065.

182. Techkarnjanaruk, S., and Goodman, A. E. (1999) Multiple genes

involved in chitin degradation from the marine bacterium Pseudoalteromonas

sp. strain S91. Microbiology. 145:925-34.

183. Thomas, K. V. (2001) The environmental fate and behaviour of antifouling

paint booster biocides: a review. Biofouling. 17:73-86.

184. Thomas, R. W. S. P, and Allsopp, D. (1983) The effects of certain

periphytic marine bacteria upon the settlement and growth of Enteromorpha, a

fouling alga. Biodeterioration 5:348-357.

185. Tillett, D., and Neilan, B.A. (2000) Xanthogenate nucleic acid isolation

from cultured and environmental cyanobacteria. J. Phycol. 36:251-258.

186. Tolker-Nielsen, T., Brinch, U. C., Ragas, P. C., Andersen, J. B.,

Jacobsen, C. S., and Molin, S. (2000) Development and dynamics of

Pseudomonas sp. biofilms. J. Bacteriol. 182:6482-6489.

References


187. Tujula, N. A. (2006) Analysis of the epiphytic bacterial community

associated with the green alga Ulva australis. PhD Thesis, University of New

South Wales, Sydney, Australia.

188. Unabia, C. R. C, and Hadfield, M.G. (1999) Role of bacteria in larval

settlement and metamorphosis of the polychaete Hydroides elegans. Mar.

Biol. 133:55-64.

189. Van Den Hoek, C., Mann, D. G., and Johns, H. M. (1997) Algae. An

Introduction to Phycology. Cambridge University Press. UK.

190. Wagner-Dobler, I., and Biebl, H. (2006) Environmental biology of the

marine Roseobacter lineage. Annu. Rev. Microbiol. 60:255–280.

191. Wahl, M., Jensen, P. R., and Fenical, W. (1994) Chemical control of

bacterial epibiosis on ascidians. Mar. Ecol. Prog. Ser. 110: 45-57.

192. Walsby, A. E. and Xypolyta, A. (1977) The form resistance of chitan fibres

attached to the cells of Thalassiosira fluviatilis Hustedt. Br. Phycol. J. 12:215-

223.

193. Wang, Q, G. M., Garrity, J. M., Tiedje, and Cole, J. R. (2007) Naïve

Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New

Bacterial Taxonomy. Appl. Environ. Microbiol. 73:5261-5267.

194. Way, J.C., Davis, M.A., Morisato, D., Roberts, D.E., and Kleckner, N.

(1984) New Tn10 derivatives for transposon mutagenesis and for construction

of lacZ operon fusions by transposition. Gene 32: 369-379.

195. Webb, J. S., Lau, M., and Kjelleberg, S. (2004) Bacteriophage and

phenotypic variation in Pseudomonas aeruginosa biofilm development. J.

Bacteriol. 186:8066-8073.

196. Wetherbee, R., Lind, J. L., and Burke, J. (1998) The first kiss:

establishment and control of initial adhesion by raphid diatoms. J. Phycol.

34:9-15.

197. Wigglesworth-Cooksey, B., and Cooksey, K. E. (2005) Use of

fluorophore-conjugated lectins to study cell-cell interactions in model marine

biofilms. Appl. Environ. Microbiol. 71:428-435.

198. Wilks, A. (2002) Heme oxygenase: evolution, structure, and mechanism.

Antioxid. Redox. Signal. 4:603-614.

References


199. Yebra, D. M., Kiil, S., and Dam-Johansen, K. (2004) Antifouling

technology-past, present and future steps towards efficient and

environmentally friendly antifouling coatings. P. Org. Coat. 50:75-104.

200. Zhu, W., Hunt, D. J., Richardson, A. R., and Stojiljkovic, I. (2000) Use

of heme compounds as iron sources by pathogenic neisseriae requires the

product of the hemO gene. J. Bacteriol. 182:439–447.

201. Zhu, W., Wilks, A., and Stojiljkovic, I. (2000) Degradation of heme in

gram-negative bacteria: the product of the hemO gene of Neisseriae is a heme

oxygenase. J. Bacteriol. 182:6783–6790.

Appendix I: Media and buffers


Appendix I

Media and buffers

Luria Broth (LB) medium (per litre)

LB 10

10 g NaCl,

10 g tryptone,

5 g yeast extract

For agar plates add 15 g agar before autoclaving

LB 15

15 g NaCl,

10 g tryptone,

5 g yeast extract

For agar plates add 15 g agar before autoclaving

XS Buffer (per 50 ml)

0.5 g potassium ethyl xanthogenate

5 ml 1M Tris-HCl, pH 7.4

2 ml 0.45M EDTA, pH 8

2.5 ml 20% sodium dodecylsulfate

10 ml 4M ammonium acetate

dH2O up to 50 ml

5 × TBE Buffer (per litre)

54 g Tris base

27.5 g boric acid

20 ml 0.5M EDTA solution (pH 8.0)



F/2 Medium (Guillard and Ryther, 1962; Guillard, 1975)

To 950 ml filtered seawater add:

Quantity Compound Stock Solution Molar Concentration

in Final Medium

1 ml NaNO3 75 g/L dH20 8.83 × 10-4 M

1 ml NaH2PO4.H20 5 g/L dH20 3.63 × 10-5 M

1 ml Na2SiO3.9H2O 30 g/L dH20 1.07 × 10-4 M

1 ml f/2 trace metal solution (see instructions below) -

0.5 ml f/2 vitamin solution (see instructions below) -

Make final volume up to around 1 L with filtered seawater and autoclave without

adding f/2 vitamin solution. Allow to cool and add filter-sterilized f/2 vitamin

solution. Sterile f/2 trace metal solution may be added after autoclaving.

F/2 Trace Metal Solution (Guillard and Ryther, 1962; Guillard, 1975)

To 950 ml filtered seawater add:


in Final Medium

3.15 g FeCl3.6H2O - 1 × 10-5 M

4.36 g Na2EDTA.2H2O - 1 × 10-5 M

1 ml CuSO4.5H2O 9.8 g/L dH2O 4 × 10-8 M

1 ml Na2MoO4.2H2O 6.3 g/L dH2O 3 × 10-8 M

1 ml ZnSO4.7H2O 22.0 g/L dH2O 8 × 10-8 M

1 ml CoCl2.6H2O 10.0 g/L dH2O 5 × 10-8 M

1 ml MnCl2.4H2O 180.0 g/L dH2O 9 × 10-7 M

Make final volume up to 1 L with dH2O and autoclave.



F/2 Vitamin Solution (Guillard and Ryther, 1962; Guillard, 1975)

To 950 ml dH2O add:


in Final Medium

1 ml Vitamin B12

(cyanocobalamin)

1.0 g/L dH2O 1 × 10-10 M

10 ml Biotin 0.1 g/L dH2O 2 × 10-9 M

200 mg Thiamine.HCl - 3 × 10-7 M

Make final volume up to 1 L with dH2O and filter sterilize.

Appendix II: Primers


Appendix II

Primers (5'- 3')

Ad1 CTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CCG

CCC GGG CAG GT

Ad2 P- ACC TGC CC -NH2

Ap1 GGA TCC TAA TAC GAC TCA CTA TAG GGC

Ap2 AAT AGG GCT CGA GCG GC

F27 GAG TTT GAT CCT GGC TCA G

R1492 ACG GTT ACC TTG TTA CGA CTT

Tn10C GCT GAC TTG ACG GGA CGG CG

Tn10D CCT CGA GCA AGA CGT TTC CCG

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Appendix IV Transposon insertion sites in the P. tunicata genome


Appendix IV

Gene Sequence (PTD2_12754- IMG locus tag or ZP_01132901 Genbank accession) >gi|88857803:493602-496676 Pseudoalteromonas tunicata D2 1099591001423,

whole genome shotgun sequence

493501 tgttaggttt ttttacttta atgatccaga taacaaaata gtacaaatag attagagaaa

493561 taagcctctt gtagaacgct acaagaggct ataaaggtgt attagtgttt cttgattctt

493621 agcgctaata gacatggcgt taaaatcaat gtcagcacgg ttgcaaaggc caagccaccg

493681 gccaccgctg tggataattg cacccaccat tgagtcgatg gtgctccaaa atcaatttga

493741 cgattaaaca aatctatatt gacctgtaaa accatcggca ttaaacctaa aatagtagtc

493801 accgttgtca gtaatacagg tcttaaacgc tgagcccccg ttcttaaaat agcttcttta

493861 gcttcaatcc cctgcttaca gagcacatta taagtatcta tcagcactat gttgttattt

493921 acgacaatgc cggcaagtga aatgacacca attccagaca tcacaattcc aaatggctgt

493981 tgtaaaatta atagacccaa aaacacccct acggtcgaga aaataaccgc acttaaaata

494041 agcaaagctt gatagaagct attaaattga gtcactaaaa taattcccat cacaaaaagc

494101 gccactaaaa atgcattttg taaaaaggtt tcggattcgt tttgctcttc attttcgccg

494161 cgaactttga gcttaactct agggtcgagg ccttgctctg taagctgcgc ttgtaaacga

494221 ggcaatgcca agctcagcaa ctcccccact ttcatatcag cattaacaga cacaactcta

494281 tggctgtcaa ctcggcgcac cgaatcaact ttttgcaccg cttggcgttc tacaaagtga

494341 gttattggaa tttggccgta ttgcgtatta actcgtaagg tatctaaacg acttaaatct

494401 cgcttatcaa aaggaaagcg aactcgaata tcaatttcat catctacatc atctggccga

494461 tattcaccga gttttaaacc attggtgatc atctgcacat tcgcgccgag catggcagca

494521 tcggcaccaa atcgagcagc gtcagcgcgg ttaagtttca gctgccattc aatacccggt

494581 tttgagccag tatcatcaac attcgtgaac gatccatcag cttcaatggc ttggcgaatc

494641 cgtcttgctt cttgattcag tacttcagga aacttagagc taagctctat tgataaatct

494701 ttaccgccgc cgggaccatt ttcatcttta cgtaactcaa tttcgacgcc cgcaattgtg

494761 ctagttaacg acatcacttt ggcaataatt tggtctgcag gttctcgctc atcccaatct

494821 tttaaattta gacgcaacgt acctactaaa tccttgcctc ctgttaatga ataaagcgtt

494881 ttaatacctg caacactcag cacttttgct tcgatttctt gcattatggt gtctttttca

494941 taaatagata aatcaccata ggaacgcact ttaatattaa ccccattagg ctcaacatca

495001 ggaaaaaact caacccccag cttagatacg ccatacccaa taaaaacgaa tactgaaaat

495061 acaatcgcgc cgaataaaac tttccaagga tgcctaatgg ctcggtcaag cacccgaaca

495121 tagccaccaa taaaaccatg taactgcgtt aagtcgcctt cctcagcatt aagtaattct

495181 tgtttttgcg tggctgaaag tggcttgacc ttaccaatta aactaccaat ggtcggcaca

495241 aaaataagcg ccataactaa agaggcactt aaggtggcca ttagtgtaat aggtaagtat

495301 ttcatgaact cccccatcat acccggccaa aaaattaagg gggcaaacgc tgcaagtgta

495361 gtcgctgttg aagcaataat tggccaagcc atgcgttttg cggctagaga ataggctttt

495421 ttacggtgca ttccttcgcc catcatacgg tcggcaaatt cagtcaccac aattgcgcca

495481 tccaccagca tacctaccgc cataattaag gcaaataaca ctacgatatt aactgtcatg

495541 ccaaataacg agatcactaa aatacccgtt aaaaacgatc ccggaattgc aaccccaact

495601 aaaaaggcgg cgcggctgcc caaaatagca ataattacaa ttaccactaa caatacagct



495661 gaaagcacat tattttgtaa atcagcaagc atttgctcaa catcaagtga catatcaccg

495721 gtgtaattaa ctttgatatg atcaggccag cgcgtgcgtg tttcttcaac cacagctttg

495781 acttgtttca cagtatcgat aatattttcg ccaacccgtt ttttaacttc aagcgatacc

495841 gctaactcac cgttaatgcg agcaatcgta ttggggtctt tataagcacg gcgaataaca

495901 gcaacatcca taaaacggac aaccttatca cccacgactt tgacaggctg ttccattaca

495961 tcttgaatcg attcaaacac tgaaggaatt tttatcgcaa aacgaccttt cccggtatca

496021 agcgtacccg cagcgattaa gcgattatta ttgctaagca actggtaaat atcattttgt

496081 tttaagccgt atgaaatcat cgctaaagga tcgacttcga tttcaaccat atcctctcta

496141 tcaccaccaa tctcaacttc taatacactt gaaattgact ccaattcatc ttttaaattg

496201 cgagcaagag tcagcaaacc acgctcaggt acattgcccg ataaggttaa tgttatggta

496261 ggctgctcgt cttccattaa tacttcatgt acttcaggct cttcagattc tgaaggtaat

496321 tttgcttttg caagcgagac tttatcgcgt acatcagcaa gggcttcttt aggatccatg

496381 cccgctaaaa actcaagtgt gactgaagca tgaccttcac ttgccactgc gctcatttct

496441 tttacgcctt caatcgaccg taattcaatt tccattgggc gtacaagcaa ccgttcggca

496501 tcctcaggtg aaataccatc atgaacaatt gagacataaa taaaaggaat tgtgacatct

496561 ggattggctt ctttggggat attttggtaa gtcacccaac cggcaataag taacaaaata

496621 aatatcgata aaccagtgcg tgtatgatga atagccgcat caatcaaatt acccataatt

496681 actcctgctc attactataa acaggttcaa cttcatcacc aatgcgcaca aacccttggc

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Gene Sequence (coding sequence 233041...578021)



>638341157.NZ_AAOH01000001 Pseudoalteromonas tunicata D2, whole genome shotgun

sequence.

233041 ataaccgagt taaaaaatcg gggttttatt atcgtcaact agggaaataa aatgtcttat

233101 caacaaaaaa aacaaccacg ttttaatcgt tcatgtcttt gcttggctat tagcagcgcg

233161 ttactttgct ttgcggcggc ggctgaacaa aatgcaccag ccgagcaagg taaaaaactg

233221 ctcgatcttg aaaaaattat cgttacaggt accacacgtc gcggccaaac caaacttgag

233281 tcttcagtgt caattaccac cttagatgca aaacaacttg aacgagagca accccttggt

233341 actgccgatt tacttgaagt cgtgcctggt ttttgggttg aagattcggg cggtgaaact

233401 aataacaatg ttgctccacg tggtcttcgt ggcggcgaag ggtttcgtta tattggcgta

233461 gaagaagatg gcttacctgt tgtatacgat ggtgtgtggg ttgattttta tcagcgtcaa

233521 gatcttagca ttcaaaacat ggaagcggta cgtggtggta cttctggttt actcaccgtc

233581 aatggccctg cggcattagt gaattttatt acccgcaaac ccgatgatat cgaagaaggc

233641 accattcgtg tatcgactgc cgattacggc atgctcaaaa ccgaactgtt ttacggcacg

233701 ccaattagcg acaattggaa aatggcggtc ggtggttttt atcgtcattc tgaaggcgtt

233761 cgagacactg agtttagtgc tgatcacggt ggccaagtgc gcctaacgtt agttcgcgaa

233821 tttgaaaaag gccagctaac gctatctgcc aagcacctca atgatcacac tactttttat

233881 gtaccaattc cattacaaga ccaacaaaat ccaaccggga tcccaggggt tgacccacaa

233941 agtggcacct tgattggcaa cgaccaacgt ttattaagct atcgcaaagc cgatggaaat

234001 tacgttacgc gcgaccttaa agatggccag catacccaat tttcaaccct tggctttaat

234061 ttagattggg agcttagtga taactggctc atgaaattgg caggacgtta ttcaaaattc

234121 gataacgaca tgtatatttt acttaacttt gataactcga ctttaatgaa tgcaaacgat

234181 cgcttagcac aacaagatgt acagggaatg ctcagccatt ttgcagatga tggcgctgtg

234241 cgagccatgt atcggtatgt gggagacgat cacataatta acgacccaag ccaacttaat

234301 ggcaatggct tagtcaccac tagttaccct ttgttttcaa gctaccaagc tgagcaattt

234361 gtaaataaag cctcgttcac ttacgaaggc gagcaaaaca atctcaccct tggctggtta

234421 tatgcttatg ttgatgccga taccctaccc gttgataaat gggaatcaca atttttaacg

234481 gaagtacgct ctaatgcccg tcgcctcgat atagttgcag tcaataacag cggtgatgtt

234541 gtgggccagc taaccgataa cggttcaaca ggttatgcgc caggctgggg ccaagccacg

234601 gcttttggta cttcaagctc acactcattt tttattaatg atgaatttca agccaccgat

234661 gatttacgcc ttgatgcggg tattcgtatt gaatggctca agttagatag cactgcatcg

234721 ggtacgcaat tcgctgtgcc aattttgggc gcctttaacg ccaatggcga tgacagcgat

234781 aacattatgg ccaataacta tgccgatatg ccatcgagta aattttataa ccaaacccgt

234841 aacgaaaccg aagccgcgtg gacagtaggg tttaattaca cttttaataa agatatggcg

234901 atgtttggcc gctatgccga cgcttttgaa atgccacgct tattaagcca tggccaaggg

234961 atccacagcg gtaaaagcgc tgactttaac gacgtggtta atttaacctt tagcgaatta

235021 ggcttacgtt attcgggtga atcaattggt acctctgcca cattatttcg taccaagttt

235081 aatgatttaa ccgagcgtaa tttcacctca agtaacggcg cagtcgccaa tcagaccata

235141 gataccatta ccgatggcgt cgagtttgaa gcggcgtggc aagcaaccga tgatttaaaa

235201 attgagctta caggtgtggt ccaagagcct aaaatgtcgg gctttgaagg tgattttaaa

235261 cattgggaaa acaaccaagt taaacgtact ccaaaaatgc aattacgcat tactcctacc



235321 tattatttta ataatggcga tgtgtattta accatgcacc atttaggcga tcgtttttca

235381 gatggtgaga ataaatttga attacccgcc tataccacat gggatgcagg agtgaactat

235441 caattcactt caagcctgcg cttacatgtc aaagccgcta acctgaccga tgaaattggt

235501 ttaaccgagg gtaatccacg tgccattaat gaccaacaag cgggttatga atactactat

235561 gctcgcccga ttttagggcg caccattagt gcctcgctca cctttgattt ttaagcttta

235621 tttttaatat ttgttcaact tcccccttga acttacccaa aacccaacgt ctgccaacgc

235681 ttgggtttta tttttctgtt ggagttatcg atgtattata aaattttgac cagtattagc

235741 gtattggtat tttcagcttg gctttgggct gcgccgtcaa caatctctat tgtgcctgaa

235801 ccgaaacaaa cgattgtttc aaaaggtgta ttttccctaa acaatcaaac aaaaataagt

235861 tacgacagcg acaaaagcaa gccgacagca accatgtttt ggcaaactat tgcgcccgtt

235921 accggttatc aacagccaat aacacagcgc tcggttgttg gaaaaaacca cattcatttt

235981 cagcttgata gcacactgac cacgccagaa agttatcagt tgagtgtctc tgtagagcag

236041 gttcgtattc gtgctgcgga cgtagccggg ttattttatg gcatgcaatc attgttgcaa

236101 ttactcccgc cagatattta tgccaatcat cccatcaacc aattaagttg ggatattcca

236161 gccgtcgaaa ttaatgacca accacgtttt agttatcgcg gcatgcacct tgatgtcagc

236221 cgccactttt ttaatgtgga ttttattaaa agctatattg attggctggc ctttcacaaa

236281 ctaaatgtat ttcaatggca tctaaccgat gatcaaggct ggaggataga aattaagacc

236341 tatcccaagc tcactgaagt tggatcaata cgtaatcaaa cggtattggg ccacacctat

236401 gactaccaac cactttttga tacgaccgct gtaaaggggt tttacactca agcacaaatc

236461 aaggacgtcg tggcctacgc ggcagcaaga catgtgatgg tcattcctga aatcgacatc

236521 ccgggccaca gcaccgctat tttagcggct tatccagaat taggttgcag cggtaagcgc

236581 cccgtagttg aagacaactt tggcattttt gaagcggttt tatgccccac cgagcaaacg

236641 tttgccttct tacaacaggt ttatcaagaa gtcgctacgc tttttcctgc cccttatatt

236701 catgttggtg gtgatgaagt gatcaaaaaa caatggctcg caagcccttt tgtgcagcaa

236761 ctaatgcaag agcttcagct tactagcaca gaacaagtac aaagttattt tattggcaga

236821 gtcagtaata tagtcaccgc gcttggcaaa aaaatgatcg gctgggatga aatattagaa

236881 ggtggcttag cccctaatgc cttagttacc agctggcgcg gcgaagatgg cggtgttgct

236941 gcagcaacac tgggtcatca agtaataatg agcccttatc aatttgttta ttttgatgcc

237001 tatcaatccc tgtcacagcg cgaacctaaa gcaattcatg gtttaaccac cctcaaagac

237061 gtgtatttat atgagcctat acctgcgcag ctgcccgctt cacaacatca tttagtccta

237121 ggcgcacaag gggctttgtg gacggaatac ataaaaaccc cgcaacaagc tcaatacatg

237181 ctttttccac gtattgctgc ttttgctgaa ggggtttgga gccaacctgc gcagcgcaac

237241 tggtctttgt tcacgcaaaa attgccgcta ttatttgcac gctaccaagc gcaaaacatc

237301 cattatgcac tcagccattt agtgcctgat attgccatta agcaagtcaa ctcggggcaa

237361 tcacaattaa ctattgccaa ccaattagat ggccaaattc aggttaaact gacaaatgaa

237421 acactggatc atatctacag tgaaccgctt ttagtcgata gtaataaaca agtggtgagt

237481 gtcagtgctc gtttatttgc gcccaaatta ggcctctatt ctttgcccgt acaagtcagt

237541 tttgcccacc acaaagcggt aggtaaacct attaccttaa aatacccggc gcaatctgat

237601 gggctacaaa aactcaatga tggtattttt gcttttgacc agttttaccg tgcagataat

237661 ttcgctattt tttatgatag cgacttagag gccgttatcg accttgaaaa caaaacgtca

237721 tttcatcaaa tagtcatggg tattgatgcc ggccgacatc gccaattaca tccacccatt



237781 gcaatatcag tgtgggtatc gaatgacaag caaaattggc agcgagtaac acaattaaat

237841 gcaagtgaaa taaacggacc actccttagc tttgcggtag gtaaacagat agcacgttat

237901 gtgaaagtac acgcagttaa tgccaaaaat agtaccgatc cacaaatccc aaaattgccg

237961 ctttacatcg atgaaattgc gattttttaa tgatgattac agataaaact aagtatggct

238021 gagttcaaaa acactcacat cgctagatag tttattttta attaacaagg caaataagca

238081 ataaaaaaag ggctctgtga gccctttaaa tcagttttat agttattaat tattgcggga

238141 tatattggta aatggcttta ccataataat tggtataagc gccaccaacc gacagcagag

238201 tattaccata ggcaaccgtt ttaaagtcac gtaaataata gctagtggta tcttgcacct

238261 caaccgcaaa cggattagcg ctaagcgttt tccattgcat taaatcaaaa tcaaattgca

238321 caaagttttt tgcacttaac aggtataact tattgcctgt aagtgtagtg tcataataaa

238381 caccggctgc aaaatcagga attaactctg aatttaaaac actttgtgtt gtagtatcaa

238441 attgacttaa cagtaattgc tcatcttgac gttggataac aaaaagcgca tcacctttgc

238501 taaaacaatg ttgtatttca ttcaaactct caggtaattc tgttgcttgc cagctgttgt

238561 ccactgtatt taaacgatta aggcgttgct gttgaccatt tttagaaaac gcaatgacct

238621 caccgttaaa tgaacaactc gcttgtagcg gattactaaa tggcgcaaaa gtcggcgctg

238681 caaacgttag ccaggttttt tcttgtgtat catacacttc catattagct tgcgcatttg

238741 ccccacttga acacgcctcg gtgccaccaa agagataaag ctttttatca acaagttctg

238801 cttgataata ataacgactc gattgcggag agctcagttc ttgccaatta ttttgtgcaa

238861 tatcccaata tgacaacctt ggagcgctcg gttcggcact tgcacacaac atcgcagagg

238921 caatcgcaaa gttatgtgtc actgcataca gatcatcacc ggttgactgg acacttaaat

238981 gcgaaccttc aaaacccggt aaatcaccga gtttaaccca agatactgca atcacttcaa

239041 tatcaaatgc aggtaaagca gcttcaaatt caccatctga gacacgaata acaatgccac

239101 tggttttacc tgcatgctga tttgaaggca taccactcaa taccccagtt acagtatcaa

239161 acgtcgccca atcaggttta ttgtcaatac taaaactcaa ttgctgcgaa tcaatatctg

239221 aagccgcggc agcaaaatga tacgtttcat tcgcttgcac agtagctgct ggagttcctg

239281 taattactgg cgcatcgtta acagccgcaa gggtaatatt aacttttacc gtgctacttt

239341 ctttagagcc tttttttagt ttaaagctaa aggtatcgtc accaaattgg ttaattgctg

239401 gagagtaagt taaagcagga tacacacccg tcacttgtcc aagcgtcggt tgatttaaca

239461 gttcaacaac tacagcttca tcaccatcag ggacttgatt agcactaata cctatcgtta

239521 atgggttatc ttcagtacca ttaaccgtga tttcaggttc gacttcaaaa atgctttggg

239581 caatcacatc aacattaaag gcgggtaaag cagcttctaa ttcaccgtca gagacacgaa

239641 taacaatgcc actggttgta cctgcatgct gatttgaagg cataccactt aataccccag

239701 ttacggtatc aaacgtcgcc caatcaggtt tattgtcaat actaaaactt aattgctcag

239761 aatcaatatc tgaagccgcg gcagcaaaat gatacgtttc attcgcttgc acagcagcag

239821 ctggagttcc tgtaattact ggcgcatcgt taacagccgc aagggtaata ttaactttta

239881 ccgtgctact ttctttagtg ccttttttta gtttaaagct aaaggtatcg tcaccaaatt

239941 ggtctgctac cggcacatag cttagatttg gatatacgcc cgatatctca ccaagcgtag

240001 gttgatctac taattctaaa actatagcct cagtaccagc ggggacttta ttggcattaa

240061 tgactacagc tgatgggcta tcttccttgc ctgtaaccgt cattactggc tggatctcaa

240121 atacactttg cactagggtc ggtgtgcttt ttttatcaga acccccacag ccggttaaca

240181 atacagcaga tatagctaaa cttaaaattt tgatcttcat aataaaacca cttccgatgt



240241 gaacagaaat ttaatgtgcc ttgcgcttct catctttacc atgcaattga cgagctatga

240301 gcaacaaaag gatagaactt cattaaatac taaagctttt tcaggtacaa gaattcatgc

240361 taattcattg taaataaatg tataaaaaaa ctgcaacacc agcaattgtc taaatttatg

240421 tttgtcacct ttaccatcac caaccaactt ttttacagct aattttataa tgaactcaat

240481 tacaccacac cgcagctttt catctttata gcttcaaacg acttaaaccc tatggtgatg

240541 gcgcttttta tgtcggtttt atcggcaaaa ttggcttgct gcgtcatttg ctctatccca

240601 ttacctaagg tgtaacgcat tttagcgctg ccatgaccgt aatatctcag ctgataagca

240661 aactgatatt ttttagtatt ggcattatac atcggctctg atatccggta ttcgagcgcg

240721 caatcattgc cgaaaaaccg ccaagcggaa taattgattt gcgctttatt ttgcgaaaag

240781 gtttcttgtg gtgtgccgcg cccttgtaaa acacggtatt caatcgctga ttgcgtagcc

240841 aaaggttggg tttcttgaaa ggctaaaaac gatgtaacca gtaaatcggg ctgaagacct

240901 agtgtctgac tttgttgggg gttgacgcca ttatgaagca caaacttgcc aactggcagt

240961 gttactttga gcttatggtg cagactcact tgctcataga tatcgacttg cccagcagta

241021 ggttcaccca caacataagc ccgtttaagg tgctgtaaag cactaacaaa acgctcagcc

241081 accacccctg tttgctcgct ggttagcaca tacaccggca catgagctcg ttttttaccg

241141 gccactcttt taagtgttgt aatgatgtcg gccttttttt taccaacttt atcaatacga

241201 tataactcag taggttgttc aaataaataa cttgcggtgt acaacaagac atcagaatcc

241261 gcattttggg ggctgcggct gtggcgcaaa tcaaaaacaa atacctcagc atccgccact

241321 tggctaaaaa gggtatcaat ttgtgtttga gtgctgtgta aatcaagttc aatcagtgct

241381 atttcatcat ttacgaatgt gatttgattc gaggccgtgg tggcagctac agcatgtgaa

241441 aggtcgggct cattcgccga tacgacaatg gagggatctt gtaacaccgc ttgcacatct

241501 tgggtcagtt tggcagcaaa gtcagcgggc tgatcaaagc ctaaatactt ataagattga

241561 tattgtttgc tgaggtgcgc tgcaattcgt ttgcctactt gtgggtcact gctgcgctgt

241621 tcaatctgta acgctaaagt attgataatt tctagctggg cttcttcgct tagtaataac

241681 gatgcttgcg agtcctcgga ataggccaac gttgctacac aagctaacat cccaacaacg

241741 agtcctttac ccatctttgc agccatccat cgcactcctt tattcgccat ttgttcctat

241801 tacacatcgg taaactagaa cattaatgca gcaggctgtc agcaaaaaaa gcacgttaat

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Gene Sequence (PTD2_02946 IMG locus tag and ZP_01132126 Genbank accession)



>gi|88857000:575410-576111 Pseudoalteromonas tunicata D2 1099591001414, whole

genome shotgun sequence

574561 aaacaaaaca agcagacaac cggctaatgt taatttttta tagaatttca tgttgggcac

574621 ctttatttgt ttgtttcatt acttgtttca taatgactag tttttttgaa atagcgttat

574681 caactaagct tggaaaaata ccattaagct tggcaaaaaa tcgctcagga aaaccaataa

574741 cagtgcgtgc aattctgcta ttgagttgtt ttactagttg ttttgccact agctcagggc

574801 tatccatgtg attgccaaga gcagtattca tagcccgttc cagcacacca ttaatttggg

574861 tatcggttgc ccttggcgca agataaagca catcaatcgg tttatcactg agttctcttt

574921 taagcgcttc agtaaaccca cgtagaccaa acttactcgc gcaataactc gcataatatg

574981 gaaaaccaat actgccaaac gcactgccaa cattaacaat tgttgcactg tctttttgtg

575041 ataattgtgc caaaaacgct tgagttaata acatcggtac caatagattc atttcaagag

575101 tttttgagat atcaaccgca ctttgatgat caaaactggc catttgattc acgccggcat

575161 tgttaataag taccgaagca ccacctaact tttgcgcttt atgtaccaaa gcatctcgcc

575221 cttctatcgt tgctaaatcg gccgtaatat agctatgttg attacccaaa cttttagaaa

575281 gttgtgccag ttttagttca ttgcggccca ccaataacaa acgataacct tgatcatgaa

575341 gtgctgcagc cattgcttgg ccaatgccac cgcttgcgcc agtcaataca cataaaggtg

575401 attttgacat cataatgaca cctcacaatc agcattgacc tgtttagttg cagttaaacc

575461 gtgaagcggg tctaagcttc gaaacatatc accatataaa tgataaaagg catttgcact

575521 tttaatgatc agttgttgct cagatgggtc tgtaatttta tccattaaac taataaagaa

575581 cttaacgtga tcttgatcta atgcaccatg ggaacgtaaa tagctaaatg ctttatttgg

575641 aagctgaaga cggttttgca cctgcgcagc ggcgttatcg gctaatgcga tactggtgcc

575701 ttcaagcaca tgcaccatgc caaaaaagca cagcggatta attctgctca cacaatcata

575761 cgcatacgat accattaatt cggtcgcaaa taatggcgtt gacttacgtg cttgttcttt

575821 atcataacca caagctgcaa tatcgtttaa tacccattct tgatggccaa gttcttcttc

575881 aatatattct gcaacttcat ttcgcagcca ttctttggat tctggtaatc gacttccaac

575941 agccattaat aaaggtgttg tatgcttaac atgatgataa gcctgctgta aaaaggcgac

576001 ataatcatca atgctaaatt gacctgcaaa acaacgttga ataatcggtg cttgtagaag

576061 atattgctgc gactgctgtg ttttttgtac tagtgtttga taaaaactca taatgactcc

576121 aaagcagaac caacaggttc agcacattgt gatttagatg cataaagggc gttaacttgg

576181 gccgcaaaat gcgtagcaat ttctgttctt tttggtcggt tgtttgcggt ataaagatta

576241 tcgatttgac tcattggtgc attgagtact tcatacgccc gtacttttgc gtaatcgggt

576301 aattgtgcat taactttatt gatggcagca ctcagattgg cctcatccat tggtttaatt

576361 ggtacaagca aagcaataca gaatggtctt tcttcaccta atacaaccgc ttggtaaaaa

576421 aggccggtcg cttgcaataa gctttcaggc cattcggggg cgatattgcg accaaagctt

576481 gaaataatca gatttttttt acgaccattg ataattaaaa agtcgtcgtt aataacagct

576541 aaatctcccg ttttgaactc tcgcgcaaac caagaatcgg gatcgttttg gtatcccaag

576601 tggcaattgc cacagacaac taattcacca tcaacttcat atacttgaca atgaggcagc

576661 actttacctg cactggcatg attacaatcc aaaggcgtat ttaggctcac caccgagcca

576721 cactccgaaa gtccatagcc ttgataaaca ggtaaaccta atctgtgggc ttcttctaat



576781 aaatcacacg cgacttttgc accaccaaca gcgataaatt tcaacgatgt cggtgtagtc

576841 caacctgcct tgattgcatt gaccaataac agcaacagtt caggtactaa aattaaagag

576901 ttcggttcag cacggctaat attcatcaag agtttatttg gctcaaccag cttagtaccg

576961 ttaaaaccta atgcttcaag ggatacaagc gtcacagtgc caccagccaa taagggcgca

577021 tacacccctg cgatattttc taacaaaacc gaaaggggta ataaacaaag gtgttttggc

577081 gcttttaaag cgatagcatc aactaatgag tgagccacta ctgtctgatt ttcaacagat

577141 aagcacaccc cttttggtaa accggtagag ccagaagtaa aggtgacttt ttgggtacca

577201 ttaggtcgca tgccaaagtc tgattgcatt aattgtgcga taaataaagt gcggccaaag

577261 ccaacaaaca cctcactacc gacagccgaa atatcattat taatctgctc actgataaaa

577321 caatcgaccg ctgagctctc aattaaatgt ttaatttggc tttcagtaaa aaataaaggc

577381 actgagataa ttgtgatatt cgcttgttgc gcagctaaat caaacaaaat ccattcaatg

577441 caattatcga tatgtagtgc cagagtttgc acgtttaaat gagttaataa gcggctcctt

577501 acatgtacgg cttctatcag ttgttcgtaa cttaattgac attcaccatc gtctaacgca

577561 atgcgagttt caaattggct tatcgaatct atcaagctca taattgagtc acctctggca

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INHIBITION OF PRIMARY COLONIZERS BY - USP …digilib.library.usp.ac.fj/gsdl/collect/usplibr1/index/...INHIBITION OF PRIMARY COLONIZERS BY MARINE SURFACE-ASSOCIATED BACTERIA By Vipra

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