Genomic Analysis Of Nitrogen Fixation In Synechococcus Sp. PCC 7002 and Chlorobium tepidum

Genomic Analysis of Nitrogen Genomic Analysis of Nitrogen fixation in fixation in SynechococcusSynechococcus sp. sp.

PCC 7002 and PCC 7002 and Chlorobium Chlorobium tepidumtepidum

Heather Jordan

Department of Biochemistry and Molecular Biology

The Pennsylvania State University

University Park, Pennsylvania 16802

March 18, 2003

Bryant Lab Research InterestsBryant Lab Research Interests

• Physiology of growth at low temperature in cyanobacteriaPhysiology of growth at low temperature in cyanobacteria• The role of multiple sigma factors in gene expression in cyanobacteriaThe role of multiple sigma factors in gene expression in cyanobacteria• The structure, function & biogenesis of PI rxn centerThe structure, function & biogenesis of PI rxn center• Alternative electron transport pathways in cyanobacteriaAlternative electron transport pathways in cyanobacteria• Photosynthetic apparatus in green sulfur bacteria (chlorosome)Photosynthetic apparatus in green sulfur bacteria (chlorosome)• Physiology & genetics of green sulfur bacteria Physiology & genetics of green sulfur bacteria C. tepidumC. tepidum & & C. vibrioformeC. vibrioforme• Bacteriochlorophyll biosynthesis in green sulfur bacterium Bacteriochlorophyll biosynthesis in green sulfur bacterium C. tepidumC. tepidum

Bryant Lab Research InterestsBryant Lab Research Interests

Chlorobium tepidumChlorobium tepidum

Synechococcus sp. PCC Synechococcus sp. PCC 70027002

What is cyanobacteria?What is cyanobacteria?• 7500 species7500 species

– Greatest diversity in Greatest diversity in fresh waterfresh water

• PigmentationPigmentation– ChlorophyllChlorophyll– CarotenoidsCarotenoids– PhycobillinsPhycobillins

• Add biomass & nitrogen to Add biomass & nitrogen to barren areasbarren areas

• Found in a wide variety of Found in a wide variety of environments ranging from environments ranging from hot springs to glacier icehot springs to glacier ice– Recently found in the Recently found in the

most barren area of most barren area of Antarctica where no other Antarctica where no other life is found.life is found.

– May also be endolithicMay also be endolithic

What is cyanobacteria?What is cyanobacteria?

• Morphological Hierarchy:Morphological Hierarchy:– Single cellsSingle cells– ColoniesColonies– FilamentsFilaments– Branched, unbranched or Branched, unbranched or

clusteredclustered

Evolutionary SignificanceEvolutionary Significance• Important source for fixed nitrogenImportant source for fixed nitrogen• Cyanobacteria most likely origin of Cyanobacteria most likely origin of

chloroplastschloroplasts– Endosymbiont TheoryEndosymbiont Theory– Supported through analysis of Supported through analysis of

photosynthetic apparatus & C photosynthetic apparatus & C assimilationassimilation

• Components (LHC) of C. tepidum Components (LHC) of C. tepidum chlorosome resembles plant chlorosome resembles plant chloroplastschloroplasts

• Mechanism of COMechanism of CO22 fixation distinct fixation distinct from Calvin cycle found in high from Calvin cycle found in high plants.plants.

• Prominent role in global carbon Prominent role in global carbon cyclingcycling

• Constitute some of the oldest Constitute some of the oldest fossils (over 2.7 billion years old)fossils (over 2.7 billion years old)– Oldest oxygen-producing organismOldest oxygen-producing organism– Profoundly changed the earth’s Profoundly changed the earth’s

atmosphere.atmosphere.

Diversity of CyanobacteriaDiversity of Cyanobacteria• Differences Differences

probably due to probably due to adaptation to adaptation to different different environmentsenvironments

• Sequencing Sequencing projects seek to projects seek to establishestablish

– Which genes are Which genes are common to allcommon to all

– Which genes are Which genes are specific to specific to ecological nichesecological niches

The Ecological Significance of CyanobacteriaThe Ecological Significance of Cyanobacteria

• Can lead to erosion Can lead to erosion in limestone & coral in limestone & coral reefsreefs– Grazing animals eat Grazing animals eat

the cyanobacteria the cyanobacteria and subsequently and subsequently reduce the amount of reduce the amount of limestone present,limestone present,

• Can lead to limestone Can lead to limestone depositsdeposits• Some marine species Some marine species precipitate CaCOprecipitate CaCO33

Nitrogen FixationNitrogen Fixation• Nitrogen is needed for the synthesis of amino acids & Nitrogen is needed for the synthesis of amino acids &

nucleotidesnucleotides

• Organic Route:Organic Route: Breakdown of proteins Breakdown of proteins

• Inorganic Route:Inorganic Route: Nitrate Reduction Nitrate Reduction• Nitrate reduced to nitrite via nitrate reductaseNitrate reduced to nitrite via nitrate reductase• Nitrite reduced to ammonia via nitrite reductaseNitrite reduced to ammonia via nitrite reductase• Ammonia incorporated into amino acidsAmmonia incorporated into amino acids

• N fixation is an energetically costly processN fixation is an energetically costly process

Nitrogen FixationNitrogen Fixation• Often have symbiotic relationships Often have symbiotic relationships

with other organismswith other organisms– Lichens, Hornworts, Azolla (fern), Cyads, Lichens, Hornworts, Azolla (fern), Cyads,

Gunnera (flowering plant)Gunnera (flowering plant)

• Heterocysts are sites for N fixationHeterocysts are sites for N fixation– Provide an anaerobic environmentProvide an anaerobic environment

– Oxygen inhibits nitrogenaseOxygen inhibits nitrogenase

– Internal membranes are thylakoids that have Internal membranes are thylakoids that have lost chlorophyll (sites for nitrogenase)lost chlorophyll (sites for nitrogenase)

– Connected to vegetative cells via Connected to vegetative cells via microplasmodesmata (pore in end wall)microplasmodesmata (pore in end wall)

Nitrogen FixationNitrogen Fixation• In non-heterocystous In non-heterocystous

cyanobacteria (i.e., cyanobacteria (i.e., SynechococcusSynechococcus), N is ), N is fixed aerobicallyfixed aerobically

• Cyanobacteria unique Cyanobacteria unique in that they can in that they can perform both N fixation perform both N fixation & oxygenic & oxygenic photosynthesisphotosynthesis

N-fixation occurs N-fixation occurs when PSII is not when PSII is not oxidizing Hoxidizing H22O to OO to O22..

Nitrogen Fixation GenesNitrogen Fixation Genes• NAR1 (NAR1 (SynechococcusSynechococcus))

• nifD (nifD (C. tepidumC. tepidum))

• Nitrogen fixation related (Nitrogen fixation related (nifnif) genes are expressed ) genes are expressed under anaerobic conditionsunder anaerobic conditions

• Nitrate Assimilation Related (Nitrate Assimilation Related (NARNAR) genes) genes

– NAR1 gene encodes a chloroplast membrane protein NAR1 gene encodes a chloroplast membrane protein involved in nitrite transportinvolved in nitrite transport

– Nitrite reduced to ammoniaNitrite reduced to ammonia

Project 1:

Genomic Analysis of Nitrogen Fixation in Synechococcus sp. PCC 7002

Optimal Growth Conditions for SynechoccusOptimal Growth Conditions for Synechoccus

• Temperature:Temperature: 38 38ooCC• Likes brackish waterLikes brackish water• Obligate Obligate

requirement for B12requirement for B12• Facultative Facultative

photoheterotrophphotoheterotroph• Tolerant of high light Tolerant of high light

intensities intensities (up to (up to 5000 5000 E mE m-2-2 s s-1-1))

Other Characteristics of Other Characteristics of SynechococcusSynechococcus

• Also known as Also known as Agmenellum Agmenellum quadrupiplicatumquadrupiplicatum strain PR-6 strain PR-6

• Isolated by Chase Van Baalen Isolated by Chase Van Baalen in 1961 from a marine mud in 1961 from a marine mud sample in Puerto Ricosample in Puerto Rico

• Naturally transformableNaturally transformable• Among fastest-growing Among fastest-growing

cyanobacteria (doubling time cyanobacteria (doubling time under opt. conditions = 3.5 under opt. conditions = 3.5 hours)hours)

• Cells 1.5-2.5 Cells 1.5-2.5 m in size.m in size.• Usually occur as single cells Usually occur as single cells

but sometimes in clustersbut sometimes in clusters• Gram negativeGram negative

Other Characteristics of Other Characteristics of SynechococcusSynechococcus

• Foundation of the marine food Foundation of the marine food webweb

• Primary producers on a global Primary producers on a global scalescale

• One of the most numerous One of the most numerous genomes on Earthgenomes on Earth

• Obligately marineObligately marine• 1/3 of open ocean isolates 1/3 of open ocean isolates

possess a type of swimming possess a type of swimming motility not seen in any other motility not seen in any other type of microbe (propel at type of microbe (propel at speeds of up to 25 mm/sec)speeds of up to 25 mm/sec)

• Motility in response to small Motility in response to small gradients of nitrogenous gradients of nitrogenous compoundscompounds

Hypothesis:Hypothesis:• Knocking out Knocking out

NAR1 should NAR1 should result in result in physiologically physiologically interesting interesting phenotypesphenotypes

•Do this by inserting a SpDo this by inserting a Sprr cassettecassette

Checking for segregation & orientationChecking for segregation & orientation

• Segregation:Segregation: Is the Is the insert present?insert present?

– YES!YES!

• Orientation:Orientation: Is the Is the insert going in the insert going in the proper direction?proper direction?

– Will find out Will find out tomorrow.tomorrow.

Project 2:

Genomic Analysis of Nitrogen Fixation in Chlorobium tepidum

Optimal Growth Conditions for C. tepidumOptimal Growth Conditions for C. tepidum

• Found in sediments, Found in sediments, muds, microbial mats muds, microbial mats and anoxic & sulfide-and anoxic & sulfide-rich watersrich waters

• Anaerobic, obligate Anaerobic, obligate autotrophsautotrophs

• Photo-oxidize reduced Photo-oxidize reduced sulfur compounds (i.e., sulfur compounds (i.e., sulfide & sulfur) sulfide & sulfur)

• Temperature:Temperature: 47 47ooCC

Other Characteristics of Other Characteristics of C. tepidumC. tepidum

• Methods for natural transformation allow for targeted gene Methods for natural transformation allow for targeted gene inactivation by homologous recombinationinactivation by homologous recombination

• More than 30 mutants have been created with specifically More than 30 mutants have been created with specifically inactivated genesinactivated genes

• Revealed information about processes pertinent to biosynthetic Revealed information about processes pertinent to biosynthetic pathways of carotenoids and bacteriochlorophylls to pathways of carotenoids and bacteriochlorophylls to chlorosome proteinschlorosome proteins

Transformation of Transformation of C. tepidumC. tepidum

• Antibiotic resistance used as markerAntibiotic resistance used as marker

– Spectinomycin, streptomycin, Ampicillin & chloramphenicolSpectinomycin, streptomycin, Ampicillin & chloramphenicol

• Can use natural transformation, chemical transformation & Can use natural transformation, chemical transformation & electroporation.electroporation.

• Most genes targeted for inactivation were chlorosomal Most genes targeted for inactivation were chlorosomal proteinsproteins

• Inactivation of nifD expressed phenotypically (inability to Inactivation of nifD expressed phenotypically (inability to grow diazetrophically)grow diazetrophically)

– Markers used to date include Spectinomycin-Streptomycin, Gentamicin Markers used to date include Spectinomycin-Streptomycin, Gentamicin & Erythromycin& Erythromycin

GenomeGenome HighlightsHighlights• 1 circular DNA molecule1 circular DNA molecule• 2,154,946 bp2,154,946 bp• G+C content 49.1%G+C content 49.1%• 2,284 ORFs2,284 ORFs

– 50% have been assigned 50% have been assigned a known functiona known function

• pAQ1 has been pAQ1 has been sequencedsequenced

• Six plasmids (pAQ1-pAQ6)Six plasmids (pAQ1-pAQ6)– 4.6, 10.0,15.9, 31.0, 4.6, 10.0,15.9, 31.0,

38.6 and 115.6 kb 38.6 and 115.6 kb respectivelyrespectively

nifD DetailsnifD Details

• nifD gene encodes a subunit of nitrogenasenifD gene encodes a subunit of nitrogenase

• Located in the middle of the nifHDK operonLocated in the middle of the nifHDK operon

Creating a nifD knockoutCreating a nifD knockout• Making pTN1CXMaking pTN1CX

– nifD knock-out construct nifD knock-out construct for C. tepidumfor C. tepidum

• 6,553 bp6,553 bp• nifD (~1100nifD (~11002550)2550)• Restriction Sites:Restriction Sites:

– AhdI (6553)AhdI (6553)– HindIII (4018, 1639, 896)HindIII (4018, 1639, 896)– ScaI (3139, 1570, 1560, ScaI (3139, 1570, 1560,

289)289)– Sty I (3378, 1390, 1377 Sty I (3378, 1390, 1377

339, 69)339, 69)– SspI (2747, 2701, 1105)SspI (2747, 2701, 1105)

Hypothesis:Hypothesis:

• Since targeted inactivation of nifD Since targeted inactivation of nifD using antibiotics as markers has using antibiotics as markers has worked successfully in the past, use worked successfully in the past, use of a slightly altered cassette of a slightly altered cassette containing the same markers should containing the same markers should work as well.work as well.

nifD Project OutlinenifD Project Outline

—————— —————— I am here.I am here.

—————— —————— Previous EndPrevious End

ReferencesReferences• Frigaard, N.U., and Bryant, D.A. (2001) Chromosomal Gene Inactivation in the Green Sulfur Frigaard, N.U., and Bryant, D.A. (2001) Chromosomal Gene Inactivation in the Green Sulfur

Baterium Chloroboum tepidum by Natural Transformation. App. & Env. Microbiol. 2538-2544.Baterium Chloroboum tepidum by Natural Transformation. App. & Env. Microbiol. 2538-2544.• Herdman, M., Janvier, M. Waterbury, J.B., Ripka, R., Stanier, R.Y., and Mandel, M. (1979a) Herdman, M., Janvier, M. Waterbury, J.B., Ripka, R., Stanier, R.Y., and Mandel, M. (1979a)

Deoxyribonucleic acid base composition of cyanobacteria. J. Gen. Microbiol. 111, 63-75.Deoxyribonucleic acid base composition of cyanobacteria. J. Gen. Microbiol. 111, 63-75.• http://geoweb.princeton.edu/research/biocomplexity/index.htmlhttp://geoweb.princeton.edu/research/biocomplexity/index.html• http://www.bact.wisc.edu/microtextbook/Metabolism/NitrogenAssim.htmlhttp://www.bact.wisc.edu/microtextbook/Metabolism/NitrogenAssim.html• http://www.bigelow.org/cytometry/Image_gallery/SYN.html• http://www.biologie.uni-hamburg.de/b-online/library/webb/BOT311/Cyanobacteria/Cyano.htmlhttp://www.biologie.uni-hamburg.de/b-online/library/webb/BOT311/Cyanobacteria/Cyano.html• http://www.bmb.psu.edu/deptpage/faculty/bryant/bryant.html• http://www.bmb.psu.edu/faculty/bryant/lab/index.htm• http://www.bom.hik.se/~njasv/disp.htmlhttp://www.bom.hik.se/~njasv/disp.html• http://www.cbs.dtu.dk/services/GenomeAtlas/Bacteria/Chlorobium/tepidum/TLS/Ctepidum.htmhttp://www.cbs.dtu.dk/services/GenomeAtlas/Bacteria/Chlorobium/tepidum/TLS/Ctepidum.htm• http://www.dsmz.de/strains/no012025.htm• http://www.er.doe.gov/production/ober/gc/omp.htmlhttp://www.er.doe.gov/production/ober/gc/omp.html• http://www.jgi.doe.gov/JGI_microbial/html/synechococcus/synech_content.htmlhttp://www.jgi.doe.gov/JGI_microbial/html/synechococcus/synech_content.html• http://www.ornl.gov/TechResources/Human_Genome/publicat/99santa/158.htmlhttp://www.ornl.gov/TechResources/Human_Genome/publicat/99santa/158.html• Roberts, T.M. and Koths, K.E. (1976) The blue-green alga Roberts, T.M. and Koths, K.E. (1976) The blue-green alga Agmenellum quadriplicatumAgmenellum quadriplicatum contains contains

covalently closed DNA circles. Cell 9, 551-557.covalently closed DNA circles. Cell 9, 551-557.• Van Baalen, C. (1962) Studies on marine blue-green algae. Bot. Mar. 4, 129-139.Van Baalen, C. (1962) Studies on marine blue-green algae. Bot. Mar. 4, 129-139.

AcknowledgementsAcknowledgements

Joel GrahamJoel Graham Niels-Ulrik FrigaardNiels-Ulrik Frigaard

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