Characterization of swarming motility in Citrobacter freundii

R E S E A R C H L E T T E R

Characterizationof swarmingmotility inCitrobacter freundiiYanguang Cong1, Jing Wang1, Zhijin Chen1, Kun Xiong1, Qiwang Xu2 & Fuquan Hu1

1Department of Microbiology, Third Military Medical University, Chongqing, China; and 2Center of Biological Wave, Third Military Medical University,

Chongqing, China

Correspondence: Fuquan Hu, Department

of Microbiology, Third Military Medical

University, Chongqing 400038, China.

Tel./fax: 186 23 6875 2834; e-mail:

[email protected]

Received 23 September 2010; revised 30

December 2010; accepted 18 January 2011.

Final version published online 1 March 2011.

DOI:10.1111/j.1574-6968.2011.02225.x

Editor: Reggie Lo

Keywords

Citrobacter freundii; swarming; motility.

Abstract

Bacterial swarming motility is a flagella-dependent translocation on the surface

environment. It has received extensive attention as a population behavior invol-

ving numerous genes. Here, we report that Citrobacter freundii, an opportunistic

pathogen, exhibits swarming movement on a solid medium surface with appro-

priate agar concentration. The swarming behavior of C. freundii was described in

detail. Insertional mutagenesis with transposon Mini-Tn5 was carried out to

discover genetic determinants related to the swarming of C. freundii. A number of

swarming genes were identified, among which flhD, motA, motB, wzx, rfaL, rfaJ,

rfbX, rfaG, rcsD, rcsC, gshB, fabF, dam, pgi, and rssB have been characterized

previously in other species. In mutants related to lipopolysaccharide synthesis and

RcsCDB signal system, a propensity to form poorly motile bacterial aggregates on

the agar surface was observed. The aggregates hampered bacterial surface migra-

tion. In several mutants, the insertion sites were identified to be in the ORF of

yqhC, yeeZ, CKO_03941, glgC, and ttrA, which have never been shown to be

involved in swarming. Our results revealed several novel characteristics of

swarming motility in C. freundii which are worthy of further study.

Introduction

Bacterial swarming is a flagella-dependent surface transloca-

tion exhibited by a wide variety of flagellated bacteria (for a

review, see Allison & Hughes, 1991; Fraser & Hughes, 1999;

Harshey, 2003; Kaiser, 2007; Kearns, 2010). This form of

locomotion, which was identified as forming a typical

swarm colony on a solid media surface characterized by

circular symmetry and regularly spaced concentric terraces,

was first described by Hauser (1885) in Proteus mirabilis

over a century ago. Both swimming and swarming motilities

depend on bacterial flagella, but they differ in many ways.

The most noticeable distinction is that swimming is an

individual behavior, whereas swarming is a movement of

bacterial populations. Moreover, the cells exhibit differen-

tiation during swarming; they are usually elongated and

hyperflagellated compared with the vegetative cells grown in

liquid media (Allison & Hughes, 1991; Harshey, 2003;

Rather, 2005). Swarming also shares features with other

surface phenomena, such as biofilm formation and host

invasion, and is associated with pathogenesis in some

organisms. For example, swarming of P. mirabilis facilitates

ascending colonization of the urinary tract and is conducive

to biofilm formation on catheters (Allison et al., 1994;

Stickler et al., 1998). Expression of flagella and virulence

factors are coordinated in P. mirabilis and Serratia liquefa-

ciens (Allison et al., 1992; Givskov et al., 1995). The flagellar

export apparatus of Yersinia enterocolitica also functions as a

secretion system for the transport of a virulence-associated

phospholipase (Young et al., 1999). In many species, swarm-

ing bacteria exhibit adaptive resistance to multiple antibio-

tics (Butler et al., 2010).

In recent years, system-screening studies in various

species have revealed numerous swarming-related genes.

These genes are involved in flagellar assembly, synthesis

of polysaccharides, chemosensors, signal regulation, and

metabolic pathways, whereas others are hypothetical

genes with unknown functions (Kearns et al., 2004; Inoue

et al., 2007; Overhage et al., 2007). However, the genetic

determinants for this special process vary among species,

indicating different swarming patterns in various swarming

bacteria. Therefore, the study of swarming motility in

various bacteria would facilitate a thorough understanding

of this special bacterial motion. Considering that many

types of genes are related to swarming motility, such a study

also provides a tractable model to study the function of

FEMS Microbiol Lett 317 (2011) 160–171c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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genes involved in bacterial differentiation, multicellularity,

and pathogenesis.

Citrobacter freundii is a motile gram-negative bacterium

living in soil and aqueous environments; it is often isolated

in clinical specimens as an opportunistic pathogen. In this

study, we demonstrated that swarming motility could be

induced in C. freundii. It was examined in detail because

little is known about this motility in C. freundii. To discover

the genetic determinants that affect swarming, the mini-Tn5

transposon mutation was used to screen swarming-

associated genes by impairing bacterial swarming ability.

Our results showed that a number of genes are related to the

swarming of C. freundii, among which several have been

newly identified.

Materials and methods

Bacterial strains and media

The following strains were used in this study: C. freundii

ATCC8090 was a gift from Dr Tomofusa Tsuchiya of

Okayama University, Japan; P. mirabilis CMCC49003 was

purchased from the China Medical Culture Collection

Center; and Escherichia coli S17-1(lpir)/pUT mini-Tn5-Km

was a gift from Dr Victor de Lorenzo of the Centro Nacional

de Biotecnologia CSIC, Spain.

M9 salts medium supplemented with 0.5% glucose was

used as the minimal medium. The swarm medium con-

tained 10 g tryptone, 10 g NaCl, and 5 g of glucose L�1 the

final agar concentration was 0.5%. The swim medium

contained the same constituents solidified with 0.3% agar.

To better visualize swarming colonies, a vital dye, triphenyl

tetrazolium chloride (TTC), was added to achieve a final

concentration of 0.05% when required. Both swim and

swarm plates were allowed to dry overnight at room temper-

ature before use. Antibiotics were added, when appropriate,

at the following concentrations: kanamycin at 100 mg mL�1

and rifampicin at 100 mg mL�1.

Light microscopy

To observe swarming motility, 1 mL culture incubated for

10 h in lysogeny broth (LB) (adjusted to 0.5 OD600 nm) was

inoculated onto a thin layer of solid swarm media in a Petri

dish (6 mL media per plate). The plates were directly

observed at � 400 magnification under an Olympus in-

verted microscope IX71 in a room heated to 30 1C. Sterile

slides were occasionally used instead of Petri dishes to

achieve better visualization. The slides were submerged in

swarm media, which was solidified with 0.5% agar, to obtain

a thin layer of media on the surface and dried at 37 1C briefly

before use. After inoculation, the bacteria on the surface of

the media were observed under the inverted microscope.

Images were recorded using a video camera.

Electron microscopy

For negative staining, formvar-coated TEM grids (copper,

75 mesh) were floated on a drop of bacterial cells suspended

in phosphate-buffered saline (PBS, pH 7.4) for 5 min to

allow the adhesion of bacterial cells. The grids were stained

for 5 min using 2% phosphotungstate. After staining, these

were rinsed with water and then air dried. For ultrathin

sectioning, bacteria were washed and suspended in PBS,

fixed in 0.2% v/v glutaraldehyde, and embedded in Spurr

resin. The specimens were examined with a transmission

electron microscope (Philips Tecnai 10).

Mutagenesis with mini-Tn5 and isolation ofmutants defective in swarming

Mutagenesis was performed according to the method de-

scribed by de Lorenzo et al. (1990). Citrobacter freundii and

E. coli S17-1 (lpir)/pUT mini-Tn5-Km were grown over-

night in LB media with rifampicin and kanamycin, respec-

tively. A 100-mL aliquot of each culture was mixed in 5 mL of

10 mM MgSO4 and filtered through a 0.45-mm cellulose

membrane filter. The filter was then placed on the surface of

an LB plate and incubated at 37 1C for 10 h. The bacteria on

the filter surface were washed and suspended in 2 mL of

10 mM MgSO4. About 100 mL of the resulting bacterial

suspensions was spread onto LB plates containing kanamy-

cin and rifampicin and incubated at 37 1C for �36 h. The

antibiotic-resistant bacteria were then transferred to swarm

agar plates and incubated at 37 1C for 12 h. All swarming-

defective colonies were selected.

Analysis of swarming defects

All swarming-defective strains were screened on swim media

to identify the mutants possessing functional flagella but

unable to swarm. Defects in flagellar function were identi-

fied by the absence of outward migration on the media; this

was then confirmed by direct observation under a light

microscope.

Identification of mutant genes

An inverse PCR method was used to amplify the sequence

flanking the inserted mini-Tn5 transposon in the chromo-

some of the swarming-defective mutants. Genomic DNA from

each mutant was isolated according to the cetyltrimethylam-

monium bromide protocol and completely digested with TaqI.

The DNA fragments were self-ligated with T4 DNA ligase and

then used as templates for inverse PCR with the primers P904

(50-GGAGAGGCTATTCGGCTATG-30) and P194c (50-GTAA

GGTGATCCGGTGGATG-30), which were designed according

to the motile sequence of Mini-Tn5-Km plasmid. The PCR

products were separated by agarose gel electrophoresis

and then purified using a gel extraction kit (Watson

FEMS Microbiol Lett 317 (2011) 160–171 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

161Swarming motility in C. freundii

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Biotechnologies Inc.). The PCR products were directly

sequenced at Shanghai GeneCore BioTechnologies. If sequen-

cing failed, the PCR products were ligated to PMD18-T vector

(Takara Co. Ltd, Dalian, China) and the sequencing was

attempted again. To identify the mutant genes, nucleotide

sequence databases were searched with the BLASTN and BLASTX

programs developed by the National Center for Biotechnology

Information (NCBI).

Isolation of flagellin

Flagellin was isolated from bacterial cells according to the

method described by DePamphilis & Adler (1971). The

bacterial cells were suspended in 0.1 M Tris-HCl buffer

(pH 7.5). Flagellar filaments were sheared with a tissue

homogenizer at maximum speed for 30 s. The bacteria were

observed microscopically to ascertain loss of motility. Cell

debris was removed from the flagella by centrifugation at

15 000 g for 15 min, and flagellar filaments were then pelleted

from the supernate by ultracentrifugation and then suspended

in 0.1 M Tris-HCl buffer (pH 7.5). Sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) was used to

analyze the purity of samples.

Preparation of flagellin antiserum

Flagellin protein dissolved in Tris-HCl buffer was emulsified

in Freund’s incomplete adjuvant (1 : 3). One rabbit was

immunized three times at intervals of 2 weeks. Serum was

collected 1 week after the final injection and stored at � 20 1C.

Analysis of flagellin production byWestern blotting

Bacteria were suspended in Tris-HCl buffer and then adjusted

to 1 OD600 nm. All the cell lysates were subjected to SDS-PAGE

electrophoresis and then transferred onto a nitrocellulose

membrane. The flagellin was visualized via Western blotting

with enhanced chemiluminescence detection (Pierce). Rabbit

polyclonal antiflagellin serum was used as the primary anti-

body. The secondary antibody was a goat anti-rabbit immuno-

globulin G conjugated with horseradish peroxidase. Detection

was performed according to the protocol of the supplier.

Hydrophilicity measurement

The surface hydrophilicity of bacterial cells was quantified

using bacterial adherence to hydrocarbon (BATH) test, ori-

ginally described by Rosenberg et al. (1980). Bacterial cells

suspended in a BATH buffer (adjusted to 1 OD, 4 mL) were

vortexed with xylene (1 mL) for 60 s. After the phases were

allowed to separate, the aqueous phase was carefully removed

and the A600 nm was measured. The results were expressed as

the percentage in OD of the aqueous phase compared with the

OD of the cell suspension without xylene.

Fluorescence staining with acridine orange

Bacterial smears were fixed with methanol and then stained

using 0.01% acridine orange in 0.05 M PBS (pH 4.8) for

5 min. The samples were viewed at � 1000 magnification

with an Olympus BX51 microscope.

Results and discussion

Differentiation from vegetativecells to swarming cells

When grown in liquid media, C. freundii cells were 0.5–2.0-

mm-long rods (mean value is 1.74� 0.18; 10 cells were

observed) with one to two polar or lateral flagella (mean

value is 1.6� 0.5; 10 cells were observed). When inoculated

onto a solid media surface, usually after 3–4 h bacterial cells

underwent a change in both shape and flagellar production.

They became hyperflagellated (mean value is 13.7� 3.5,

Po 0.05; 10 cells were observed) and slightly elongated (mean

value is 4.55� 0.79, Po 0.05; 10 cells were observed) (Fig. 1a

and b). They also displayed a special form of translocation, i.e.

swarming, on the media with appropriate agar concentration.

Citrobacter freundii cells exhibited swarming motility optimally

on 0.5–0.7% agar and not on agar with concentrations over

1%. On these high concentration agars, the decreased water

content inhibited the bacterial motility. When inoculated on

0.5% agar surface, after 3–4 h of stationary phase, bacterial cells

differentiated into swarming cells and then moved rapidly and

colonized the entire surface in 6–8 h with an expansion rate of

0.44–0.58 cm h�1 (Fig. 1c). The flagellin of C. freundii isolated

from swarming cells grown on swarming media and from

vegetative cells grown in liquid media possess the same

molecular mass (�47.5 kDa) based on their respective migra-

tion distances in SDS-PAGE electrophoresis (Fig. 2a).

Effect of nutrient composition on theswarming motility of C. freundii

Besides agar concentration, nutrient composition in the

medium served as another critical factor affecting swarming

motility. Citrobacter freundii cells were unable to swarm on

the M9 minimal media, although they had grown well and

displayed normal swimming motility in M9 liquid media.

Swarming requires the presence of certain inducers in the

swarm agar plates. Usually, casamino acids satisfy the

requirement for swarming. Proteus mirabilis and Pseudomo-

nas aeruginosa have been shown to respond to single amino

acids as inducers of swarming motility (Allison et al., 1993;

Kohler et al., 2000). However, in this study, C. freundii did

not swarm on the minimal media M9 supplemented with

either each of 20 amino acids or a mixture of amino acids

(casamino acids) until tryptone or peptone was added into


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the media, indicating that the swarming stimulus for

C. freundii is likely to be a certain oligopeptide. Although

tryptone alone was enough to support swarming, the

addition of carbon sources facilitated motility. Various

carbon sources, including glucose, glycerol, aminoglucose,

sucrose, propionate, succinate, pyruvate, and citrate, facili-

tated the swarming of C. freundii. Based on these observa-

tions, the formulation of swarming medium was modified

to contain 10 g tryptone, 10 g NaCl, 5 g glucose, and 5 g agar

L�1. This medium was used in subsequent tests.

TTC was added to the media to visualize the swarming

colonies better. TTC reacts with the respiratory chain via

Fig. 1. (a) Electron micrograph of vegetative cells collected from the inoculation site. Scale bar = 1mm. (b) Electron micrograph of swarming cells

collected from the swarming region. Scale bar = 1 mm. (c) Swarming colonies of Citrobacter freundii wild-type strain ATCC8090 stained in situ with TTC.

A 1-mL sample of bacteria in fresh LB culture (adjusted to 0.5 OD600 nm) was inoculated into the center of the swarm media with 0.05% TTC and then

incubated at 37 1C for 12 h. (d) When the same swarming colony was incubated at 37 1C for 48 h, the margin of the colonies turned red. (e) Another

swarming colony of C. freundii was incubated for 96 h on swarm media with an agar concentration of 0.6%. (f) After 6 h of incubation, the swarming

colony was observed directly at �400 magnification under an Olympus inverted microscope. The pictures were taken at 1-min intervals.



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cytochrome oxidase and is reduced to formazan, an insolu-

ble red pigment, in the cells (Boker-Schmitt et al., 1982).

Because it stains cells in situ, TTC is commonly used to aid

in the examination of bacterial colonies (Parrington et al.,

1993; Semmler et al., 1999). Figure 1c–e shows that TTC

stains swarming colonies in situ and discriminates the

different regions composed of swarming and vegetative cells,

as reported in a previous study (Falkinham & Hoffman,

1984). Bacteria in the red zones (inoculation sites) consisted

of vegetative cells with normal morphologies and rare

flagella (Fig. 1a), whereas those in the lightly staining zones

consisted of swarming cells with elongated bodies and dense

flagella (Fig. 1b). A similar phenomenon was observed in P.

mirabilis and alternate color cycles of red (consolidation)

and lightly stained (swarming) areas were visible in the

stained colonies (Supporting Information, Fig. S5).

It is evident from the color alteration of the bacterial

colonies that the vegetative cells in red zones had a high

aerobic respiration rate and might have obtained energy

mainly from the tricarboxylic acid (TCA) cycle. In compar-

ison, the swarming cells in the light zones had a low aerobic

respiration rate and perhaps primarily obtained their energy

from sugar fermentation. This assumption is supported by a

previous study. In E. coli, three components of the TCA cycle

and aerobic respiration, sdhCDAB (succinate dehydrogen-

ase), cyoABCDE (cytochrome o ubiquinol oxidase), and gltA

(citrate synthase), were demonstrated to be downregulated

by the transcriptional regulatory complex FlhD/FlhC, a

global regulator involved in many cellular processes (Pruss

et al., 2003). High-level FlhD/FlhC is induced in swarming

colonies and then apparently results in deduced expression

of the abovementioned enzymes as well as inhibition of TCA

cycle and respiratory process. The repressed aerobic respira-

tion in swarming cells could explain the staining character-

istics of the swarming colonies observed in this study. Our

results indicated that TTC is a suitable dye for staining

swarming colonies that are difficult to distinguish.

Microscopic features of swarmingmotility in C. freundii

As observed under the inverted microscope, the swarming

colonies of C. freundii consisted of one tier of cells on the agar

surface (Fig. 1f and Video S1). Swarming cells seemed to form a

wet environment on the agar surface, which likely provided

enough space for the bacteria to rotate their flagella. Single

bacteria were not found moving on the surface of the media,

although these bacteria certainly possessed the same functional

flagella. The moist milieu of swarming colonies could be due to

bacterial polysaccharide structures that extract and contain

water from the media. The significant role of the polysacchar-

ide structure on swarming has been revealed in previous studies

(Toguchi et al., 2000; Inoue et al., 2007). To support this point,

swarming cells of C. freundii were observed to be more

hydrophilic compared with vegetative cells (0.961 for swarming

cells; 0.814 for vegetative cells, Po 0.05; Fig. S2) in this work.

In swarming colonies, C. freundii cells moved actively.

However, the cells at the periphery of colonies were less active

due to the decreased moisture capacity in areas where the cells

moved out and back occasionally, or were pushed by the

actively moving cells in the central region. As a result, the edge

of colonies expanded outward continuously. Citrobacter

freundii did not display alternating cycles of swarming and

consolidation during the development of swarming colonies

as in P. mirabilis. Once differentiation occurred at the

inoculation site, swarming cells spread continuously, until

they occupied the entire agar surface. Even when inoculated

onto a large plate with a diameter over 20 cm, alternating

cycles of swarming and consolidation were not observed.

Transposon mutagenesis and screening ofmutants with defective swarming motility

Transposon mutagenesis involving the use of Mini-Tn5 on a

suicide plasmid pUT was carried out to further understand

the genetic determinants of swarming motility in C. freundii.

Fig. 2. (a) SDS-PAGE of purified flagellin samples

isolated from Citrobacter freundii wild-type strain

ATCC8090 incubated in two different media. 1,

Incubated in broth; 2, incubated in swarm medium

with 0.5% agar; M, molecular size marker. (b)

Flagellin expression of different mutants incubated

in swarm medium with 0.5% agar. Aliquots

of OD600nm-equalized cells were analyzed by

Western blotting using antiflagellin antibodies

as described in Materials and methods.


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A total of 85 swarming-defective mutants were screened

from approximately 6000 transconjugants; of the 85 mu-

tants, 53 were defective in both swimming and swarming.

The remaining 32 mutants were defective in swarming but

not swimming. The mutants with normal swimming pat-

tern were subjected to further sequence analysis to deter-

mine the insertionally mutated gene. Given that swarming is

dependent on functional flagella, as demonstrated in pre-

vious studies, of the 53 swimming-defective mutants, only

five randomly selected mutants were further subjected to

sequence analysis. As a whole, sequences produced valid

results with only four exceptions (CF407, CF415, CF701,

and CF711). In most cases, the most similar genes obtained

through the homology searches usually belonged to Citro-

bacter koseri ATCC BAA-895, a species of Citrobacter with

complete genome sequence information. The results of the

homology searches are listed in Tables 1 and 2 and are also

described in the following two sections on genes that have

been previously characterized in other species and those first

identified in this study.

Previously characterized genes

As many as 16 swarming-related genes identified in our

study have already been characterized previously in other

species. The underlying causes for the defective swarming

motility of the mutants are listed in Table 1. However, some

of them are worthy of further discussion.

Swarming motility of C. freundii is dependenton flagella

As expected, flhD, motA, and motB mutants were identified

among the five mutants found to be defective in both

swarming and swimming motilities. Because the flhDC

operon controls flagellar biosynthesis (Liu & Matsumura,

1994), no flagellum was produced in the flhD mutants (Fig.

2b). On the other hand, although the motA and motB

mutants produced flagella, they were still unable to move

because MotA and MotB formed a proton channel that

transferred proton-motive force to drive the flagella (Asai

et al., 2003); either motA or motB gene mutations resulted in

the production of nonfunctional flagella (Figs 2b and 3c).

These data demonstrate that the swarming of C. freundii is

dependent on functional flagella, as in other swarming

bacteria (Kearns, 2010).

Genes related to lipopolysaccharide synthesis

The largest gene cluster identified in our study is involved in

the synthesis of lipopolysaccharide. Altogether, 13 mutants

were isolated, of which six mutated genes – wzx, rfaL, rfbX,

rfaJ/CKO_05084, rfaJ/CKO_05086, and rfaG – were identi-

fied. The swarming ability of these mutants was dramatically

decreased (two of them are shown in Fig. 3g and h as

examples). As observed directly under inverted microscope,

only a few bacterial cells were actively motile in the swarm-

ing colonies of these mutants and these were mainly

distributed at the edges. In the central region, most cells

formed aggregates that scarcely moved (Videos S2 and S3).

In contrast, in wild-type colonies, all swarming cells were

actively motile (cells in the edge of colonies were less active)

and no aggregation was observed (Video S1).

The hydrophilicity of these mutants was decreased com-

pared with the wild type (Fig. S2), which could have led to

the aggregation. In a previous study, many transposon

swarming mutants isolated in Salmonella enterica serovar

Typhimurium have been shown to have mutations in the

lipopolysaccharide biosynthetic pathway (Toguchi et al.,

2000). The authors suggested that the O antigen directly or

indirectly improved the surface wettability required for

swarm colony expansion. Our observation showed that the

polysaccharide structure on the cell surface had important

role not only in overcoming friction between bacterial cells

and media surface, but also in reducing intercellular inter-

action. The poorly motile aggregates formed with bacteria

on the agar surface because of the O antigen defects could

account for the defective swarming in addition to the

decreased wettability of the agar surface.

The rcsC-rcsD-rcsB system

rcsC and rcsD mutants were identified in this study, and both

mutants displayed defective swarming behavior (Fig. 3a and

b). The products of rcsC and rcsD, together with RcsB,

constitute the regulator of the capsule synthesis (Rcs)

phosphorelay system. The regulator RcsB is activated by the

transfer of a phosphate group from its cognate sensor, RcsC,

through a histidine-containing phosphotransmitter (Hpt)

domain intermediate called RcsD (previously called YojN;

Takeda et al., 2001). The Rcs system has been implicated in

the regulation of bacterial responses to osmotic and other

kinds of membrane stress, growth at low temperatures in the

presence of glucose and zinc, and growth on solid surfaces

(Carballes et al., 1999; Majdalani & Gottesman, 2005; Wang

et al., 2007). Mutations in rcsC and rcsD affect the temporal

regulation of swarming motility and result in precocious

behavior in E. coli and P. mirabilis (Belas et al., 1998; Takeda

et al., 2001). Francez-Charlot et al. (2003) have shown that

the RcsCDB system negatively regulates the flhDC operon in

E. coli and that the exaggerated swarming behavior of the

rcsC and rcsD (yojN) mutants is probably the consequence of

the higher basal expression of the flhDC operon in the rcs

mutants, leading to a higher expression of genes, including

those required for the synthesis of flagellin. In contrast, in

our study, the colonies of C. freundii rcsC and rcsD mutants



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Tab

le1.

Swar

min

gm

uta

nts

with

muta

tion

inpre

viousl

ych

arac

terize

dgen

es

Cat

egories

Muta

nts

Stan

dar

dnam

eof

gen

es/s

ynonym

sof

CK

O�

Funct

ion

of

the

pro

duct

s

Poss

ibili

tyof

pola

ref

fect

/poss

ible

gen

esor

pat

hw

ayaf

fect

edw

Rea

son

for

dec

reas

edsw

arm

ing

motilit

yor

refe

rence

Flag

ella

rfu

nct

ion

CF4

30,C

F604

flhD

/CK

O_0

1056

Tran

scriptional

activa

tor

Yes

/flhC

No

flag

ella

rpro

duct

ion;Li

u&

Mat

sum

ura

(1994)

CF5

09,C

F519

motB

/CK

O_0

1059

Flag

ella

rm

oto

rpro

tein

MotB

No

Nonfu

nct

ional

flag

ella

;A

saie

tal

.(2

003)

CF7

03

motA

/CK

O_0

1058

Flag

ella

rm

oto

rpro

tein

MotA

Yes

/motB

LPS

bio

synth

esis

CF4

08,C

F202,

CF4

14

wzxz

O-a

ntigen

flip

pas

eYes

/LPS

bio

synth

esis

Dec

reas

edsu

rfac

ew

etta

bili

ty;To

guch

iet

al.

(2000)

CF4

18,C

F402,

CF4

24

rfaJ

/CK

O_0

5084

LPS

1,2

-glu

cosy

ltra

nsf

eras

eYes

/LPS

bio

synth

esis

CF4

32,C

F603,

CF7

02

rfaL

/CK

O_0

5081

Lipid

Aco

re–

O-a

ntigen

ligas

eYes

/LPS

bio

synth

esis

CF6

05,C

F719

rfaJ

/CK

O_0

5086

LPS

1,2

-glu

cosy

ltra

nsf

eras

eYes

/LPS

bio

synth

esis

CF4

12

rfbX

/CK

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166 Y. Cong et al.

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were not precocious (Fig. 3a and b). As observed directly

under the inverted microscope, similar to those lipopoly-

saccharide mutants, mutants of rcsD and rcsC formed

aggregates in the swarming colonies (Video S4). As the

regulator of capsule synthesis, mutations in RcsD and RcsC

certainly lead to a decrease in bacterial surface hydrophili-

city, which was supported by our results of BATH measure-

ment (Fig. S2).

New swarming genes identified in our study

Aside from the previously characterized genes, several new

swarming-related genes were identified in the present study.

A novel yqhC gene

Four mutants were identified as having yqhC gene muta-

tions that formed small colonies on the swarm plate (Fig.

3d). The product of the yqhC gene is a putative AraC-family

transcriptional regulatory protein, as annotated in the

NCBI. Most members of the AraC-XylS proteins are positive

transcriptional regulators involved in the control of many

important processes related to carbon metabolism, stress

responses, and pathogenesis (Egan, 2002). The flagellar

production of yqhC mutant was comparable to that of the

wild type (Fig. 2b), suggesting that the decrease of swarm

ability of yqhC mutant was not due to a disruption of

flagellar synthesis. However, the high output of yqhC

mutants in our study indicates a close relationship between

swarming motility and the function of the yqhC gene. In a

recent study, yqhC gene in E. coli has been shown to regulate

the transcription of the adjacent genes, yqhD and dkgA, that

encode NADPH-dependent oxidoreductases with broad-

substrate ranges that include furfural and methylglyoxal

(Perez et al., 2008; Turner et al., 2010). As 0.5% glucose was

added into the swarm media, the concentration of aldehydes

was inevitably increased in bacterial cells of the yqhC mutant

due to the lack of expression of the yqhD and dkgA genes.

The high concentration of aldehydes was harmful to the

bacterial cells and may have interrupted swarming in some

unknown ways.

A novel yeeZ gene

Among the mutants, the yeeZ mutant was notable because it

displayed an elongated shape whether grown in liquid media

or on the surface of the solid plate (Fig. 4a–c). In liquid

media, elongated bacteria formed aggregates that were even

deposited at the bottom of the tube (Fig. 4d). The product

of yeeZ is a putative nucleoside-diphosphate-sugar epimer-

ase that has a possible role in polysaccharide synthesis.

However, no direct functional studies of yeeZ have been

undertaken until now. Based on the results of the fluores-

cence staining with acridine orange, the bacterial cells of

yeeZ mutant were multinucleate (Fig. 4e), and the bacterial

cell walls were intact, as revealed by electron microscopy

(Fig. 4f). Hydrophilicity of the mutant decreased compared

with the wild type (Fig. S2) and the insertional mutation in

yeeZ gene also resulted in dramatic low growth rate (Fig.

S3). These features suggest that the function of yeeZ gene

may be associated with bacterial cell division. However, the

downstream gene, CKO_00769, which encodes a putative

LysR-type transcriptional regulator, overlaps in sequence

with the yeeZ gene. So the possibility that the novel features

of CF204 may be due to the polar effect of transposon on the

expression of the CKO_00769 must be considered.

In liquid media, the mutant bacteria were motile but less

active than the wild type even though the flagellin level of

the yeeZ mutant was comparable to that of the wild type

(Fig. 2b and Video S5). Cell elongation has been previously

Table 2. Swarming mutants with mutation in previously uncharacterized genes

Mutants

Standard name of genes/

synonyms of CKO� Function of the products

Possibility of polar effect/possible

genes or pathway affectedw

CF426, CF1001,

CF405, CF717

yqhC/CKO_04404 Transcriptional regulator, AraC family K00567 Yes/CKO_04405, CKO_04406(yqhD)

and dkgA

CF204 yeeZ/CKO_00768 Hypothetical protein, nucleoside-diphosphate

sugar epimerases

Yes/CKO_00769

CF520 CKO_03941z Hypothetical protein, oligoketide

cyclase/lipid transport protein

Yes/CKO_03940 with unknown

function

CF1002 ttrA‰ Tetrathionate reductase subunit A No

CF101 glgC/CKO_04849 Glucose-1-phosphate adenylyltransferase;

ADP-glucose pyrophosphorylase

Yes/glycogen synthesis

�CKO: Citrobacter koseri ATCC BAA-895.wThe possibility of polar effect was judged by the analysis of the adjacent genomic context to the mutated gene.zThere is no standard gene name.‰The sequence of mutated gene has the most similarity with the ttrA gene of Salmonella enterica ssp. enterica serovar Agona str. SL483 and there is no

synonym in CKO.



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suggested as a key factor for swarming process. Some

swarming null mutants and crippled mutants of P. mirabilis

have been identified previously as defective in swarming cell

elongation (Belas et al., 1991). However, in C. freundii, our

results indicated that an elongated shape was not always

advantageous for swarming motility.

Genes related to metabolic pathways

Three of the new swarming-related genetic loci were found

to be involved in different metabolic pathways, and the

mutation of these genes resulted in a moderately defective

swarming (Fig. 3j–l). CKO_03941 encodes a putative poly-

ketide cyclase/dehydrase family protein that has an unclear

function. Its role in swarming motility has yet to be

determined.

The glgC gene encodes an ADP-glucose pyrophosphor-

ylase, which catalyzes the first rate-limiting step in glycogen

biosynthesis. Glycogen is widespread in enteric genera as a

major energy storage compound. Glycogen reserves are

important for biofilm formation, virulence in Salmonella

enteritidis (Bonafonte et al., 2000), and sporulation in

Clostridium and Bacillus (Preiss, 1984). Based on our results,

the growth rate of the glgC mutant was less than that of the

Fig. 3. Swarming colonies of Citrobacter freundii mutants incubated for 12 h. (a) CF518(rcsC); (b) CF201(rcsD); (c) CF703(motA); (d) CF426(yqhC);

(e) CF204(yeeZ); (f) CF604(flhD); (g) CF408(wzx); (h) CF511(rfaG); (i) CF510(rssB); (j) CF520(CKO_03941); (k) CF1002(ttrA); and (l) CF101(glgC).


168 Y. Cong et al.

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parent strain (Fig. S3). The growth rate change may be

reflected in the defective swarming.

The ttrA gene encodes tetrathionate reductase subunit A.

The ability to respire tetrathionate is a characteristic of

certain genera of Enterobacteriaceae, including Citrobacter,

Salmonella, and Proteus (Hensel et al., 1999). Although no

exogenous tetrathionate was added to the swarming media

used in the study, the protein digests in the complex media

were shown to contain thiosulfate, which was readily

oxidized to tetrathionate (Barrett & Clark, 1987). The role

for tetrathionate reduction in swarming is somewhat diffi-

cult to imagine; however, the extreme pH changes associated

with the metabolism of tetrathionate might be used in the

manipulation of the proton gradient across the cell mem-

brane (Barrett & Clark, 1987), which serves as the energy

source for driving flagellar rotation.

In summary, the swarming motility of C. freundii has

been described in this work. Our results demonstrated that

the nutritional requirement for swarming motility in

C. freundii is quite high. A mixture of amino acids was

found to be unable to induce swarming of C. freundii,

although they could induce swarming in some other swarm-

ing bacteria such as P. mirabilis, P. aeruginosa, and S. enterica

serovar Typhimurium (Allison et al., 1993; Kohler et al.,

2000; Toguchi et al., 2000). In swarming colonies, C. freundii

cells became hyperflagellated and slightly elongated com-

pared with the vegetative cells grown in liquid media.

To date, many species have been found to possess the ability

to swarm on agar surfaces. However, the genes required

specifically for this type of motility are not completely

understood and vary among species. In this work, numerous

swarming genes have been identified in our attempt to

screen the genetic determinants for C. freundii swarming.

Among the mutants with mutations that have been mapped

to previously characterized genes, there are several unique

characteristics in C. freundii. For example, the mutants

related to lipopolysaccharide synthesis and the RcsCDB

signal system showed a propensity to form less motile

aggregates in the swarming colonies, and the rcsD and rcsC

mutants do not display precocious swarming phenotype as

in other bacteria. Moreover, insertion mutation in the five

genetic loci, which have not been demonstrated to be

involved in swarming, have been identified to result in

defective swarming behavior in C. freundii. Some of these

have interesting phenotypes; for example, the yeeZ mutant

displayed an elongated shape, which may provide a clue for

studying the function of related genes. Our results indicate

that swarming motility is more complicated than currently

known; in addition, its features vary among swarming

bacteria. Thus, further studies on swarming in different

bacteria are needed to achieve a complete understanding of

this special motility.

Fig. 4. Bacterial cell morphology and structure.

(a) The bacterial cells of CF204(yeeZ) were grown

overnight on swarm media at 37 1C (Gram stain,

� 1000 magnification). (b) The bacterial cells of

wild-type strain were grown overnight on swarm

media at 37 1C (Gram stain, � 1000 magnifica-

tion). (c) The bacterial cells of CF204(yeeZ) were

grown overnight in liquid LB media at 37 1C

(Gram stain, � 1000 magnification). (d) The

bacterial cells of CF204(yeeZ) formed aggregates

in liquid LB, usually deposited at the bottom of

the tube. This picture was taken after shaking the

tube. (e) CF204(yeeZ) (fluorescent stain with

acridine orange, �1000 magnification). (f)

CF204(yeeZ) (electron micrograph with ultrathin

section, scale bar = 0.2mm).



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Acknowledgements

We thank Tomofusa Tsuchiya of Okayama University, Japan,

for providing strain C. freundii. We also gratefully acknowl-

edge Victor de Lorenzo of Centro Nacional de Biotecnologia

CSIC, Spain, for providing Mini-Tn5 transposon.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. Electron micrograph of bacterial cell collected from

LB plate with 1.5% agar; scale bar = 2mm.

Fig. S2. Bacterial surface hydrophilicities measured by

BATH method, as described in the Materials and methods.

Fig. S3. Growth curves of the mutant and wild-type strains.

Fig. S4. SDS-PAGE of lipopolysaccharide profiles.

Fig. S5. Swarming colonies of Proteus mirabilis CMCC49003

stained in situ with TTC.

Video S1. Movement of wild type cells on swarm media.

Video S2. Movement of wzx mutant cells on swarm media

(episode 1).

Video S3. Movement of wzx mutant cells on swarm media

(episode 2).

Video S4. Movement of rcsD mutant cells on swarm media.

Video S5. Movement of yeeZ mutant cells in liquid LB

media.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.



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Characterization of swarming motility in Citrobacter freundii

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