JOURNAL OF BACTERIOLOGY, Feb. 2007, p. 950–957
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 3
Genome-Wide Screening of Genes Required for Swarming
Motility in Escherichia coli K-12?†
Tetsuyoshi Inoue,1‡* Ryuji Shingaki,1‡ Shotaro Hirose,1Kaori Waki,1Hirotada Mori,2
and Kazuhiro Fukui1
Department of Oral Microbiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences,
Okayama University, Okayama, Japan,1and Graduate School of Biological Sciences,
Nara Institute of Science and Technology, Ikoma, Nara, Japan2
Received 16 August 2006/Accepted 14 November 2006
Escherichia coli K-12 has the ability to migrate on semisolid media by means of swarming motility. A
systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used
to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene
mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants
displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifi-
cally associated with the swarming phenotype. The swarming-associated genes were classified into various
functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of
cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acqui-
sition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface compo-
nents, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccha-
ride and the enterobacterial common antigen may be important surface-acting components that contribute to
the reduction of surface tension, thereby facilitating the swarm migration in the E. coli K-12 strain.
Swarming is a flagellum-dependent form of bacterial motil-
ity that facilitates migration of bacteria on viscous substrates,
such as semisolid agar surfaces, which has been observed for a
variety of motile bacteria (13). In order to swarm, cells first
differentiate into a specialized state (swarmer cells) character-
ized by an increase in flagellum number and the elongation of
cells and then move as multicellular rafts across surfaces (10,
12, 13). This is in contrast to swimming motility, which repre-
sents individual cell motility in aqueous environments. In ad-
dition to the morphological changes, it is known that the
swarmer cells produce extracellular materials (wetting agents),
such as surfactants and exopolysaccharides, to increase surface
wetness and thus facilitate movement (13). In view of these
features of swarming, it is very likely that various cellular
activities are involved in this type of motility.
Recent genome-scale approaches have disclosed that
swarmer differentiation coincides with the regulation of a wide
range of cellular activities. Wang et al., using DNA microarray
analysis, demonstrated that surface-growing Salmonella en-
terica serovar Typhimurium cells had physiologies markedly
different from those of Salmonella cells grown in broth (49). In
addition, the proteome analysis by Kim and Surette indicated
that the metabolic pathways in the swarmer cells of Salmonella
were dynamically shifted compared with those in the swimmer
cells (20). However, little is known about the genes that are
required for the swarming phenotype in Escherichia coli. In the
case of E. coli K-12 strains, swarming requires media solidified
with Eiken agar (12). The requirement for this special agar is
currently explained by the O-antigen-defective lipopolysaccha-
rides (LPSs) of these strains and the particular wettability of
the Eiken agar surface (13). However, the involvement of
wetting agent-like materials in the swarming motilities of these
strains has not been explored.
Recently, a set of single-gene knockout mutants of all the
nonessential genes in E. coli K-12 (the Keio collection) was
constructed (1). In the present study, we used this mutant
collection to identify the genes required for swarming motility
in E. coli and found that a wide variety of genes are implicated
in this form of motility. In addition, we propose the cell surface
components that are required for swarming motility in this
strain, possibly as wetting agent-like materials. To our knowl-
edge, this is the first report of a comprehensive analysis of
swarming-related genes using a systematic, gene-deleted mu-
MATERIALS AND METHODS
Single-gene knockout mutant collection (the Keio collection). The E. coli K-12
strain W3110 was used as a wild-type strain for the swarming motility assay. In
this study, we used mutants from a systematic, single-gene knockout mutant
collection (the Keio collection) of all the nonessential genes of BW25113, a
strain derivative of W3110. These mutants were created by Baba et al. (1) by
replacing the open reading frame coding regions with a kanamycin resistance
cassette, according to the method of Datsenko and Wanner (7). Glycerol stocks
of the collection dispensed in a 96-well microplate format were maintained at
?30°C and ?70°C.
Screening of the swarming-associated genes. The media used for the swarming
assay was Luria-Bertani (LB) medium containing 0.5% (wt/vol) glucose and
0.6% (wt/vol) Eiken agar (Eiken Chemical Co., Tokyo, Japan), which was dis-
pensed into OmniTray dishes (Nalge Nunc International, NY). Typically, these
* Corresponding author. Mailing address: Department of Oral Mi-
crobiology, Graduate School of Medicine, Dentistry, and Pharmaceu-
tical Sciences, Okayama University, 2-5-1, Shikata, Okayama 700-8525,
Japan. Phone: 81-86-235-6656. Fax: 81-86-235-6659. E-mail: inouet
† Supplemental material for this article may be found at http://jb
‡ These authors contributed equally to this work.
?Published ahead of print on 22 November 2006.
dishes, which are termed swarm plates, were air dried for 10 min before being
used. Precultured bacteria grown in LB broth containing kanamycin (30 ?g/ml)
and 1.0% (wt/vol) agar in 96-well microplates were carefully inoculated with a
96-pin metal replicator (Copyplate; Tokken Inc, Chiba, Japan) onto the surfaces
of the swarm plates. The plates were then wrapped with Saran Wrap to prevent
dehydration. The plates were incubated at 33°C for 18 to 20 h. Swarming motility
was assessed by examining the colony sizes and the branch-spreading patterns on
the semisolid agar medium. Every mutant was classified into one of three cate-
gories: the strongly repressed phenotype, the moderately repressed phenotype,
and the normal swarming phenotype (Fig. 1). The swarming assay was performed
at least three times for each mutant in the Keio collection. The function of each
swarming-associated gene was assigned by referring to the EchoBASE (http:
//www.ecoli-york.org/) (30) and EcoCyc (http://www.ecocyc.org/) (18) databases.
Mutants exhibiting the strongly repressed swarming phenotype were tested for
their swimming abilities. Each strain was inoculated on LB medium solidified
with 0.3% (wt/vol) Eiken agar and incubated at 33°C for 15 to 16 h.
Electron microscopy. The bacterial strains were grown overnight at 33°C on
the swarm plates. The cells were taken up with toothpicks from the edges of the
colonies and transferred into 10 mM ammonium acetate (pH 7.2). Five micro-
liters of the cell suspension was placed on collodion membrane-coated grids, and
the excess liquid was removed with filter paper. The cells on the grids were
negatively stained with 1% phosphotungstic acid (pH 7.5) for 1 min and observed
using a Hitachi H-800 transmission electron microscope at an accelerating volt-
age of 100 kV.
RESULTS AND DISCUSSION
Screening of the genes involved in swarming motility. The
swarming motility of E. coli K-12 strains can usually be ob-
served on semisolid Eiken agar medium (Fig. 1). The genome
of the E. coli K-12 strain comprises 4,390 open reading frames
(1). The Keio collection contains 3,985 nonessential gene mu-
tants, in each of which a kanamycin resistance cassette has
been used to create a highly targeted single-gene disruption (1,
7). Mutants were screened for swarming defects on LB soft
agar (0.6% agar) containing 0.5% glucose. On inspection of
the mutant library, 294 gene mutants (7.4% of the nonessential
genes) were found to express strong repression of the spread-
ing and branching colony morphologies (the strongly repressed
phenotype) (Fig. 1). In addition, another 510 mutants (approx-
imately 13% of the nonessential genes) exhibited partially re-
duced colony spreading (the moderately repressed phenotype)
(Fig. 1) (see Table S2 in the supplemental material).
Swimming ability was tested for the mutant displaying the
strongly repressed phenotype, using LB solidified with 0.3%
agar. Of the 294 mutants, 216 showed no significant defects in
swimming motility, thereby indicating that the 216 genes were
specifically associated with the swarming phenotype (Table 1).
The remaining 78 mutants (the atpA, atpB, atpC, atpD, atpE,
atpF, atpG, atpH, cheA, cheB, cheR, cheW, cheY, cheZ, cmk,
dnaK, dsbA, fabH, fis, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgI,
flgJ, flgK, flgL, flgN, flhA, flhB, flhC, fliA, fliC, fliD, fliF, fliG, fliI,
fliJ, fliK, fliM, fliN, fliO, fliP, fliQ, fliS, folB, folP, galU, gmhA,
hflD, motA, motB, mtn, pgm, priA, rcsC, rfaH, rho, rluD, rplA,
rrmJ, sufC, tap, tolB, tolR, ubiH, ubiX, waaC, waaD, waaE,
ycjW, yfgA, yhbG, yhcA, and yjeQ mutants) that were clearly
defective in swimming ability had deletions in genes for flagel-
lar function (the che, flg, flh, fli, and mot genes) and energy
production (the ATP synthase and ubiquinone genes) (the
function of each gene product is described in Table S3 in the
supplemental material). For the other mutants with impaired
swimming abilities, this impairment may be due to defects in
flagellar function or energy production for active growth and
The 216 swarming-associated genes were classified into clus-
ters of orthologous groups (COGs) (1, 46), suggesting that they
belong to a variety of functional categories (Table 1) (the
function of each gene product is described in Table S1 in the
supplemental material). The results imply that a wide variety of
genes are associated with the swarming motility of E. coli.
Compared to other swarming bacteria, E. coli K-12 strains are
known to be relatively fastidious in their requirements for
swarming. This is because the LPSs of the K-12 strains lack the
O-antigen, and thus, the swarming ability of this bacterium is
dependent upon the special Eiken agar that provides a partic-
ularly wettable surface. It might therefore be assumed that
swarming in K-12 is more sensitive to any mutation affecting
cell surface properties, thus resulting in a relatively large num-
ber of swarming-defective mutants. Additionally, it might be
possible that due to polar effects in the case of genes arranged
in operons, the current assessment of swarming motility results
in an overestimation of the number of swarming-related genes.
However, the available evidence suggests that E. coli K-12 cells
deploy numerous and various cellular functions to facilitate
migration over viscous surfaces and thereby expand their grow-
ing area. In the following sections, we focus on several func-
tional groups of gene mutants with the strongly repressed
swarming phenotype and no significant swimming defects, and
we discuss the roles of the gene functions in swarming.
TCA cycle and glucose metabolism. It has been suggested
that swarming is a process that requires a large amount of
energy (20). Consistent with this, there are several genes as-
sociated with energy production, such as those coding for ATP
synthase, respiratory chain components, and tricarboxylic acid
(TCA) cycle enzymes, with strongly repressed swarming mo-
tilities (Table 1; also see above). Of these, two TCA cycle
enzymes, succinate dehydrogenase (Sdh) and 2-ketoglutarate
dehydrogenase (Suc), are notable because these gene mutants
displayed significant swarming defects but normal swimming
motilities. The reactions catalyzed by these enzymes yield re-
ducing power, which might supply a significant amount of en-
ergy required for swarming movement or increased production
of cellular components for facilitating swarm migration (de-
scribed below). With regard to this, Kim and Surette presented
FIG. 1. Swarming motility phenotypes. Bacterial cells were inocu-
lated onto LB medium containing 0.5% (wt/vol) glucose and 0.5%
(wt/vol) Eiken agar and incubated overnight at 33°C. Results are
shown for the wild-type strain (WT; normal swarming type), the phoQ
mutant (moderately repressed type), and the fliC mutant (strongly
VOL. 189, 2007SWARMING-ASSOCIATED GENES IN E. COLI951
data indicating that the expression of these two enzymes as
well as ATP synthase components was upregulated in the
swarmer cells compared to that observed in the swimmer cells.
From these findings, it seems likely that swarming is a more
energy-consuming process than swimming. Recently, it was
proposed that the entire TCA cycle is exploited during the
swarm migration of Salmonella (20). However, our results
demonstrated that certain TCA cycle enzymes, including isoci-
trate dehydrogenase (Icd), malate dehydrogenase (Mdh), and
citrate synthase (GltA), were dispensable for swarming; this
indicated that the total activity of the TCA cycle is not neces-
sary for swarming in E. coli.
In Salmonella, the addition of glucose to LB agar medium is
required for swarmer cell differentiation (12, 19, 21). This is
probably because glucose metabolism provides the required
energy for swarmer cells. In the case of E. coli, we observed the
requirement of glucose for swarm migration (data not shown).
Our results for E. coli suggested that glucose utilization is one
of the key factors for the swarming motility of this bacterium.
For example, several genes whose products are involved in the
uptake of glucose (ptsH, ptsI) and in glycolysis (pgi, gpmI) are
listed in Table 1. Furthermore, in addition to energy produc-
tion, the requirement of the zwf gene product may suggest an
alternative means by which glucose utilization plays important
roles in swarming differentiation. The zwf gene encodes glu-
cose-6-phosphate dehydrogenase, which catalyzes the conver-
sion of glucose-6-phosphate into 6-phosphogluconolactone,
which is the first step of the pentose pathway (50). The pathway
may be important for swarming by supplying the precursors for
the biosynthesis of various cellular constituents, particularly
cell surface materials, such as LPS and the enterobacterial
common antigen (ECA), that are required for swarming as
Lipopolysaccharide and enterobacterial common antigen.
The medium used for testing bacterial swarming motility is
generally solidified by the addition of agar to a final concen-
tration of 0.5% to 2.0%. Owing to the lack of surface moisture,
a semisolid surface on which the swimming motilities of indi-
vidual bacterial cells are inhibited is created (13). Under such
conditions, swarming bacteria are considered to produce ex-
TABLE 1. E. coli genes whose mutations caused strongly repressed swarming with no significant defects in swimminga
Information storage and processing
Translation, ribosomal structure and biogenesis (3/170)
DNA replication, recombination and repair (6/237)
deaD, efp, truA
argP, cysB, envR, hipB, marA, rof, uxuR, ybdO, ycjZ, yfeR, yfeT
dps, fimB, idaB, ogt, xerC, xerD
Cell division and chromosome partitioning (1/34)
Posttranslational modification, protein turnover,
Cell envelope biogenesis, outer membrane (20/219)
ahpF, grxB, hflC, hscA, htpG, htpX, osmC, ppiB, ppiD, tpx, trxB
MasmA, dgkA, etk, hlpA, mrcB, ompA, pal, rffH, tolC, tsx, waaB,
waaF, waaG, waaI, waaJ, waaQ, wcaE, wecA, wecE, wecG
fimA, fimD, fimF, fliR, tolQ
chaC, cvrA, feoA, feoB, fepA, fepB, fepD, fepG, fes, narH, ppk,
trkA, yheL, yheM, yheN
atoS, barA, cpxA, crp, cusR, evgS, gmr, hnr, ompR
Cell motility and secretion (5/151)
Inorganic ion transport and metabolism (15/253)
Signal transduction mechanisms (9/187)T
Energy production and conversion (24/281)CackA, acnA, aldA, aldH, cydD, cyoA, fdrA, fdx, hyaA, hyaB, iscU,
lpdA, nuoF, rnfG, sdhA, sdhB, sdhC, sucA, sucB, sucD, ybdH,
yeiA, ygfH, ynfG
fucU, galM, gatY, gpmI, mglB, mgsA, nagC, pgi, ptsH, ptsI, rpiA,
treA, yhcH, yqaD, zwf
argG, argR, aroE, asnB, csdA, dapF, glnA, gmhB, metL, mpaA,
guaB, ndk, purC, purK, pyrF
hemE, lipA, pabC, pdxJ, pdxY, ubiF
entB, entE, entF, yrbE
Carbohydrate transport and metabolism (17/355)G
Amino acid transport and metabolism (12/390)E
Nucleotide transport and metabolism (5/83)
Coenzyme metabolism (6/144)
Lipid metabolism (1/96)
Secondary metabolite biosynthesis, transport and
General function prediction only (11/448)
S, U, V
cvpA, nlpI, prpD, trmE, wcaH, wzxE, ycdY, ydeE, yfeH, ygeD, ygfZ
sapF, ybaK, ybeD, ybiA, ydcZ, ydgA, yfgL, yneE, yqaA
No COG assignment (48/1,214)arpB, dnaT, dsbB, envC, eutS, fadK, fimC, fimH, galE, gapC, iscS,
macA, mdoC, mdtH, ninE, osmB, pldB, ppdC, prmB, rep, trpL,
ubiC, ubiE, waaP, waaS, waaY, waaZ, wecB, wecC, wecD,
wecF, yaiW, yccK, yccV, yciG, ydaT, ydbA, ydcX, ydfT, yehP,
yfbJ, yfgJ, yfjN, yfjP, yigG, ymfA, yncH, yncN
aGenes are classified into COG categories. The numbers in parentheses represent the number of swarming-related genes out of the number of genes belonging to
each COG category. The function of each gene product is described in Table S1 in the supplemental material.
952 INOUE ET AL. J. BACTERIOL.
tracellular wetting agents consisting of polysaccharides and
surfactants, etc., that extract water from the agar and thereby
increase their surface wetness; this, in turn, facilitates the
flagellar-driven bacterial motility on the agar plates (13, 51). In
the present study, a comprehensive analysis using the Keio
collection demonstrated that the genes involved in the biosyn-
thesis of two cell surface components, LPS and ECA, were
required in bulk for E. coli K-12 swarming motility but they
were not required for swimming motility. Here, we describe
the results and discuss the roles of these amphipathic sub-
stances in the swarming motility of E. coli K-12.
The contribution of LPS, particularly its O-antigen, to
swarming motility has been reported for several gram-negative
bacteria (11, 29, 47, 49). In addition, it appears to be an indis-
pensable component for the flagellum-independent surface
translocation in Vibrio cholerae (4) and also for the social
motility in Myxococcus xanthus (3). Presumably, LPS acts by
increasing the wetness of both the bacterial cell surface itself
and the surrounding environment. The outer-membrane-an-
chored form of LPS, through its outer extruded core oligosac-
charide and O-antigen polysaccharide, confers hydrophilicity
to the cell surface, whereas the extracellular form of LPS acts
as a surfactant, reducing the surface tension and decreasing the
frictional resistance between the agar and cell surface (13).
In the E. coli K-12 strain used in this study, swarming mo-
tility was significantly repressed in many of the LPS biosynthe-
sis-related gene disruptants as shown in Table 2. The E. coli
K-12 strain carries a mutation in the O-antigen biosynthesis
TABLE 2. Swarming and swimming of mutants in genes associated with biosynthesis of ECA and LPSa
Status of indicated ability
Gene product and function
Undecaprenyl-phosphate ?-N-acetylglucosaminyl transferase
Chain length modulation protein
UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase
dTDP-glucose 4,6-dehydratase 2
dTDP-glucose pyrophosphorylase 2
Lipid III flippase
UDP-N-acetyl-D-mannosaminuronic acid transferase
LPS biosynthesis CoregmhB
D,D-heptose 1,7-bisphosphate phosphatase
D-sedoheptulose 7-phosphate isomerase
Fused heptose 7-phosphate kinase/heptose 1-phosphate
Lipopolysaccharide core biosynthesis protein
Heptosyl transferase I
Protein involved in KdoIII attachment during core biosynthesis
Lipopolysaccharide core biosynthesis protein
Lipopolysaccharide core biosynthesis protein
Core heptose phosphorylation protein
Lipopolysaccharide core biosynthesis protein
NTRegulator of length of O-antigen component of
Lipopolysaccharide biosynthesis protein
Lipopolysaccharide PST transporter
a?, strongly repressed; ?, moderately repressed; ?, normal motility; NT, not tested.
VOL. 189, 2007SWARMING-ASSOCIATED GENES IN E. COLI 953
gene locus that results in the inability to assemble mature
O-antigen (26). The results also suggest that all the genes that
were involved in core oligosaccharide synthesis, but not those
involved in O-antigen synthesis, were required for swarming.
The necessity of all the genes involved in the core oligosaccha-
ride synthesis is presumably indicative of the fact that core
oligosaccharide not only plays a role in conferring hydrophi-
licity to the cell surface but also serves as an acceptor for
additional polysaccharides other than the O-antigen. In addi-
tion to the swarming defects, the disruption of the gmhA, waaE
(hldE), waaD (hldD), and waaC genes led to impaired swim-
ming motility (Table 2). The products of these genes are re-
sponsible for the construction of the inner core of LPS (34),
thereby suggesting that the inner core may be necessary for
flagellar assembly and function. Although GmhB is also a
member of the inner-core-synthesizing pathway, the gmhB mu-
tation did not result in swimming deficiency. This may be
explained by the presence of an additional enzyme that par-
tially compensates for the gmhB mutation as proposed by
Kneidinger et al. (22).
In addition to LPS, swarming was also strongly repressed in
most of the ECA biosynthetic gene mutants listed in Table 2.
ECA is one of the cell surface components of enteric bacteria,
and its carbohydrate portion is a linear heterosaccharide con-
taining a trisaccharide repeat unit, 33)-?-D-Fuc4NAc-(134)-
?-D-ManNAcA-(134)-?-D-GlcNAc-(13 (38). Three forms of
ECA have been reported: ECAPG, ECALPS, and ECACYC.
ECAPGand ECALPSare the major and a minor forms of ECA
in which the trisaccharide repeats are linked to the outer mem-
brane phosphoglyceride and the core region of LPS, respec-
tively (38). ECACYCis another water-soluble cyclic form of
ECA (38), and in the E. coli K-12 strain, it has been demon-
strated to consist of four uniformly repeating units (9). The
structure and assembly of ECA are now fairly well understood
due to exhaustive research efforts (17, 38, 39, 45); however,
with the exception of its contribution to resistance against
organic acids (2) and bile salts (36), little is known about the
function of this molecule. Swarming has been reported for
various species of enterobacteria (13). Although the require-
ment for ECA in swarming motility may be obvious in E. coli
K-12 strains lacking the O-antigen, which is an important pre-
requisite for semisolid surface translocation in many species of
gram-negative bacteria, the findings presented here indicate
that ECA plays an important role, possibly as an additional
wetting agent, in swarming motility in E. coli. Additionally, the
findings provide a new insight into the ECA molecule itself
and establish the importance of conserving ECA and its wide
With regard to the other polysaccharide materials, it is con-
sidered that capsular polysaccharide also contributes to a cer-
tain extent in the swarming motility of E. coli K-12. The re-
spective genes are listed in Table 1 and include the cholanic
acid capsule synthesis genes wcaE and wcaH. Furthermore,
disruption of the wcaB, wcaF, and wcaM genes resulted in
moderate swarming repression (see Table S2 in the supple-
mental material). Although the extracellular polysaccharide
was reported to play a role in the social motility of M. xanthus
(24, 27), the capsular polysaccharide is not generally consid-
ered to be crucial for swarming motility in other swarming
bacteria. However, in the case of E. coli K-12 strains lacking
the O-antigen, the assemblage of available extracellular poly-
saccharides may be necessary in order to facilitate swarm mi-
Type 1 fimbriae. The type 1 fimbriae in E. coli are peritric-
hously expressed filamentous surface structures and are one of
the virulence factors in uropathogenic E. coli (43). The follow-
ing eight genes in the fim gene cluster are involved in the
production of type 1 fimbriae: fimB, fimE, fimA, fimC, fimD,
fimF, fimG, and fimH (43). Of these, the fimA (major fimbrial
subunit), fimB (recombinase), fimC (periplasmic chaperone),
fimD (outer membrane usher), fimF (adaptor), and fimH (ad-
hesin) mutants exhibited strongly repressed swarming motili-
ties but no significant repression of swimming motility (Table
1). The fimA, fimC, and fimD gene products are essential for
constructing the fimbrial fiber, while the fimF and fimH gene
products that are located in the fibrillar tip are not required for
this. However, the fimF and fimH mutants were reported to
have markedly reduced numbers of fimbriae per cell (40, 42).
To examine the roles of fimbriae in swarming motility, we
used electron microscopy to observe the expression of fimbriae
and flagella in the wild-type strain and the fimA mutant. While
the swimmer and swarmer cells of the wild-type strain pos-
sessed many flagella, almost all of these cells did not express
fimbriae (Fig. 2A and C). This unexpected finding may suggest
that swarming movement requires the participation of fimbrial
genes but does not require the fimbrial fiber structure. In the
swimming cells, there was no significant difference in flagellar
number between the wild-type and the fimA mutant (Fig. 2A
and B). However, when the cells are grown on the swarm
plates, the number of flagella per cell in the fimA mutant was
very small compared with that in the wild-type strain (Fig. 2C
and D). From these observations, it seems most likely that the
swarming defect in the fimA mutant is caused by a decrease in
the number of flagella and not by the lack of fimbrial fiber. The
reduced production of flagella in the fimA mutant might be
due to defects in the induction of flagella expression in the
fimA mutant during swarmer cell differentiation. Recently,
Ko ¨hler et al. reported a similar observation in Pseudomonas
aeruginosa, i.e., a mutation in the fimbrial subunit gene pilA
caused complete swarming inhibition; however, there was no
significant effect on swimming motility (23). Interestingly, the
presence of pilus-like structures was not obvious in the
swarmer cells of the P. aeruginosa strain. An alternate report
by Latta et al. suggested that pili are expressed at specific times
during the development of swarming colonies in Proteus mira-
bilis (25). Our findings that fimbrial genes are required for
swarming but that no fimbriae were observed on the swarmer
cells might suggest that the expression of fimbrial and flagellar
genes is tightly controlled during swarmer cell differentiation
in E. coli. A more detailed analysis is necessary for clarifying
the roles of fimbrial genes in swarming motility.
Signal transduction systems. Flagella and chemotaxis sys-
tems have been demonstrated to play important roles in
swarmer cell differentiation on semisolid agar surfaces. In ad-
dition to these factors, swarming motility in E. coli appears to
require several two-component signaling systems consisting of
sensor and regulator proteins; in most cases, a mutation in
either the sensor or the regulator gene affected swarming mo-
tility. Of these, the Cpx-signaling system consisting of CpxA
(sensor kinase) and CpxR (response regulator) is considered to
954INOUE ET AL.J. BACTERIOL.
respond to envelope stress (35). Additionally, the Cpx-signal-
ing pathway was suggested to be involved in the regulation of
adhesion-induced gene expression (32). With respect to flagel-
lar gene expression, the Cpx system negatively regulates the
level of motABcheWA gene expression (8). However, in our
study, the cpxA gene mutation was observed to abolish swarm-
ing, whereas the cpxR mutant exhibited a normal swarming
phenotype, thereby suggesting a specialized role for the cpxA
product in swarming activity. The exclusive requirement for
cpxA without cpxR was also reported for the regulation of
HilA, an activator of invasion gene expression in S. enterica
serovar Typhimurium (16, 31). These cases seem curious but
may be due to cross-talk among two-component systems (14).
Chaperones and protein-folding catalysts. The mutant for
the gene encoding the molecular chaperone HscA exhibited a
normal swimming but a remarkably repressed swarming phe-
notype (Table 1). HscA (Hsc66) is a DnaK-like chaperone that
interacts with the iron-sulfur (Fe-S) cluster assembly protein
IscU (15). Both the hscA and the iscU genes belong to the isc
operon, encoding proteins involved in the biosynthesis of the
Fe-S cluster (48). Analysis of our data revealed that the iscS
and fdx genes in this operon were also associated with swarm-
ing (Table 1), suggesting that the Fe-S cluster, which is a
cofactor incorporated into several proteins, is essential for
swarming activity. Other molecular chaperons, including HtpX
(a putative membrane-bound zinc metalloprotease) (41),
HtpG (function unknown) (44), and the protein-folding cata-
lysts PpiB and PpiD (peptidylproryl-cis-trans-isomerases) (6),
were also implicated in swarming motility. As described above,
swarming appears to involve a wide range of cellular processes.
Therefore, it is reasonable to expect that chaperones and pro-
tein-folding catalysts are required for the correct folding and
degradation of many swarming-related proteins.
Iron acquisition. Iron is an essential factor for the growth of
bacteria; however, it is not readily available in natural environ-
ments. To obtain this metal, bacteria have developed multiple
iron acquisition systems. In our study, mutations in most of the
genes involved in the utilization of enterobactin (enteroche-
lin), a well-known siderophore in E. coli, significantly influ-
enced colony spreading by swarming motility (Table 1). In
addition, other genes coding for iron uptake are included in
the list of partially repressed swarming mutants (see Table S2
in the supplemental material). The results indicate that iron
acquisition systems are indeed required for E. coli swarming on
LB agar plates. The ent and fep genes listed are enterobactin
biosynthesis and transport genes, respectively (5, 37). Muta-
tions in these genes had no significant effects on the E. coli
growth in liquid LB medium (1), supporting the contention
that iron is not limiting in LB medium. However, our data may
imply that E. coli cells growing on LB agar swarm plates are in
an iron-starved state, which might promote swarmer cell dif-
ferentiation. In this regard, McCarter and Silverman (28) have
demonstrated that iron limitation is a second signal for the
expression of the lateral flagella that is responsible for swarm-
ing motility in Vibrio parahaemolyticus. They reported that
iron-regulated outer membrane proteins were also produced in
FIG. 2. Electron microscope images of bacterial cells negatively stained with 1% phosphotungstic acid. (A) Wild-type swimmer cells. (B) fimA
mutant swimmer cells. (C) Wild-type swarmer cells. (D) fimA mutant cells grown on the swarm plate. Bars, 1 ?m.
VOL. 189, 2007 SWARMING-ASSOCIATED GENES IN E. COLI 955
V. parahaemolyticus cells grown on agar plates containing iron-
rich heart infusion; this strongly suggests that the heart infu-
sion agar-grown cells were iron deprived. A possible explana-
tion for iron starvation on nutrient-rich agar media is based on
the limitation of iron diffusion into cells on agar surfaces (28).
Consistent with this explanation, it has been reported by Wang
et al. that growth of Salmonella on swarming agar induced the
expression of iron metabolism-related genes, which included
genes for the biosynthesis and transport of enterobactin and
the hydroxamate-dependent iron uptake system (49). There-
fore, it may be presumed that subsistence of swarm plate-
grown cells occurs in virtually iron-limited conditions even in
nutrient-rich media, which may be an important factor in fa-
cilitating swarming motility. Additionally, this may be a com-
mon feature among various swarming bacteria.
In this study, we used a complete set of single-gene knockout
mutants from E. coli K-12 for the screening of swarming-
associated genes in this strain. The results illustrate that the
production of energy and surface materials, such as LPS and
ECA, significantly affects swarming motility on semisolid agar
surfaces. Although E. coli K-12 swarming has been reported to
be highly dependent on the wettability of the agar, the require-
ment of LPS and ECA suggests that wetting agents, which are
bacterial products that reduce surface tension, are important in
facilitating swarm migration in this strain, which is similar to
that in other swarming bacteria. In particular, the biochemical
properties of ECA have been extensively studied; however, the
function of this molecule is largely unknown. Therefore, it is of
interest that the present data suggest a novel function for ECA.
Another intriguing result of this study is that numerous
genes, up to one-fifth of the genes on the genome, are involved
in swarming motility. This leads us to infer that swarming is a
highly organized mode of motility in viscous environments that
requires various cellular functions in addition to the flagellar
function. Among the swarming-associated genes, there are
many genes whose functions are currently unknown. Studies of
the roles of these uncharacterized genes that may be impli-
cated in swarming would lead to an understanding of the func-
tional properties of the gene products and new discoveries of
the molecular mechanisms involved in swarming. Some of the
swarming-associated genes are expected to be induced during
swarmer cell differentiation. In this regard, a recent study by
Wang et al. is notable in that nearly one-third of the genes on
the Salmonella genome were demonstrated to be differentially
regulated between surface and liquid growth (49). In viscous
surface environments, a single bacterial cell would be unable to
expand its growing space by the swimming mode. In order to
overcome such stressful conditions, cells probably activate var-
ious metabolic and synthetic pathways to develop into swarmer
cells and move as a group despite the high energy cost.
To date, it has been demonstrated that a variety of motile
bacteria have the ability to swarm on semisolid agar, which may
imply that bacterial cells frequently encounter viscous surfaces
in their natural environments. It is considered that bacteria are
capable of sensing environmental viscosity with their flagella
and that they determine their mode of motility, swimming or
swarming, depending on the perceived viscosity. For patho-
genic bacteria, swarmer cell differentiation may represent one
of the physiological states during the infection process. Thus
far, swarming has been reported to be associated with viru-
lence in several bacteria (10, 13, 33). Furthermore, some vir-
ulence factors were shown to be coregulated with swarmer cell
differentiation. Although the involvement of swarming motility
during the infection process remains to be elucidated, it is
expected that bacterial pathogens would be exposed to viscous
environments, such as mucus, during the early stages of infec-
tion; under these circumstances, they may preferentially dif-
ferentiate into swarmer cells to increase their growing area. In
the future, using genetically well-characterized E. coli and S.
enterica serovar Typhimurium as model systems, a global net-
work of gene expressions in viscous environments may be un-
raveled; this may contribute to our understanding of both bac-
terial signal transduction systems and pathogenic mechanisms
within the host.
We thank Yasuhiro Kasahara, Hokkaido University, for his help
with the maintenance of the Keio collection. We are grateful to Hi-
royuki Ohta, Ibaraki University, for helpful comments.
We thank the National BioResource Project (NIG, Japan): E. coli
for their support of the distribution of the Keio collection.
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