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.
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