Screening of the genes involved in swarming motility.
The swarming motility of E. coli
K-12 strains can usually be observed on semisolid Eiken agar medium (Fig. ). The genome of the E. coli
K-12 strain comprises 4,390 open reading frames (1
). The Keio collection contains 3,985 nonessential gene mutants, in each of which a kanamycin resistance cassette has been used to create a highly targeted single-gene disruption (1
). 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 spreading and branching colony morphologies (the strongly repressed phenotype) (Fig. ). In addition, another 510 mutants (approximately 13% of the nonessential genes) exhibited partially reduced colony spreading (the moderately repressed phenotype) (Fig. ) (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 ). 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 flagellar 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 motility.
E. coli genes whose mutations caused strongly repressed swarming with no significant defects in swimminga
The 216 swarming-associated genes were classified into clusters of orthologous groups (COGs) (1
), suggesting that they belong to a variety of functional categories (Table ) (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 particularly 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 number 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 growing area. In the following sections, we focus on several functional 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 associated with energy production, such as those coding for ATP synthase, respiratory chain components, and tricarboxylic acid (TCA) cycle enzymes, with strongly repressed swarming motilities (Table ; 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 reducing power, which might supply a significant amount of energy required for swarming movement or increased production of cellular components for facilitating swarm migration (described below). With regard to this, Kim and Surette presented 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
). However, our results demonstrated that certain TCA cycle enzymes, including isocitrate 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 necessary for swarming in E. coli
, the addition of glucose to LB agar medium is required for swarmer cell differentiation (12
). 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
) and in glycolysis (pgi
) are listed in Table . Furthermore, in addition to energy production, 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 glucose-6-phosphate dehydrogenase, which catalyzes the conversion 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 described below.
Lipopolysaccharide and enterobacterial common antigen.
The medium used for testing bacterial swarming motility is generally solidified by the addition of agar to a final concentration of 0.5% to 2.0%. Owing to the lack of surface moisture, a semisolid surface on which the swimming motilities of individual bacterial cells are inhibited is created (13
). Under such conditions, swarming bacteria are considered to produce extracellular 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
). In the present study, a comprehensive analysis using the Keio collection demonstrated that the genes involved in the biosynthesis 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 substances in the swarming motility of E. coli
The contribution of LPS, particularly its O-antigen, to swarming motility has been reported for several gram-negative bacteria (11
). In addition, it appears to be an indispensable component for the flagellum-independent surface translocation in Vibrio cholerae
) and also for the social motility in Myxococcus xanthus
). Presumably, LPS acts by increasing the wetness of both the bacterial cell surface itself and the surrounding environment. The outer-membrane-anchored form of LPS, through its outer extruded core oligosaccharide 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 motility was significantly repressed in many of the LPS biosynthesis-related gene disruptants as shown in Table . The E. coli
K-12 strain carries a mutation in the O-antigen biosynthesis 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 oligosaccharide synthesis is presumably indicative of the fact that core oligosaccharide not only plays a role in conferring hydrophilicity to the cell surface but also serves as an acceptor for additional polysaccharides other than the O-antigen. In addition to the swarming defects, the disruption of the gmhA
), and waaC
genes led to impaired swimming motility (Table ). The products of these genes are responsible 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
mutation did not result in swimming deficiency. This may be explained by the presence of an additional enzyme that partially compensates for the gmhB
mutation as proposed by Kneidinger et al. (22
Swarming and swimming of mutants in genes associated with biosynthesis of ECA and LPSa
In addition to LPS, swarming was also strongly repressed in most of the ECA biosynthetic gene mutants listed in Table . ECA is one of the cell surface components of enteric bacteria, and its carbohydrate portion is a linear heterosaccharide containing a trisaccharide repeat unit, →3)-α-d
). Three forms of ECA have been reported: ECAPG
, and ECACYC
are the major and a minor forms of ECA in which the trisaccharide repeats are linked to the outer membrane phosphoglyceride and the core region of LPS, respectively (38
is another water-soluble cyclic form of ECA (38
), and in the E. coli
K-12 strain, it has been demonstrated 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
); 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 requirement for ECA in swarming motility may be obvious in E. coli
K-12 strains lacking the O-antigen, which is an important prerequisite 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 distribution.
With regard to the other polysaccharide materials, it is considered that capsular polysaccharide also contributes to a certain extent in the swarming motility of E. coli
K-12. The respective genes are listed in Table and include the cholanic acid capsule synthesis genes wcaE
. Furthermore, disruption of the wcaB
, and wcaM
genes resulted in moderate swarming repression (see Table S2 in the supplemental material). Although the extracellular polysaccharide was reported to play a role in the social motility of M. xanthus
), the capsular polysaccharide is not generally considered 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 polysaccharides may be necessary in order to facilitate swarm migration.
Type 1 fimbriae.
The type 1 fimbriae in E. coli
are peritrichously expressed filamentous surface structures and are one of the virulence factors in uropathogenic E. coli
). The following eight genes in the fim
gene cluster are involved in the production of type 1 fimbriae: fimB
, and fimH
). Of these, the fimA
(major fimbrial subunit), fimB
(periplasmic chaperone), fimD
(outer membrane usher), fimF
(adaptor), and fimH
(adhesin) mutants exhibited strongly repressed swarming motilities but no significant repression of swimming motility (Table ). The fimA
, and fimD
gene products are essential for constructing the fimbrial fiber, while the fimF
gene products that are located in the fibrillar tip are not required for this. However, the fimF
mutants were reported to have markedly reduced numbers of fimbriae per cell (40
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 possessed many flagella, almost all of these cells did not express fimbriae (Fig. ). 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. ). 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. ). 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, Köhler 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 mirabilis
). 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.
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 μm. (more ...) Signal transduction systems.
Flagella and chemotaxis systems have been demonstrated to play important roles in swarmer cell differentiation on semisolid agar surfaces. In addition 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 motility. Of these, the Cpx-signaling system consisting of CpxA (sensor kinase) and CpxR (response regulator) is considered to respond to envelope stress (35
). Additionally, the Cpx-signaling pathway was suggested to be involved in the regulation of adhesion-induced gene expression (32
). With respect to flagellar 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 swarming, 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
was also reported for the regulation of HilA, an activator of invasion gene expression in S. enterica
serovar Typhimurium (16
). These cases seem curious but may be due to cross-talk among two-component systems (14
Iron is an essential factor for the growth of bacteria; however, it is not readily available in natural environments. 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 (enterochelin), a well-known siderophore in E. coli
, significantly influenced colony spreading by swarming motility (Table ). 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
genes listed are enterobactin biosynthesis and transport genes, respectively (5
). Mutations 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 differentiation. 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 swarming motility in Vibrio parahaemolyticus
. They reported that iron-regulated outer membrane proteins were also produced in V. parahaemolyticus
cells grown on agar plates containing iron-rich heart infusion; this strongly suggests that the heart infusion agar-grown cells were iron deprived. A possible explanation 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
). Therefore, 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 facilitating swarming motility. Additionally, this may be a common 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 requirement 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 implicated in swarming would lead to an understanding of the functional 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 various 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 pathogenic 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 virulence in several bacteria (10
). Furthermore, some virulence 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 infection; under these circumstances, they may preferentially differentiate 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 network of gene expressions in viscous environments may be unraveled; this may contribute to our understanding of both bacterial signal transduction systems and pathogenic mechanisms within the host.