Biosci. Biotechnol. Biochem., 67 (8), 1802–1805, 2003

Note

Flagellum-independent Trail Formation of Escherichia coli on Semi-solid AgarHajime FUKUOKA,1,2,† Michio HOMMA,3 and Shigeyuki ICHIHARA1,2
1The Agricultural High-Tech Research Center, Meijo University, Tenpaku-Ku, Nagoya 468-8502, Japan
2Laboratory of Microbiology, School of Agriculture, Meijo University, Tenpaku-Ku, Nagoya 468-8502, Japan
3Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602,

Japan
Received November 25, 2002; Accepted April 25, 2003

Escherichia coli can form linear trails and move in a‰agellum-independent manner on semisolid agar
containing carbon sources. Trail formation seemed toor carbon
correlate with the growth speed and W
metabolism. Cell morphology in linear trails changed
into larger cell sizes. We speculate that the ‰agellumindependent trail formation is a new mechanism for
migration of E. coli cells.
Key words:

Escherichia coli ; cell morphology; ‰agella; migration; growth

Bacteria can sense contact to their growth surface
and adapt to their environment by changing theirgrowth mode. Several bacteria can translocate on thetheir growth surface in an inherent manner. Surface
translocation of bacteria is relevant to symbiosis andpathogenicity to their host.1–5) Translocation is also
important for ˆnding nutrients or colonization sites
within the host and for avoiding toxic substrates.
Several diŠerent types of bacterial surface translocation have been observed and classiˆed into thefollowing six types: (i) swimming, (ii) swarming, (iii)
gliding, (iv) twitching, (v) sliding and (vi) darting.6)
Each of these modes of surface translocation requiresspeciˆc structures such as ‰agella, pili, surfactants,slime, or capsules. Flagella, which contribute toswimming and swarming ability, are the best characterized locomotive organelles. On the other hand,
there are many cases of ‰agellum-independent
surface translocation. Serratia marcescens secretes
an extracellular surfactant, called serrawettin, whichenables cells to spread on a semisolid agar plate byreducing the friction between the cell and the semisolid agar surface.7,8) Mycobacterium smegmatis, anon-‰agellated Gram-positive bacterium, can also
spread on semisolid agar plates.9,10) Spreading of M.
smegmatis requires glycopeptidelipid (GPL), which
exists in the outermost layer of the cell wall.

In Escherichia coli, surface translocation has been
described during cell diŠerentiation, when the cells
are hyper‰agellated and elongated, similar to swarming cells.11) Flagellum-independent surface translocation of E. coli has been reported to be an activeprocess.12) In this regard, we identiˆed ‰agellumindependent surface translocation in non-‰agellated
strains of E. coli, which form linear trails of cells on
soft agar containing either glucose or other carbon
sources which E. coli can metabolize.
When the CSH26 (ara thi D(lac-pro) )13) ‰agellated
strain was inoculated onto ×0.5LB (-NaCl) soft agar
plates (0.3z agar) and incubated at 379C, CSH26
formed a halo in the soft agar (Fig. 1D). This translocation phenotype is based on the ability of cells toswim by making use of ‰agella.6) The strain MC4100( relA D(argF-lac)U169 deoC araD rpsL thiA ptsF‰hD tonA),14) on the other hand, did not form a halo(Fig. 1B), because this strain is defective in the ‰hDgene, the master gene for ‰agellar gene expression.
However, we did observe that MC4100 cells formedsome white trails and spread on soft agar containing
150 mM glucose, despite the absence of ‰agella(Fig. 1A). Similar trails were observed in CSH26 cells
on soft agar containing 150 mM glucose (Fig. 1C).
Although CSH26 is a ‰agellated strain, ‰agella synthesis would be repressed by high concentrations ofglucose. These results suggest that trail formation is a‰agellum-independent mode of surface translocation.
Initially, the MC4100 and CSH26 strains grew at
the inoculation position on the surface of the plate.
After 3 hours, we could observe trails that began toextend from the original point and spread over theentire plate after 24 hours. The trails are probably
formed by the grown cells. The MC4100 strain did
not form trails in the absence of glucose, even after
48 hours. The trails extended on the soft agar plate,appearing to sink into grooves. In the early stage of† To whom correspondence should be addressed. Tel: ＋81-52-789-2991; Fax: ＋81-52-789-3001; E-mail: 4hajime＠bunshi3.bio.nagoya-u.ac.jp; Present address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya
464-8602, Japan

Flagellum-independent Trail

Fig. 1. E. coli Trail Formation on Soft Agar.
E. coli cultures (1 ml) were inoculated onto 0.3z soft agar
plates in the presence (A, C) or absence (B, D) of 150 mM glucose and incubated at 379C for 24 hours (A, B, C) or 8 hours(D). A, B; MC4100, the ‰agella-defective mutant (the ‰hDmutant). C, D; CSH26, the ‰agellated strain.

incubation, a transparent region appeared aroundthe trails, but not around colonies that formed
without trail formation (data not shown).
To conˆrm whether trail formation is due to a
spontaneous mutation, cells were picked up from thetrail and spotted on a soft agar plate in the presenceor absence of glucose. A new trail formed on plates
containing glucose, but not on plates lacking glucose(data not shown). This indicates that trail formation
is not the result of a mutation, but is rather aresponse to environmental stimuli.
We investigated whether trail formation is strainspeciˆc or a common phenotype among E. coli. We
used JM103 15) or YK410 16) as well as derivative
strains defective in one of the ‰agellar genes. JM103
formed trails on soft agar plates containing glucose.
On the other hand, the YK strains, YK410 (WT),YK4130 ( ‰iC ), YK4131 ( ‰hD ), and YK4136 ( ‰hC ),did not form trails (data not shown).
Various environmental factors such as viscosity,
growth temperature, osmolarity, and nutrients aŠect
bacterial phenotype. We studied whether trails
formed under various growth conditions. First, weinvestigated the agar concentration of the plate. Aculture of the MC4100 strain was spotted onto 0.3z
–0.8z agar containing 150 mM glucose, and incubated at 379
C. Trail formation was observed at 0.3z
agar, but not when agar was higher than 0.4z. In0.4z agar in the absence of glucose, the CSH26
strain was able to swarm, by ‰agellum-dependenttranslocation, although the swarm size was smaller

1803

than in 0.3z agar. This result suggests that thehardness of the plate is critical for trail formationand ‰agellum-dependent translocation. Second, theMC4100 strain was incubated at 259C, 309C, andC on 0.3z soft agar containing 150 mM glucose.
379
Compared to incubation at 379C, branching and trail
length were reduced at 309C. At 259
C, trail formation was not observed even with a longer incubation
time (72 hours). This result suggests that the trail
formation may correlate with growth speed, because379
C is the optimal growth temperature for E. coli.
Third, the glucose concentration of the soft agar
plates was changed. The MC4100 strain was incubated at 379
C on 0.3z soft agar containing 150 mM,
15 mM, 5 mM, and 1.5 mM glucose. Trail formation
was observed on agar containing at least 5 mM
glucose, but not on agar containing 1.5 mM glucose.
This indicates that the lower threshold glucose
concentration for trail formation was between 5 mM
and 1.5 mM. Trails were also noted to be straighterand shorter at lower glucose concentrations.
We also tested the involvement of cAMP and thecAMP receptor protein (CRP) on the trail formation,using the TB100 (WT), TB102 (crp ), and TB105 (cya)strains (provided by Dr. H. Aiba).17) Although trail
shape appeared to be diŠerent from MC4100, trail
formation was observed in all the strains on plates
containing glucose (data not shown). Thus, cAMPand cAMP receptor protein are not likely to berequired for trail formation.
We investigated whether trail formation wasinduced by carbon sources other than glucose.
Galactose, maltose, trehalose, pyruvate, or glycerol
induced trail formation while fructose, lactose,
sucrose, and cellobiose did not. E. coli cannot
metabolize sucrose and cellobiose. In addition, theMC4100 strain cannot metabolize lactose, due to amutation in the lac gene. When MC4100 was grownin a medium containing fructose as the sole carbon
source, the growth rate was very slow compared to
growth in media supplemented with glucose at thesame concentration. These results suggest that trail
formation correlates with the growth speed and Wormetabolic activity of E. coli.
E. coli produces acetic acid when grown in medium
containing high concentrations of glucose.18) The pHof the medium around the cells was measured withpH test papers. In almost all media, pH was reduced
to 4–5 from the original pH of 7 around the trails
except in the case of pyruvate (pH 9). On the otherhand, the pH of the medium around the colony that
could not form trails was 7. Therefore, low pH
appears to not be directly correlated with trailformation.
When bacteria swarm on soft agar, cells have been
observed to develop excessive numbers of ‰agella andto elongate their cell bodies.12) We investigated themorphology of cells that form trails (Fig. 2). Cells

1804

H. FUKUOKA et al.

Fig. 2. Microscopic Analysis of MC4100 Cells in Trails.
Cells from the plates in Fig. 1-A and Fig. 1-B, from trails in0.3z soft agar plates containing 150 mM glucose (A), and fromcolonies in 0.3z soft agar plates containing no glucose (B),respectively, were picked up by pipette and observed under amicroscope (OLYMPUS BX60). Photographs were taken with adigital camera (OLYMPUS DP10). Bar, 10 mm.

forming trails (on agar containing 150 mM glucose)
appeared larger than cells forming colonies (on agar
containing no glucose): about 10 mm long and 2 mmwide (maximal size) for trails and about 2 mm longand 1 mm wide for colonies. We observed that cells
were enlarged anywhere in trails and in the original
inoculation point. Cells grown in liquid media withor without 150 mM glucose have the same morphology. There is a possibility that a change in cellmorphology contributes to trail formation. Little is
known about the mechanism and regulation of thisputative morphological transformation.
The E. coli MC4100, non-‰agellated strain formed
trails on soft agar containing glucose. The trail
formation is probably the movement of whole cells
and we do not know about the movement of individual cells. In other words, the trail formation may bethe expansion of the cell population by cell division.
Trail formation seemed to correlate with the growth
speed in MC4100 because it was greatest at 379C, theoptimal growth temperature for E. coli and, inaddition, trail formation was only observed on agar
containing carbon sources that can be metabolized byE. coli. Flagellum-independent migration of E. coli
has been reported, but the described migration
proˆles are very diŠerent from one another.12)
Currently, we do not know whether these phenotypes
are due to a similar mode of migration. Many ‰agella-independent migration phenomena have been
observed in various species. In ‰agellum-independent
surface translocation, secreted surfactants or slimematerials are thought to be essential for reducing
friction between the attached surface and the cell surface.8,19) We observed a transparent region aroundthe trails at an early stage of incubation. From thisresult, there is a possibility that E. coli also secretesmaterials that might assist in trail formation.
In ‰agellum-dependent translocation of Proteus
mirabilis and Salmonella typhimurium, mutants
defective in the biosynthesis of polysaccharide( Proteus ) or lipopolysaccharide ( Salmonella) cannot
swarm.19–21) Similarly, lipopolysaccharide has beenshown to be important for ‰agella-independent
surface translocation of Vibrio cholerae,12) andglycopeptide lipids (GPL), a component of mycobacteria cell walls, are required for sliding.9,10) The
biosynthesis of lipopolysaccharide is known to beaŠected by environmental signals.22) Thus, the structure and W
or the components of bacterial cell walls
may be essential for surface translocation in both‰agellum-dependent and independent translocation.
During trail formation, changes in cell morphology were observed. These phenotypes might beinduced by carbon catabolism on the soft agar. We
have shown that E. coli can migrate by trail formation in an environment without the locomotive
organelle, the ‰agella. This translocation ability canbe expressed in the host and extended to a new,nutrient-rich environment. To further elucidate themechanism of trail formation or ‰agellum independent migration, we are presently trying to isolatetransposon mutants defective in trail formation.

Acknowledgments
We thank Dr. Kutsukake for providing the strain
YK410, and Dr. Aiba for providing for the starin
TB100, TB102, and TB105. This study was supported by the Agricultural High-Tech Research Center,
Meijo University, under the ``Environmental Control
through the Functions of Microorganisms'' project.

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