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, 181 (9), 2823-33

The Yersinia Enterocolitica Motility Master Regulatory Operon, flhDC, Is Required for Flagellin Production, Swimming Motility, and Swarming Motility

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The Yersinia Enterocolitica Motility Master Regulatory Operon, flhDC, Is Required for Flagellin Production, Swimming Motility, and Swarming Motility

G M Young et al. J Bacteriol.

Abstract

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.

Figures

FIG. 1
FIG. 1
Growth conditions affect Y. enterocolitica motility. Each panel shows an image of Y. enterocolitica JB580v grown at 26°C for 20 h on medium solidified with 0.35% Difco agar. The plates were inoculated by spotting 2 μl of culture into the center. LB medium (LB), T medium (T), T medium plus yeast extract (TYE), T medium plus NaCl (TN), T medium plus lactose (TL), and T medium plus glucose (TG) plates are shown.
FIG. 2
FIG. 2
Swarming motility is influenced by growth conditions. Each panel shows an a image of Y. enterocolitica grown at 26°C for 24 h on T medium solidified with 0.6% Difco agar and supplemented with carbon sources (indicated above each panel) at a final concentration of 100 mM. The plates were inoculated by spotting 2 μl of an overnight culture of Y. enterocolitica JB580v into the center.
FIG. 3
FIG. 3
Images of colony and cellular morphology of swarming bacteria suggest that Y. enterocolitica differentiates into cells that cooperatively migrate over agar surfaces. (A) The top row shows images of the morphology of an advancing colony of bacteria grown on a TG plate at 26°C, conditions that induce swarming motility. (Top left) Image of a colony at ×4 magnification; S indicates the location of the slime layer preceding the front of migrating colony; T1 indicates the first terrace of migrating cells in the swarming colony; T2 indicates the location of the second terrace of cells that appear to overlay cells forming the first terrace. (Top middle) Image of the same colony at ×20 magnification, showing rafts of cells that often advance ahead of a colony. (Top right) Image of elongated cells resuspended from a swarming colony taken at ×60 magnification. The bottom row shows images (at ×60 magnification) of bacteria grown on LB plates, T plates, or T plates containing 100 mM glucose and 1% NaCl (TGN) at 26°C, conditions that influence swimming motility but do not induce swarming motility. (B) Electron micrographs of bacteria isolated under conditions that influence motility. Bacteria were isolated from agar plates grown for 16 h at 26°C. (Top left) Cells isolated under conditions that induce swimming motility on T medium. (Top middle) Cells isolated under swarm-induced conditions on TG medium. (Top right) A single swarm cell that appears to be dedifferentiating. (Bottom left) Cells isolated from LB medium, which does not induce motility. (Bottom middle) cells isolated from TGN medium, which does not induce motility. (Bottom right) A single elongated swarm cell isolated under inducing conditions on TG medium. Bar, 1 μm.
FIG. 4
FIG. 4
Detection of flagellin proteins produced by cells isolated from media that influence the expression of swimming and swarming motility. Proteins were detected by immunoblot analysis with an anti-flagellin-specific monoclonal antibody. Each lane contains cells at 0.25 OD600 units per ml, isolated from 0.6% agar plates incubated for 18 h at 26°C. Lanes: TYE, T medium supplemented with yeast extract; LB, LB medium; TN, T medium supplemented with 1% NaCl; TGN, TG medium supplemented with 1% NaCl; TG, TG medium; T, T medium. The bracket at the left of the figure indicates the location of the detected flagellin.
FIG. 5
FIG. 5
Genetic and functional analysis of the Y. enterocolitica chromosomal region encompassing the motility master regulatory operon. (A) Schematic representation of the flhDC locus. Shaded boxes indicate the locations and directions of the ORFs identified by DNA sequence analysis, and relevant restriction sites are shown above the diagram. The series of plasmid deletions are shown below. Heavy black lines indicate the DNA present in each clone, and thin black lines indicate internal regions that were deleted. Plasmids pGY11 to pGY18 are derived from pGY10. Plasmids pGY19 and pGY20 contain a fragment of DNA subcloned from pGY10 into pWKS130 and pVLT33, respectively. The box at the right shows the results of functional complementation by each plasmid for the chromosomal flhDC mutations described in this study. A positive result (+) for swimming motility or swarming was determined as migration extending away from the point of inoculation on TG medium containing 0.35 or 0.6% agar, respectively. A negative result (−) was determined as bacterial growth at the point of inoculation but no apparent migration. An intermediate result (+/−) was determined as significantly reduced levels of migration. All of the functional assays were compared to the swimming and swarming motility of the wild-type strain, JB580v. (B) The DNA sequence of the chromosomal region upstream of flhD. The upstream nucleotide sequence is shown in standard type, and the bold type shows the first 18 nucleotides of flhD. Above the DNA sequence, single bold letters show the first 6 amino acid residues predicted for FlhD based on the DNA sequence; convergent dashed lines indicate palindromic sequences that may serve as cis-acting regulatory sites; the double dashed line indicates the location of the insertion for the chromosomally integrated suicide vector in Y. enterocolitica GY357; italic type indicates the locations of relevant restriction sites.
FIG. 6
FIG. 6
Extracellular protein production, swimming motility, and swarming motility are affected by the levels of flhDC when expressed under the control of a ptac promoter. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5% polyacrylamide) of extracellular protein samples stained with Coomassie brilliant blue showing the production of flagellins and several other extracellular proteins (arrowheads) induced by increased levels of IPTG in the growth medium. Lanes: 1, wild-type strain JB580v grown in T broth; 2 to 4, GY357/pVLT33 grown in T broth with 0, 5, and 50 μM IPTG, respectively; 5 to 7, GY357/pGY20 grown in T broth with 0, 5, and 50 μM IPTG, respectively; 8, GY357/pMG600 grown in T broth. Unlabeled lanes contain protein molecular mass standards with their corresponding size in kilodaltons indicated on the left of the gel. (B) Swimming motility (right) and swarming motility (left) for GY357/pGY20. Cells were grown on TG medium with 0, 5, and 50 μM IPTG (indicated on the left of the figure). All the plates were inoculated by spotting 2 μl of an overnight culture in the center and were incubated at 26°C for 24 h. (C) Swimming (right) and swarming (left) motility of GY357/pMG600 on TG medium with no IPTG. The culture conditions were the same as those described for panel B.

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