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, 184 (2), 547-55

Lateral Flagella and Swarming Motility in Aeromonas Species

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Lateral Flagella and Swarming Motility in Aeromonas Species

Sylvia M Kirov et al. J Bacteriol.

Abstract

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility. Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for approximately 8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30 degrees C) for Aeromonas spp. The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined.

Figures

FIG. 1.
FIG. 1.
Schematic representation of the genetic organization of the A. caviae Sch3 lateral flagellin locus. Flagellar genes and open reading frames (ORFs) are indicated by shaded horizontal arrows and indicate the direction of transcription. ORFs are named after their homologues in other bacterial species. The open vertical arrows indicate the site of insertion of the kanamycin resistance cassette in the single mutants. The solid vertical arrow shows the site of insertion of the kanamycin cassette in the single mutant AAR58 and the site of insertion of the chloramphenicol resistance cassette in the tandem mutant AAR6.
FIG. 2.
FIG. 2.
Light microscopic detection of flagella. Bacteria were grown on TSAY at 37°C for 6 h and stained with Leifson’s stain. (A) P. mirabilis, showing peritrichous flagella (arrow). (B) V. parahaemolyticus strain ACM 2776, showing lateral flagella (arrow). (C) A. caviae strain Sch3, showing lateral flagella (arrow). Bar, 5 μm.
FIG. 3.
FIG. 3.
Transmission electron microscopy of flagella (negative staining; 0.5% phosphotungstic acid, 10 s). Bars, 1 μm. (A) Sheathed polar flagellum of V. parahaemolyticus. The inset is a high-power magnification (boxed section) showing the double membrane structure of the flagellar sheath. Inset bar, 50 nm. (B) Numerous unsheathed lateral flagella of V. parahaemolyticus strain NCTC 10884. Inset bar, 100 nm. (C) Unsheathed polar flagellum of A. veronii biovar sobria strain BC88. (D) Numerous unsheathed lateral flagella of A. veronii biovar sobria strain CA25. Inset bar, 100 nm.
FIG. 4.
FIG. 4.
Southern blot (2-h exposure) of PstI-digested chromosomal DNA of Aeromonas isolates probed with the 1.0-kb PstI digoxigenin-labeled fragment of the lateral flagella genes lafA1 and lafA2. Lanes 1 to 13 are A. hydrophila strain DJ188, A. veronii biovar sobria FA132, A. caviae strain Sch3, and A. veronii biovar sobria strains BC88, CA17, CA110, BC96, CA112, CA113, EA49, EA57, EA42, and EA60, respectively. Isolates were from diarrheal feces except for strain FA132, which was isolated from a chicken carcass rinse, and strains EA49, EA57, EA42, and EA60, which were isolated from water samples. Positions of molecular size standards (λ HindIII digest) are indicated on the left (in kilodaltons).
FIG. 5.
FIG. 5.
Aeromonas swarming on Eiken agar. (A) Strong swarming response. (B) No swarming, but growth at the inoculum site. The inset (boxed section) shows a raft of bacteria surrounded by flagella taken from the swarm edge (glutaraldehyde-fixed sample, Difco flagella stain). Bar, ≈1 μm.
FIG. 6.
FIG. 6.
FESEM. (A and B) V. parahaemolyticus (strain ACM 2776) on TSAY (37°C, 6 h), showing intertwining networks of lateral flagella linking bacteria on the agar surface. (C and D) A. caviae strain Sch3 on agar as above, showing similar filamentous structures linking bacteria. Bars: (A and C) 2 μm; (B and D) 500 nm.

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