Several mycoplasma species are known to glide in the direction of the membrane protrusion (head-like structure), but the mechanism underlying this movement is entirely unknown. To identify proteins involved in the gliding mechanism, protein fractions of Mycoplasma mobile were analyzed for 10 gliding mutants isolated previously. One large protein (Gli349) was observed to be missing in a mutant m13 deficient in hemadsorption and glass binding. The predicted amino acid sequence indicated a 348,758-Da protein that was truncated at amino acid residue 1257 in the mutant. Immunofluorescence microscopy with a monoclonal antibody showed that Gli349 is localized at the head-like protrusion's base, which we designated the cell neck, and immunoelectron microscopy established that the Gli349 molecules are distributed all around this neck. The number of Gli349 molecules on a cell was estimated by immunoblot analysis to be 450 ؎ 200. The antibody inhibited both the hemadsorption and glass binding of M. mobile. When the antibody was used to treat gliding mycoplasmas, the gliding speed and the extent of glass binding were inhibited to similar extents depending on the concentration of the antibody. This suggested that the Gli349 molecule is involved not only in glass binding for gliding but also in movement. To explain the present results, a model for the mechanical cycle of gliding is discussed.Mycoplasmas are parasitic, small-genome bacteria that lack a peptidoglycan layer (28). Several mycoplasma species, including Mycoplasma pneumoniae, M. genitalium, M. pulmonis, M. gallisepticum, and M. mobile, have distinct cell polarity and exhibit gliding motility, a smooth translocation of cells across solid surfaces in the direction of the tapered end (17,22,36). The gliding motility of mycoplasmas is believed to be involved in pathogenicity, but the mechanisms underlying gliding motility have not been investigated well (19). Mycoplasmas have no surface structures, such as flagella or pili, or any homologs of genes that encode such structures. Neither do they have genes related to other bacterial motility systems or to eukaryotic motor proteins (5,11,13,21). These facts suggest that mycoplasmas glide by an entirely unknown mechanism.M. mobile, isolated in the early 1980s from the gills of a freshwater fish, is the fastest-gliding mycoplasma (18). M. mobile glides on glass in the direction of its tapered end, where its so-called head-like structure is. Its average speed is 2.0 to 4.5 m/s, about 3 to 7 times its cell length per second (32), and its maximum force can reach as high as 27 pN (23). It binds easily to glass and glides smoothly without pausing regardless of its growth stage. These distinct characteristics have allowed for detailed analyses of its gliding (9, 23-25, 32, 33) as well as for the isolation of gliding mutants, which are characterized by reduced or deficient gliding or by enhanced speed (26). However, no proteins related to gliding have been identified. In this study, we identified a huge protein that is truncated i...

Precise regulation of the number and placement of flagella is critical for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. We have shown previously that the number of polar flagella is positively regulated by FlhF and negatively regulated by FlhG. We now show that DflhF cells are non-flagellated as are most DflhFG cells; however, some of the DflhFG cells have several flagella at lateral positions. We found that FlhF-GFP was localized at the flagellated pole, and its polar localization was seen more intensely in DflhFG cells. On the other hand, most of the FlhG-GFP was diffused throughout the cytoplasm, although some was localized at the pole. To investigate the FlhF-FlhG interaction, immunoprecipitation was performed by using an anti-FlhF antibody, and FlhG co-precipitated with FlhF. From these results we propose a model in which FlhF localization at the pole determines polar location and production of a flagellum, FlhG interacts with FlhF to prevent FlhF from localizing at the pole, and thus FlhG negatively regulates flagellar number in V. alginolyticus cells.

The number and location of bacterial flagella vary with the species. The Vibrio alginolyticus cell has a single polar flagellum, which is driven by sodium ions. We selected mutants on the basis of reduced swarming ability on soft agar plates. Among them, we found two mutants with multiple polar flagella, and named them KK148 and NMB155. In Pseudomonas species, it is known that FlhF and FleN, which are FtsY and MinD homologs, respectively, are involved in regulation of flagellar placement and number, respectively. We cloned homologous genes of V. alginolyticus, flhF and flhG. KK148 cells had a nonsense mutation in flhG; cells expressing transgenic flhG recovered the swarming ability and had a reduced number of polar flagella. NMB155 cells did not have a mutation in either flhF or flhG. In wild-type cells, expression of flhF increased the number of polar flagella; in contrast, expression of flhG reduced both the number of polar flagella and the swarming ability. These results suggest that FlhG negatively regulates the number of polar flagella in V. alginolyticus. KK148 cells expressing both flhF and flhG exhibited fewer polar flagella and better swarming ability than KK148 cells expressing flhG alone, suggesting that FlhG acts with FlhF.

Mycoplasma mobile has a flask-shaped cell morphology and glides toward its tapered end at a rate of 3-7 cell lengths per s (2?0-4?5 mm s "1 ) by an unknown mechanism. Gliding requires that the surface of the cell is in contact with a solid substrate, such as glass or plastic. In order to characterize the nature of the outer surface of M. mobile, monoclonal antibodies were raised against intact cells and screened for their ability to recognize surface proteins. Four antibodies were identified and their protein targets were determined. One antibody recognized the Gli349 protein, which is known to be involved in glass binding and gliding. This antibody was also able to displace attached M. mobile cells from glass, suggesting that Gli349 is the major adhesion protein in M. mobile. The other three antibodies recognized members of the Mvsp family of proteins, which are presumably the major surface antigens of M. mobile. Immunofluorescence studies were performed to localize these proteins on the surface of M. mobile cells. Gli349 localized to the proximal region of the tapered part of the cell (the 'neck'), while the various Mvsp family members showed several distinct patterns of subcellular localization. MvspN and MvspO localized to the distal end of the tapered part of the cell (the 'head'), MvspK localized to the main part of the cell (the 'body'), and MvspI localized to both the head and body but not the neck. This analysis shows that M. mobile surprisingly expresses multiple versions of its major surface antigen at once but differentiates its surface by differential localization of the various paralogues.

ABSTRACT. Campylobacter jejuni is the major cause of human gastroenteritis worldwide. Under stress conditions, C. jejuni can enter a viable but non-culturable (VBNC) state. We found that the C. jejuni was able to enter a VBNC state by prolonged incubation at 4C. The standard isolation methods using pre-enrichment steps in Bolton broth or Preston broth could not detect the VBNC cells in spiked chicken meat. The transcription levels of virulence-associated genes (flaA, flaB, cadF, ciaB, cdtA, cdtB and cdtC) were expressed in VBNC cells but in low levels. The VBNC cells retained the ability to invade Caco-2 human intestinal epithelial cells in vitro. In most cases, VBNC cells failed to resuscitate in Caco-2 cells, but in some experiments, they formed colonies after co-incubation with host cells. Collectively, C. jejuni enters into a VBNC state at 4C and the VBNC C. jejuni remains virulent which may possibly lead to disease in humans. C. jejuni in VBNC state is a potential concern for food safety.KEY WORDS: Campylobacter jejuni, resuscitation, viable but nonculturable state, virulence.