Identification of a 349-Kilodalton Protein (Gli349) Responsible for Cytadherence and Glass Binding during Gliding of Mycoplasma mobile

Abstract

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...

Spike Structure at the Interface between GlidingMycoplasma mobileCells and Glass Surfaces Visualized by Rapid-Freeze-and-Fracture Electron Microscopy

“…At all stages of growth, M. mobile glides smoothly and continuously on glass at an average speed of 2.0 to 4.5 m/s, or about 3 to 7 times the length of the cell per s (27), exerting a force of up to 27 piconewtons (pN) (20,21). These distinct characteristics enabled detailed analyses of gliding (6,(20)(21)(22)(26)(27)(28) and isolation of gliding mutants that were characterized by reduced or deficient gliding or enhanced speed (23,28). …”

Attachment organelle ultrastructure correlates with phylogeny, not gliding motility properties, in Mycoplasma pneumoniae relatives

“…The M. pneumoniae motility mechanism has been addressed through characterization of mutants with differences in colony-spreading phenotypes, resulting in the identification of a moderate number of genes of both known and unknown function (Hasselbring et al, 2006b). In Mycoplasma mobile, a distant relative whose adherence, motility and terminal organelle components are unrelated to those of M. pneumoniae (Miyata, 2005), gliding is proposed to involve the cyclical binding and release by the adhesin Gli349 (Uenoyama et al, 2004) of carbohydrate moieties present on a wide variety of host-cell proteins, including those deposited on the surface of the microscope slide from serum in vitro (Nagai & Miyata, 2006). A similar bindingand-release process might occur in M. pneumoniae, albeit through the use of an unrelated set of adhesins.…”

Protein Kinase/Phosphatase Function Correlates with Gliding Motility in Mycoplasma pneumoniae

“…Accordingly, their distinct functions in gliding motility are difficult to define by mutagenesis alone. Given that the M. pneumoniae genome exhibits no homology to elements of defined gliding mechanisms (2,3), including those of other gliding mycoplasmas (11)(12)(13)(14), it was necessary to perform saturating transposon mutagenesis in order to identify the components specific to gliding (15). Transposon insertions in the genes encoding the cytoskeletal proteins P41 and P65 (MPN311 and MPN309, respectively) (16), which are known to localize to the terminal structure of M. pneumoniae (7), produce gliding-deficient phenotypes (15,17), but the defect in each of them is not clearly associated with an actual gliding motor and suggests little about structure and function of the motor.…”