Mutant Analysis Reveals a Specific Requirement for Protein P30 inMycoplasma pneumoniaeGliding Motility

Abstract

The cell-wall-less prokaryote Mycoplasma pneumoniae, long considered among the smallest and simplest cells capable of self-replication, has a distinct cellular polarity characterized by the presence of a differentiated terminal organelle which functions in adherence to human respiratory epithelium, gliding motility, and cell division. Characterization of hemadsorption (HA)-negative mutants has resulted in identification of several terminal organelle proteins, including P30, the loss of which results in developmental defects and decreased adherence to host cells, but their impact on M. pneumoniae gliding has not been investigated. Here we examined the contribution of P30 to gliding motility on the basis of satellite growth and cell gliding velocity and frequency. M. pneumoniae HA mutant II-3 lacking P30 was nonmotile, but HA mutant II-7 producing a truncated P30 was motile, albeit at a velocity 50-fold less than that of the wild type. HA-positive revertant II-3R producing an altered P30 was unexpectedly not fully wild type with respect to gliding. Complementation of mutant II-3 with recombinant wild-type and mutant alleles confirmed the correlation between gliding defect and loss or alteration in P30. Surprisingly, fusion of yellow fluorescent protein to the C terminus of P30 had little impact on cell gliding velocity and significantly enhanced HA. Finally, while quantitative examination of HA revealed clear distinctions among these mutant strains, gliding defects did not correlate strictly with the HA phenotype, and all strains attached to glass at wild-type levels. Taken together, these findings suggest a role for P30 in gliding motility that is distinct from its requirement in adherence.Mycoplasmas are cell-wall-less prokaryotes with minimal genomes and limited biosynthetic capabilities, dictating a strict dependence on host species for survival in nature (43). Mycoplasma pneumoniae is a human pathogen primarily colonizing the respiratory tract. While the most common clinical manifestations of infection are tracheobronchitis and atypical or "walking" pneumonia (7,12,14,30), recent studies indicate a strong correlation with asthma (5, 24, 38), and extrapulmonary complications are not uncommon (53). Adherence of M. pneumoniae cells to host respiratory epithelium (cytadherence) is required for colonization and pathogenesis (20) and is mediated largely by a differentiated terminal organelle (9, 39). This well-defined apical structure is a membrane-bound extension of the mycoplasma cell distinguished ultrastructurally by an electron-dense core (4), which is a major constituent of the M. pneumoniae cytoskeleton (17, 35).M. pneumoniae cells exhibit gliding motility, with the terminal organelle always the leading end (6), but details regarding the biological significance and the mechanism of gliding are largely unknown. Although the M. pneumoniae genome has been sequenced and twice annotated (11, 19), close inspection reveals no homology to proteins known to be involved in bacterial motility of any type in walled bact...

Terminal organelle development in the cell wall-less bacterium Mycoplasma pneumoniae

“…1A). Of the 296 cells examined, 69 had 1 P30-YFP focus; nearly 90% of these were gliding, similar to published values (20). A new P30-YFP focus appeared adjacent to the single existing focus and coincident with cessation of gliding in 38% of the motile cells during the observation period ( Fig.…”

“…Protein P30 is a terminal organelle component required for cytadherence and gliding motility (19,20). A recombinant P30 fusion with yellow fluorescent protein (YFP) introduced by transposon delivery localizes to the terminal organelle and restores cytadherence and gliding to a mutant lacking P30 (20); transformants with the recombinant transposon in an intergenic site exhibit a phenotype essentially indis-tinguishable from wild-type M. pneumoniae producing P30-YFP. As P30-YFP also yielded the strongest signal of the fluorescent protein fusions examined here (data not shown), we focused initially on this fusion.…”

“…Protein P30 is a terminal organelle component required for cytadherence and gliding motility (19,20). A recombinant P30 fusion with yellow fluorescent protein (YFP) introduced by transposon delivery localizes to the terminal organelle and restores cytadherence and gliding to a mutant lacking P30 (20); transformants with the recombinant transposon in an intergenic site exhibit a phenotype essentially indis-…”

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

“…A similar bindingand-release process might occur in M. pneumoniae, albeit through the use of an unrelated set of adhesins. In support of this hypothesis, the adhesins P30 (Hasselbring et al, 2005) and P1 (Seto et al, 2005a) have both been implicated in gliding motility of M. pneumoniae. Alternative hypotheses have focused on the electron-dense core as the major component of the M. pneumoniae motor, citing differences in fine features of individual M. pneumoniae cores (Henderson & Jensen, 2006) or postulating that the core is capable of twisting within the attachment organelle (Hegermann et al, 2002).…”

Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence

“…In addition, the TO is the leading end as mycoplasma cells glide (Radestock & Bredt, 1977) and it constitutes the mycoplasma gliding motor (Hasselbring & Krause, 2007). Motility and cytadherence are closely related in mycoplasmas and many cytadherence-related proteins are also required for cell gliding (Burgos et al, 2007;Hasselbring et al, 2005;Hasselbring & Krause, 2007;Seto et al, 2005). However, the existence of a cytadherence-independent set of motility-related proteins has been disclosed recently for the slow gliding mycoplasmas M. genitalium (Pich et al, 2006a) and Mycoplasma pneumoniae (Hasselbring et al, 2006).…”