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Abstract
Mycoplasma pneumoniae exhibits a novel form of gliding motility along solid/liquid interfaces, which may be a virulence factor, necessary for colonization of the respiratory epithelium of human hosts. Given that genes known to be involved in gliding in other organisms are absent in the M. pneumoniae genome, random transposon mutagenesis was employed to generate mutants with gliding-deficient phenotypes. Transposon insertions in the only annotated ser/thr kinase (prkC; MPN248) and its cognate phosphatase (prpC; MPN247) in M. pneumoniae resulted in significant and contrasting effects on gliding frequencies. prkC mutants glided at approximately half the frequency of wild-type cells, while prpC mutants glided more than twice as frequently as wild-type cells. The combined application of Western immunoblotting and Pro Q Diamond phosphoprotein staining identified several proteins as potential targets for PrkC phosphorylation. These included HMW1 and HMW2, which localize to the M. pneumoniae terminal organelle, are essential for the assembly of this structure, and have been previously shown to contain phosphoserine and phosphothreonine, as well as the major adhesin protein P1. In order to confirm the correlation between phosphorylation / dephosphorylation and gliding frequency, the prkC and prpC mutants were complemented with wild-type copies of their respective disrupted alleles by transposon delivery. Gliding frequencies and phosphorylation levels returned to the wild-type standard in the complemented prpC mutant. Surprisingly, the recombinant wild-type prkC allele dramatically increased gliding frequency in the prkC mutant to a level approximately three-fold greater than that in wild-type. Experiments in which phosphate was removed from the gliding medium showed that gliding frequency was reduced in wild-type cells, but not affected in prpC and prkC mutants, while the replacement of glucose with glycerol as a carbon source suggested that the regulation of gliding is tied to the phosphotransferase system. Collectively these data suggest that PrkC and PrpC work in opposition in M. pneumoniae to influence gliding frequency.