Stability of cytadherence-related proteins P140/P110 in Mycoplasma genitalium requires MG218 and unidentified factors.

BACKGROUND

Tip-mediated cytadherence in Mycoplasma genitalium requires the structural and functional stability of the P140 adhesin, its operon-related protein P110, and the high molecular weight protein MG218 (190-kDa). Disruption mutants of mg218 unable to express MG218 exhibit both a non-cytadhering phenotype and P140/P110 instability, while disruption mutants that synthesize a truncated MG218 (160 kDa) retain the stability of P140/P110 and are >95% cytadhering. However, the origin of the MG218 truncated protein in these mutants is unclear. Therefore, we attempted to identify the origin of the truncated MG218 protein and to evaluate whether this truncated protein possessed the C-terminal part of MG218. In addition, we used spontaneous mutants lacking P140 to assess the role of MG218 in the stability of P140/P110.

METHODS

RNA from M. genitalium mutant producing truncated MG218 was subjected to primer extension analysis to identify the origin of expression of truncated MG218. Extracts of this mutant were examined for the presence of the C-terminal region of MG218 by immunoblot. In addition, pulse-chase analysis was performed to assess the role of MG218 in the stability of P140/P110 in spontaneous p140 mutants.

RESULTS

Primer extension analysis identified a transcriptional start point adjacent to the gentamycin-resistance gene in disrupted mg218 mutants. Antibodies directed against the C-terminal region (amino acids 1651-1666) of MG218 bound to truncated MG218 protein from mutants. Spontaneous p140 mutants subjected to pulse chase analysis indicated that solely class I mutants exhibited instability of P140/P110 in the presence of intact MG218.

CONCLUSIONS

Expression of truncated MG218 in M. genitalium mg218 mutants appears to be due to the presence of a putative promoter upstream to the point of mg218 disruption; this truncated protein possesses the C-terminal region of MG218. However, pulse chase results from spontaneously arising, non-cytadhering P140-deficient M. genitalium mutants suggest that the stability of P140 and P110 requires not only MG218 but also additional factors.