Previous serological data have demonstrated cross-reactive antigens between two pathogenic species of mycoplasmas, M. pneumoniae and M. genitalium. Preliminary analysis of sera and monoclonal antibodies (MAbs) to protein antigens of these species showed an immunodominance of adhesin P1 (165 kilodaltons [kDa]) of M. pneumoniae in mice and hamsters and a 140-kDa protein of M. genitalium in mice and experimentally infected chimpanzees. To further characterize these two proteins, we assayed multiple anti-Pl and anti-140-kDa protein MAbs by enzyme-linked immunosorbent assay, immunoblot, and radioimmunoprecipitation techniques. The 140-kDa M. genitalium protein was shown to be surface accessible and insensitive to levels of trypsin which readily degrade protein P1. Peptide mapping was used to identify a unique class of MAbs which bound a cross-reactive molecule common to both the major adhesin protein P1 of M. pneumoniae and the 140-kDa protein of M. genitalium. MAbs generated against both M. pneumoniae and M. genitalium which were reactive with this determinant blocked M. pneumoniae attachment to chicken erythrocytes. Mycoplasma pneumoniae and Mycoplasma genitalium are pathogens which share common ultrastructural, morphological, and serological features (3, 6, 9, 16, 17, 22, 25). In recent studies in our laboratory, various proteins of M. pneumoniae were identified as ligands mediating M. pneumoniae attachment to host cells (1, 2, 11, 13, 20). A trypsin-sensitive surface protein designated P1 (165 kilodaltons [kDa]) appears to be a primary ligand mediating cytadherence consistent with its dense clustering at the terminal nap region of the mycoplasma attachment organelle and its absence in specific nonadhering mycoplasma mutants (1, 8, 10, 12, 13). Moreover, monospecific and monoclonal antibodies (MAbs) to protein P1 of M. pneumoniae have been shown to block mycoplasma attachment to erythrocytes (RBCs) and respiratory epithelium (11, 20). Recent studies by Tully et al. (22, 23) have demonstrated that M. genitalium not only shares the flask shape of M. pneumoniae, but also possesses a predominant surface nap extending distally from the tip organelle. This structure appears to mediate the adherence of M. genitalium to Vero monkey kidney cell monolayers (22), thus implying that a process similar to that of M. pneumoniae regulates M. genitalium cytadherence. This study characterizes an immunodominant protein of M. genitalium with a molecular weight of 140,000. MAbs directed against both the 140-kDa protein and protein P1 of M. pneumoniae were used to examine the antigenic relatedness of these two proteins. Using this approach, a crossreactive epitope(s) on the two molecules was identified. MATERIALS AND METHODS Mycoplasma strains and culture conditions. Virulent, hemadsorbing M. pneumoniae M129-B16 was grown in 32-oz (946-ml) prescription bottles in 70 ml of Edward medium (7) at 37°C for 72 h. Glass-adherent mycoplasmas were washed four times with phosphate-buffered saline (PBS; pH 7.2) and collected by centrifugation (9,5...

Monoclonal antibodies directed against Mycoplasma pneumoniae surface components were examined for their ability to block mycoplasma attachment to chicken erythrocytes. Purified preparations of antibodies which recognize the major mycoplasma ligand mediating cytadherence (protein P1, 165 kilodaltons) inhibited attachment by more than 85% of the control values. Monoclonal antibodies reactive with two other surface proteins of 110 and 32 kilodaltons also blocked attachment. Surprisingly, monoclonal antibodies specific for M. pneumoniae lipids (J. Morrison-Plummer, D. H. Jones, and J. B. Baseman, J. Immunol. Methods 64:165-178, 1983) enhanced mycoplasma-erythrocyte binding. All antibodies examined had no effect on thymidine incorporation by M. pneumoniae.

Using an immunological probe, we have determined that the synthesis of the Bacillus subtlis RNA polymerase promoter specificity determinant (729 is a developmentally regulated event. a29 is absent from vegetatively growing cells but is abundant in sporulating cells for a restricted (2-h) period during differentiation (hour 2 to hour 4 into the sporeforming process). The narrowness of this period suggests that 2 is a regulatory factor that directs the transcription of a subpopulation of genes at a precise, intermediate stage of spore formation. This view predicts that a29 should be dispensable for early sporulation events. We verified this prediction by an analysis of 29 accumulation in mutants that are blocked at different stages of sporulation in which we show that cells can advance to at least an intermediate point in development (stage III) in the absence of detectable 29. Lastly, our anti-o29 antibody probe detected a second, previously unrecognized protein in Bacilus cell extracts that may be a precursor to a29. This protein, P31 (molecular weight, 31,000) is synthesized earlier in sporulation than is oJ29. It has a peptide profile that is similar to (729 and is present in all Bacillus subtilis Spo-mutants that were tested and found to still be able to accumulate a29. Endospore formation in Bacillus subtilis is a simple form of cellular differentiation. In response to nutrient deprivation, the bacterium undergoes a sequential alteration in morphology and physiology, ultimately transforming itself into a new cell form, the dormant spore. The mechanisms by which sporulation genes are activated in the correct sequence to drive the program of differentiation properly are unknown. Although there are over 30 genetic loci whose products are required for successful endospore development, no regulatory factor has as yet been identified as the product of any of these genes (18, 22, 23). Biochemical studies, in which cloned segments of the B. subtilis chromosome were used as templates for in vitro transcription, have implicated RNA polymerase as a possible regulatory molecule in B. subtilis gene control. Five forms of B. subtilis RNA polymerase have been isolated which transcribe unique populations of cloned vegetative and sporulation genes in vitro. Each enzyme has the subunit composition of the core RNA polymerase (g13'ci2) plus an additional protein (sigma factor) of 55,000 (or), 37,000 (a37), 32,000 (r 2), 29,000 (J29), or 28,000 (Cr28) daltons that is responsible for the in vitro promoter specificity of the resulting holoenzyme. cr55 is the predominant sigma factor that is found in association with the RNA polymerase isolated from vegetatively growing cells (26). The-r55_ containing RNA polymerase enzyme complex (E-a55) is likely to direct most of the RNA synthesis during vegetative growth. (X37, cr32 and o28 are also found in association with vegetative cell RNA polymerase; however, holoenzymes carrying these subunits compose only a minor fraction of the extractable polymerase population (9, 14, 31). In vitro, E-...

Filamentous structures designated Triton shells were obtained from virulent Mycoplasma pneumoniae by treatment with Triton X-100. Monoclonal antibodies directed against M. pneumoniae were used in conjunction with radioimmunoprecipitation and Western blotting to detect immunologically reactive polypeptides in Triton shells. The major adhesin, protein P1, was associated with these structures.

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