Insights into the function of Mycoplasma pneumoniae protein P30 from orthologous gene replacement

Abstract

The attachment organelles of bacterial species belonging to the Mycoplasma pneumoniae phylogenetic cluster are required for host cytadherence, gliding motility and virulence. Despite being closely related, these bacteria possess distinct cellular morphologies and gliding characteristics. The molecular mechanisms for most attachment organelle phenotypes, including shape and ability to power motility, are obscure. The attachment organelle-associated P30 protein of M. pneumoniae is implicated in both adherence and motility, with mutations negatively impacting cell morphology, adherence, gliding and virulence. To test whether the P30 alleles of different mycoplasma species confer species-specific attachment organelle properties, we created an M. pneumoniae strain in which the Mycoplasma genitalium P30 orthologue, P32, was substituted for the native P30. Selected clones were visualized by scanning electron microscopy to assess morphology and by indirect immunofluorescence microscopy to localize P32. Cytadherence ability and gliding motility were assessed by haemadsorption assay and phase-contrast microcinematography, respectively. Cell and attachment organelle morphologies were indistinguishable from wild-type M. pneumoniae as well as M. pneumoniae II-3 expressing a C-terminally 6×His-tagged P30 construct. P32 was localized to the tip of the attachment organelle of transformant cells. Although a specific role for P30 in species-specific phenotypes was not identified, this first test of orthologous gene replacement in different mycoplasma species demonstrates that the differences in the M. pneumoniae and M. genitalium proteins contribute little if anything to the different attachment organelle phenotypes between these species.

Fig. 1.

Comparison of P30 orthologues from M. pneumoniae strain M129 and M. genitalium strain G37. Shaded amino acids are conserved between the two proteins. Amino acid sequences were aligned using clustal x software.

Fig. 2.

Constructs generated for this study. PCR was used to amplify the genes of interest from genomic DNA of either wild-type M. pneumoniae strain M129 or wild-type M. genitalium strain G37. Amplicons were cloned in the TA cloning vector pCR2.1, sequenced and subcloned into the Tn4001-containing vector pMT85 for delivery of the constructs into M. pneumoniae II-3 or M. pneumoniae M129. Constructs in pOO12 (a), pOO17 (b), pOO30 (c) and pOO24 (d). The construct in pOO37 is a derivative of that which is in pOO24; it contains the original MPN453 translational stop codon (the position of which is indicated by the vertical arrow) and does not include the 6×His epitope tag.

Fig. 3.

Immunoblot confirmation that the 6×His antibody does not cross-react with any proteins in non-transformed M. pneumoniae. Wild-type M. pneumoniae strain M129 (WT) total cell lysate was probed with the 6×-His antibody alongside the transformant M. pneumoniae 24-A. Molecular mass markers are indicated to the left.

Fig. 4.

Immunoblot analysis for the visualization of P30, P30_His, P32_His and P65. Whole-cell lysates of wild-type M. pneumoniae strain M129 (WT), M. pneumoniae II-3 and the transformant strains M. pneumoniae 30-C, 12-A, 17-B, 24-A and 37-D were probed with anti-P30 (a), anti-6×His (b) or anti-P65 (c) antibodies. Molecular mass markers are indicated to the left.

Fig. 5.

Morphology of strains used in this assay. SEM reveals that transformants containing either P30 or P32, with or without the C-terminal 6×His tag, are indistinguishable from wild-type M. pneumoniae M129 (WT). Strains are indicated in the upper left of each panel, with G37 indicating M. genitalium. White arrowheads indicate attachment organelles and the black arrowhead indicates a trailing filament. Bar, 1 µm.

Fig. 6.

P30_His and P32_His localize to the attachment organelle tip, the site of localization of native P30. Whole cells grown on glass coverslips (red channel) were probed with a monoclonal anti-6×His antibody and an FITC-conjugated secondary antibody (green channel). Strains are indicated in the upper left of each panel, except that NC indicates wild-type (WT) cells processed with only secondary antibody as a negative control. Bar, 2 µm.