Functional analysis of the Mycoplasma genitalium MG312 protein reveals a specific requirement of the MG312 N-terminal domain for gliding motility

Abstract

The human pathogen Mycoplasma genitalium is known to mediate cell adhesion to target cells by the attachment organelle, a complex structure also implicated in gliding motility. The gliding mechanism of M. genitalium cells is completely unknown, but recent studies have begun to elucidate the components of the gliding machinery. We report the study of MG312, a cytadherence-related protein containing in the N terminus a box enriched in aromatic and glycine residues (EAGR), which is also exclusively found in MG200 and MG386 gliding motility proteins. Characterization of an MG_312 deletion mutant obtained by homologous recombination has revealed that the MG312 protein is required for the assembly of the M. genitalium terminal organelle. This finding is consistent with the intermediate-cytadherence phenotype and the complete absence of gliding motility exhibited by this mutant. Reintroduction of several MG_312 deletion derivatives into the MG_312 null mutant allowed us to identify two separate functional domains: an N-terminal domain implicated in gliding motility and a C-terminal domain involved in cytadherence and terminal organelle assembly functions. In addition, our results also provide evidence that the EAGR box has a specific contribution to mycoplasma cell motion. Finally, the presence of a conserved ATP binding site known as a Walker A box in the MG312 N-terminal region suggests that this structural protein could also play an active function in the gliding mechanism.

FIG. 1.

Homologous recombination at the MG_312 gene. (A) Schematic representation of a double-crossover recombination between pΔmg312 and the MG_312 gene and gene replacement by the tetM438 selectable marker. Hatched boxes represent flanking regions of the MG_312 gene in suicide plasmid pΔmg312. Black, tetM438 selectable marker. Sizes of fragments resulting from the HindIII (H) digestion of DNAs from the WT strain and several pΔmg312 transformants are also indicated. (B) Southern blot hybridization profiles of genomic DNA from the WT strain and pΔmg312 transformants using the MG_312 flanking regions as a probe.

FIG. 2.

Protein profile of the WT strain and ΔMG_312 and ΔMG_312 derivative mutants. The protein profile was analyzed by 8% SDS-PAGE (A) and Western immunoblotting using anti-HMW1, anti-HMW3, and anti-MG217 antibodies (B). Arrowheads show expression of the different ΔMG_312 derivatives.

FIG. 3.

SEM analyses of the WT strain and ΔMG_312 and ΔMG_312 derivative mutants. All pictures are shown at the same magnification.

FIG. 4.

Colony morphology of the WT and ΔMG_312 and ΔMG_312 derivative mutants. Cells from the WT strain and ΔMG_312 and ΔMG_312 derivative mutants were attached to cell culture dishes and covered with SP-4 medium containing 0.5% low-melting-point agarose. All pictures are shown at the same magnification.

FIG. 5.

Schematic representation of the modular structure of the MG312 protein (A) and the construction designed to obtain the MG_312 deletion derivatives (B). The EAGR box is indicated in black. The Walker A box is underlined. Hatched boxes represent the two large identical repeats found in the central domain of MG312 protein; the small repeating motifs are indicated by gray boxes. Amino acid sequences corresponding to the EAGR box and the small repeating motifs are also indicated. Regions deleted in MG_312 deletion derivatives are represented by discontinuous lines.

FIG. 6.

PCR amplification from DNA of the M. genitalium WT strain and the ΔMG_312 mutant. The primers 5′BEKOMG312 (P1) and 5′BDKOMG312 (P2) were used in both PCR amplifications. In addition to the expected 3.4-kb band, corresponding to the MG_312 gene product, we also detected a 2.5-kb product, suggesting the presence within the WT population of specific MG_312 deletion mutants. As a control, a single 2.2-kb product was amplified from ΔMG_312 mutant DNA.

FIG. 7.

Partial time-dependent surface proteolysis of M. genitalium WT strain. Intact cells were treated with proteinase K, and reactions were stopped by adding 1 mM PMSF after 5, 10, 15, 30, 45, and 60 min. Protein profiles from cells that underwent proteolysis were analyzed by Western blotting using anti-P140 (A) and the HMW1 antiserum (B). C1 corresponds to the protein profile of untreated cells, and C2 corresponds to the protein profile resulting when PMSF was added to the cells prior to proteinase K. M, apparent molecular mass prestained marker (Invitrogen).