HMW1 is required for stability and localization of HMW2 to the attachment organelle of Mycoplasma pneumoniae

Abstract

The cytoskeletal proteins HMW1 and HMW2 are components of the terminal organelle of the cell wall-less bacterium Mycoplasma pneumoniae. HMW1 is required for a tapered, filamentous morphology but exhibits accelerated turnover in the absence of HMW2. Here, we report that a reciprocal dependency exists between HMW1 and HMW2, with HMW2 subject to accelerated turnover with the loss of HMW1. Furthermore, the instability of HMW2 correlated with its failure to localize to the attachment organelle. The C-terminal domain of HMW1 is essential for both function and its accelerated turnover in the absence of HMW2. We constructed HMW1 deletion derivatives lacking portions of this domain and examined each for stability and function. The C-terminal 41 residues were particularly important for proper localization and function in cell morphology and P1 localization, but the entire C-terminal domain was required to stabilize HMW2. The significance of these findings in the context of attachment organelle assembly is considered.

FIG. 1.

Schematic representation of HMW1 and recombinant HMW1 constructs. (A) HMW1 with the sequence of the C-terminal 112 amino acids shown above and paired Ser residues underlined. Regions affected by deletion are represented by patterned boxes. The numbers above each correspond to the four paired Ser residues. Amino acid positions corresponding to each region are indicated below the diagram, and the relative position of the BamHI site is shown for reference (not to scale). (B) HMW1 deletion derivatives, with protein designation indicated to the right for each. (C and D) Schematic of relevant regions of plasmids pKV74 and pKV37 (not to scale). Restriction sites: E, EcoRI; B, BamHI; K, KpnI; Gm^r, gentamicin resistance gene.

FIG. 2.

(A) Western immunoblot analysis of HMW2 and P1 steady-state levels in wild-type and mutant M6 M. pneumoniae. Mycoplasma samples at the indicated protein concentrations were subjected to SDS-PAGE and Western immunoblotting with anti-HMW2 serum (1:1,000) and anti-P1 serum (1:1,000). Lanes: WT, 75, 25 or 12.5 μg of wild-type M. pneumoniae protein, as indicated; M6, 75 μg of protein from mutant M6; M6 + reHMW1, 75 μg of M6 transformant producing full-length recombinant HMW1. Arrowheads indicate HMW2 and P1, while the 200-kDa protein mass marker is shown to the left. (B) Pulse-chase analysis of HMW2 synthesis and turnover in wild-type (WT), mutant M6, and mutant I-2 M. pneumoniae. The arrowheads indicate HMW1, HMW2, pre-P1, and mature P1; time points are given at the top in hours.

FIG. 3.

Western immunoblot analysis of HMW2 steady-state levels in wild-type (WT) and mutant M6 M. pneumoniae and M6 producing the indicated HMW1 deletion derivatives (see Fig. 1). Mycoplasma samples were prepared as described above, with equal amounts of protein used for each sample. The arrowhead indicates HMW2.

FIG. 4.

Analysis of HMW2-GFP localization in M. pneumoniae mutants I-2 (A) and M6 (B). The left and right panels for each pair are images by merged phase-contrast and fluorescence microscopy, and fluorescence microscopy only, respectively. HMW2-GFP complements the hmw2 gene defect in mutant I-2 (5). Bar, 2 μm.

FIG. 5.

Western immunoblot analysis of the stability of HMW1 deletion derivatives in mutant M6 M. pneumoniae. Two independent transformants are shown for each HMW1 derivative. Equal amounts of protein were loaded per lane, separated by electrophoresis, transferred to nitrocellulose, and probed with anti-HMW1 serum (1:10,000). WT, untransformed wild-type M. pneumoniae; M6, untransformed mutant M6; M6 +, M6 M. pneumoniae transformants producing the indicated HMW1 derivative. The large arrowhead indicates HMW1, while smaller arrowheads indicate recombinant HMW1 derivatives.

FIG. 6.

Phase-contrast-immunofluorescence analysis of HMW1 localization in wild-type M. pneumoniae (A) and mutant M6 producing HMW1 Δ1-4 (B), HMW1 Δ4 (C), HMW1 Δ1 (D), or reHMW1 (E). For each pair, the merged immunofluorescence and phase-contrast images are shown on the left and the corresponding immunofluorescence images alone are shown on the right. Bar, 2.0 μm.

FIG. 7.

Phase-contrast-immunofluorescence analysis of P1 localization in wild-type M. pneumoniae (A), mutant M6 (B), and mutant M6 producing HMW1 Δ1-4 (C), HMW1 Δ4 (D), HMW1 Δ1 (E), or reHMW1 (F). For each pair of images, the merged phase-contrast and immunofluorescence images are shown on the left, and the corresponding immunofluorescence images alone are shown on the right. Bar, 2.0 μm.

FIG. 8.

Phase-contrast-immunofluorescence analysis of HMW2 localization in wild-type M. pneumoniae (A), mutant M6 (B), and M6 transformants producing HMW1 Δ1-4 (C), HMW1 Δ4 (D), HMW1 Δ1 (E), or reHMW1 (F). For each pair of images, the merged phase-contrast and immunofluorescence images are shown on the left and the corresponding immunofluorescence images alone are shown on the right. Bar, 1.0 μm.