Proteins P24 and P41 function in the regulation of terminal-organelle development and gliding motility in Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae is a major cause of bronchitis and atypical pneumonia in humans. This cell wall-less bacterium has a complex terminal organelle that functions in cytadherence and gliding motility. The gliding mechanism is unknown but is coordinated with terminal-organelle development during cell division. Disruption of M. pneumoniae open reading frame MPN311 results in loss of protein P41 and downstream gene product P24. P41 localizes to the base of the terminal organelle and is required to anchor the terminal organelle to the cell body, but during cell division, MPN311 insertion mutants also fail to properly regulate nascent terminal-organelle development spatially or gliding activity temporally. We measured gliding velocity and frequency and used fluorescent protein fusions and time-lapse imaging to assess the roles of P41 and P24 individually in terminal-organelle development and gliding function. P41 was necessary for normal gliding velocity and proper spatial positioning of new terminal organelles, while P24 was required for gliding frequency and new terminal-organelle formation at wild-type rates. However, P41 was essential for P24 function, and in the absence of P41, P24 exhibited a dynamic localization pattern. Finally, protein P28 requires P41 for stability, but analysis of a P28(-) mutant established that the MPN311 mutant phenotype was not a function of loss of P28.

FIG. 1.

Organization of the P65 operon and recombinant P24 and P41 alleles thereof. (A) The gene products are indicated below each open reading frame of the P65 operon (3, 17). The horizontal arrow indicates the promoter for the operon, while transposon insertions corresponding to residues 22 and 161 of P41 are shown as inverted solid triangles. P28 is a product of internal translation initiation of the MPN310 transcript in the same reading frame as HMW2; antibodies directed against the C-terminal domain of HMW2 recognize P28 (4). The bracket above MPN310 indicates a region deleted in P28⁻ mutant C1R1 (1, 4). (B) Recombinant MPN311 and MPN312 alleles with or without translational fusion to the EYFP gene were engineered in transposon vector Tn4001cat (7). B, BamHI; E, EcoRI; N, NcoI; R, BsrGI; S, StuI.

FIG. 2.

Western immunoblot analysis of recombinant P41 and P24. M. pneumoniae strains and recombinant alleles they carry are indicated across the top. Protein size standards are shown to the left, with specific protein bands labeled to the right. P1 was included as an internal control. Protein profiles are representative of multiple filter clones of transformants of MPN311-22 and MPN311-161 (TOD). Horizontal lines indicate where the membrane was cut prior to probing with primary antisera. WT, wild type.

FIG. 3.

Role of P24 and P41 in gliding motility parameters. Mean gliding frequency and velocity were determined for at least 250 cells of the indicated strains. Gliding velocities were calculated as the total distance traveled by a cell divided by total time of the field interval minus the amount of time spent in resting periods (i.e., corrected gliding velocity, as described in detail previously [9]). Gliding frequencies were calculated as the percentage of cells exhibiting gliding in each observation interval (11). Means and standard errors were determined from clonal transformants of MPN311-22 and MPN311-161 mutants (TOD). Error bars, standard errors of the means. WT, wild type.

FIG. 4.

Terminal-organelle location in P41⁻ and P24⁻ mutants. (A) Localization of terminal organelles in the indicated M. pneumoniae strains by using P30-YFP as a marker. P41 was required for terminal-organelle localization predominantly at a cell pole (polar) as in the wild type (WT) (8), rather than at sites along the cell body (lateral) as described previously for the parental P41⁻/P24⁻ TOD mutants (10). (B) Images from time-lapse phase-contrast/fluorescence microscopy over 2-h intervals were used to establish the relative locations of newly synthesized terminal organelles. In the presence of P41, new terminal organelles formed primarily adjacent to an existing structure at one cell pole, as described previously (8). In contrast, in the absence of P41, new terminal organelles frequently appeared at lateral sites as defined above and not adjacent to an existing structure. (C) The frequency of terminal-organelle development was determined based on the percentage of cells forming a new terminal organelle over 2-h intervals for the indicated strains, from images obtained by time-lapse phase-contrast/fluorescence microscopy. Error bars, standard errors of the means.

FIG. 5.

Localization of P41 and P24 relative to P30 in wild-type (WT) and mutant M. pneumoniae. The positions of YFP-P24 and YFP-P41 were analyzed relative to P30-CFP as a terminal-organelle marker by using phase-contrast/fluorescence microscopy in the absence of P24 (A and B) or P41 (C and D). (A) P30-CFP (top panels), YFP-P41 (middle panels), and merged fluorescence images (bottom panels). In the absence of P24 there was no qualitative difference in the relative positions of P41 and P30 compared to the wild type, with some cells having an unpaired P41 focus proximal to a paired P41/P30 focus, as expected (8) (white arrowheads). (B) Quantitation of the relative positions of P41 and P30 in the absence of P24 (n > 50). Top panel, P30-CFP distribution in the absence of P24; bottom panel, P41 distribution in the absence of P24. (C) P30-CFP (top panels), YFP-P24 (middle panels), and merged fluorescence images (bottom panels). In the absence of P41, detached terminal organelles containing P30-CFP were common, as expected (10) (top and bottom panels, green arrowheads), although many had little detectable P24. YFP-P24 foci were distributed along the length of the mycoplasma cell, often unpaired with an obvious P30 (yellow arrowheads). Blue arrowheads, P30 foci paired with P24. (D) Quantitation of the relative positions of P24 and P30 in the absence of P41 (n > 50). Top panel, P30 distribution in the absence of P41 (10); bottom panel, P24 distribution in the absence of P41. Scale bars, 1 μm (A) and 2 μm (C). Error bars, standard errors of the means.

FIG. 6.

Time-lapse analysis of P24 localization in the absence of P41. Top panels, phase-contrast images; middle panels, YFP-P24 fluorescence images; bottom panels, three-dimensional representation of fluorescence intensities from the middle panels. Time points for time-lapse analysis are given at the top. At 0 min, eight major YFP-P24 foci were observed (numbered and yellow arrowheads). At 10 min, one focus had appeared to increase in intensity (red numbers and arrowheads) and seven foci to decrease in intensity (white numbers and arrowheads), while three new foci had emerged (green numbers and arrowheads). By 20 min, two foci had increased in intensity (red arrowheads) and nine decreased in intensity (white arrowheads), while two new foci had emerged (green arrowheads). Bar, 1 μm.