Identification and complementation of frameshift mutations associated with loss of cytadherence in Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae cytadherence is mediated by a specialized, polar attachment organelle. Certain spontaneously arising cytadherence mutants (designated class I) lack HMW2, fail to localize the adhesin protein P1 to the attachment organelle, and exhibit accelerated turnover of proteins HMW1, HMW3, and P65. Insertional inactivation of hmw2 by Tn4001 results in a phenotype nearly identical to that of the class I mutants, suggesting that the latter may result from a defect in hmw2. In this study, the recombinant wild-type hmw2 allele successfully complemented a class I mutant when introduced by transposon delivery. Synthesis of recombinant HMW2 at wild-type levels resulted in reacquisition of hemadsorption and normal levels of HMW1, HMW3, and P65. Low-level production of HMW2 in some transformants resulted in only an intermediate capacity to hemadsorb. Furthermore, full restoration of HMW1 and P65, but not that of HMW3, was directly proportional to the amount of recombinant HMW2 produced, reflecting the importance of proper stoichiometry for certain cytadherence-associated proteins. The recombinant class I hmw2 allele did not restore cytadherence, consistent with a defect in hmw2 in this mutant. A frameshift was discovered in different oligoadenine tracts in hmw2 from two independent class I mutants. Finally, protein P28 is thought to be the product of internal translation initiation in hmw2. A transposon excision-deletion mutant produced a truncated HMW2 but no P28, consistent with this conclusion. However, this deletion mutant was hemadsorption positive, indicating that P28 may not be required for cytadherence.

FIG. 1

(A) Restriction map of the M. pneumoniae P65 operon (crl) region. Open reading frames encoding P65, HMW2, P41, and P24 (orfp65, hmw2, orfp41, and orfp24, respectively) are indicated. The EcoRI fragment used in Southern blot hybridizations is indicated by the bar above the map, and the arrow denotes the predicted transcriptional initiation site based on primer extension studies (21). (B) The recombinant Tn4001mod transposons engineered to contain the PstI-BglII or BstEII-BglII fragment from the P65 operon in the SmaI site of IS256L. Plasmids containing the various recombinant transposons are indicated on the right, and the arrows above each indicate the orientation of the cloned fragment, relative to those of the P_in and P_out promoters. Bs, BstEII; Bg, BglII; E, EcoRI; P, PstI; S, SmaI; Sc, ScaI.

FIG. 2

Western immunoblot analysis of M. pneumoniae mutant I-2 transformants for production of HMW2 (A) and P28 (B). Mycoplasma protein (20 μg per well) was separated by SDS-PAGE (4.5% [A] or 12% [B] polyacrylamide separating gels), blotted to nitrocellulose, and probed with serum against the C-terminal region of HMW2 (21) at a 1:1,000 dilution. Lanes: WT, wild-type M. pneumoniae; I-2, mutant I-2 transformed with pISM2062 (vector control); I-2/HMW2 and I-2/P65-HMW2, I-2 transformants with pKV134 and pKV136, respectively. HMW2 and P28 are indicated by arrowheads, and protein size standards are shown on the left in kilodaltons.

FIG. 3

Western immunoblot analysis of M. pneumoniae mutant I-2 transformants for production of HMW1 (A) and HMW3 (B). Approximately equal amounts of total protein were loaded per well, electrophoresed on a 4.5% polyacrylamide separating gel, blotted to nitrocellulose, and probed with specific antiserum to HMW1 or HMW3 (34, 35). The lanes are the same as indicated in the legend to Fig. 2. HMW1 and HMW3 are indicated by arrowheads, and protein size standards are shown to the left in kilodaltons.

FIG. 4

Western immunoblot analysis of M. pneumoniae mutant I-2 transformants for production of P65 (A), P41 (B), or P24 (C). Approximately equal amounts of total protein were loaded per well, electrophoresed on a 12% polyacrylamide separating gel, blotted to nitrocellulose, and probed with specific antiserum to P65, P41, or P24 (21, 29). The lanes are the same as indicated in the legend to Fig. 2. P65, P41, and P24 are indicated by arrowheads, and protein size standards are shown to the left in kilodaltons. Previous studies indicated that P24 migrates anonymously (21), but in the current study P24 migrated at the expected size.

FIG. 5

Identification of frameshift mutations in class I mutants I-2 and I-10. The nucleotide (nt) positions of the start and end of hmw2 and of the first adenine nucleotides of the six oligo(A) tracks (filled arrows) are given according to the numbering of the P65 operon (21). The corresponding nucleotide positions in the M. pneumoniae genome (14) are given in brackets. The extra adenine nucleotide in each isolate is shown in bold lettering. WT, wild type.

FIG. 6

Western immunoblot of M. pneumoniae wild type (WT), mutant I-2, and C1R1. Approximately equal amounts of mycoplasma total protein were electrophoresed on an SDS–5 to 15% polyacrylamide gradient gel, transferred onto nitrocellulose, and probed with antibody against the C-terminal region of HMW2 (1:1,000 dilution). HMW2, truncated HMW2 (ΔHMW2), and P28 are indicated by arrowheads; size standards are indicated in kilodaltons.