A role for the class A penicillin-binding protein PonA2 in the survival of Mycobacterium smegmatis under conditions of nonreplication

Abstract

Class A penicillin-binding proteins (PBPs) are large, bifunctional proteins that are responsible for glycan chain assembly and peptide cross-linking of bacterial peptidoglycan. Bacteria in the genus Mycobacterium have been reported to have only two class A PBPs, PonA1 and PonA2, that are encoded in their genomes. We report here that the genomes of Mycobacterium smegmatis and other soil mycobacteria contain an additional gene encoding a third class A penicillin-binding protein, PonA3, which is a paralog of PonA2. Both the PonA2 and PonA3 proteins contain a penicillin-binding protein and serine/threonine protein kinase-associated (PASTA) domain that we propose may be involved in sensing the cell cycle and a C-terminal proline-rich region (PRR) that may have a role in protein-protein or protein-carbohydrate interactions. We show here that an M. smegmatis Delta ponA2 mutant has an unusual antibiotic susceptibility profile, exhibits a spherical morphology and an altered cell surface in stationary phase, and is defective for stationary-phase survival and recovery from anaerobic culture. In contrast, a Delta ponA3 mutant has no discernible phenotype under laboratory conditions. We demonstrate that PonA2 and PonA3 can bind penicillin and that PonA3 can partially substitute for PonA2 when ponA3 is expressed from a constitutive promoter on a multicopy plasmid. Our studies suggest that PonA2 is involved in adaptation to periods of nonreplication in response to starvation or anaerobiosis and that PonA3 may have a similar role. However, the regulation of PonA3 is likely different, suggesting that its importance could be related to stresses encountered in the environmental niches occupied by M. smegmatis and other soil-dwelling mycobacteria.

FIG. 1.

Comparison of the class A PBPs of M. smegmatis. (A) Domain organization. The calculated molecular mass of each of the three proteins is indicated below the designation. The amino acids at the beginning and the end of each domain are shown above the domain. TM, transmembrane (as determined by TMHMM); TG (n-PB), transglycosylase; TP (PB), transpeptidase; PASTA, PBP and serine/threonine kinase (STK)-associated domain; PRR-C, C-terminal proline-rich region; PRR-N, N-terminal proline-rich region. (B) Levels of identity (ID) and similarity (SIM) (identical and conserved) for the M. smegmatis PonA3 protein and the PonA1 and PonA2 proteins. FL, full length. The various annotations of ponA1 of M. tuberculosis and M. smegmatis in the GenBank database do not agree with each other. The original annotation for the M. smegmatis gene (GenBank accession no. AAG13121) is too short, and the translation product starts in the middle of the transmembrane domain. This annotation was apparently used for subsequent assignment of the open reading frame (ORF) in M. tuberculosis. The ORF was changed in the M. smegmatis genome project (accession no. ABK70639), and a new start codon was located 81 codons upstream of the start site in the original annotation, which allowed identification of the entire transmembrane region. We reannotated the M. smegmatis ponA1 gene and placed the start site of the ORF 42 codons upstream of the revised annotation in the M. smegmatis genome project. This revealed all of the C-terminal proline-rich regions in the PonA1 proteins of both mycobacterial species and showed that the sizes of all of the class A PBPs are similar.

FIG. 2.

Clustal W alignments of domains in the class A PBPs of M. smegmatis and M. tuberculosis. (A) PASTA domains of PonA2 and PonA3, showing the three β-strands and the single α-helix that make up the PASTA fold. Bold type indicates residues that are highly conserved in many PBPs and serine/threonine kinases. Note the proximity of the PRR-C domain, which is underlined. (B) PRR-C domains of all three class A proteins. Bold type indicates the proline residues, and the PxxP core motifs are underlined. (C) PRR-N domains of PonA1 proteins. Bold type indicates the proline residues, and the PxxP motifs are underlined. Asterisks indicate identical residues, while colons and periods indicate conserved and semiconserved residues, respectively.M.tb., M. tuberculosis; M.sm., M. smegmatis.

FIG. 3.

Penicillin (Bocillin FL) binding assays with purified M. smegmatis membranes showing PonA2 labeling. (A) Assay results for membranes prepared from wild-type strain PM1482 (ponA2⁺) mid-log-phase cultures (lanes 1 and 2) and stationary-phase cultures (lanes 5 and 6) or prepared from PM1536 ΔponA2 mid-log-phase cultures (lanes 3 and 4). Each strain was assayed using duplicate cultures. The ∼80-kDa protein present in all wild-type samples is indicated by an asterisk. (B) Membrane assays for two independent mid-log-phase cultures of the ΔponA2/pMV261 vector control strain (lanes 1 and 2) and stationary-phase (lanes 3 and 4) and mid-log-phase (lanes 5 and 6) cultures of the ΔponA2 mutant complemented with the wild-type ponA2 gene carried on a multicopy plasmid (PM1576 ΔponA2/ponA2⁺). The ∼80-kDa protein not present in the membranes prepared from the mutant is present in the membranes prepared from the complemented strain. (C) Results for membranes from the ΔponA2 mutant with the plasmid bearing the His₆-PonA2 gene (lane 1), the positive complementation strain PM1576 ΔponA2/ponA2⁺ (lane 2), and the wild-type strain PM1482 (lane 3).

FIG. 4.

Bocillin FL binding assays with purified M. smegmatis membranes, showing PonA3 labeling. The membrane profile of the PM1864 (ΔponA3) strain (lane 3) is the same as that of wild-type strain PM1482 (ponA2⁺ ponA3⁺) (lane 1). Deletion of ponA3 in the ΔponA2 background did not change the PBP profile compared to that of the ΔponA2 parent (compare lanes 2 and 4). However, overexpression of ponA3⁺ on a multicopy vector in the ΔponA2 mutant (lane 6) resulted in appearance of a new ∼80-kDa protein that was not present in the vector control strain (ΔponA2VC) (lane 5).

FIG. 5.

Survival of the M. smegmatis mutants in stationary phase. The results are expressed in CFU/ml. The values are the averages and standard deviations of duplicate determinations using two independent cultures. (A) ΔponA2 strains. Open diamonds, PM1536 (ΔponA2); open squares, PM1578 (ΔponA2 vector control); filled diamonds, PM1482 (ponA2⁺); filled squares, PM1576 (ΔponA2 ponA2⁺). *, P value for statistical analysis of the ΔponA2 mutant compared to the wild type and the ΔponA2 vector control and the ΔponA2 ponA2⁺ complemented strain. (B) ΔponA3 and ΔponA2 ΔponA3 strains. Filled diamonds, PM1482 (ponA2⁺ponA3⁺); open diamonds, PM1536 (ΔponA2); filled circles, PM1864 (ΔponA3); multiplication signs, PM1865 (ΔponA2 ΔponA3). *, P value for statistical analysis of the ΔponA2 mutant compared to the wild type and the ΔponA2 ΔponA3 mutant compared to the ΔponA3 mutant.

FIG. 6.

Light microscopy (A, B, D, and E) and TEM (C and F) of 8-day-old stationary-phase cultures demonstrating the loss of the characteristic bacillary shape of the ΔponA2 mutants. (A and C) PM1482 (ponA2⁺); (D and F) PM1536 (ΔponA2); (B) PM1576 (ΔponA2 ponA2⁺); (E) PM1578 (ΔponA2 vector control).

FIG. 7.

Lysozyme cellular aggregation assay: optical density scans over time for assays with wild-type strain PM1482 (asterisks), the complemented ΔponA2 mutant PM1576 (filled circles), and the ΔponA2 mutant PM1536 (filled triangles). (Inset) Suspensions after incubation at room temperature for 1 h. Tube 1, wild-type strain PM1482; tube 2, ΔponA2 mutant PM1536; tube 3, complemented ΔponA2 mutant strain PM1576.

FIG. 8.

Anaerobic survival of the ΔponA2, ΔponA3, and ΔponA2 ΔponA3 strains. The viable counts recovered after anaerobic incubation are expressed as log(N/N₀), where N₀ is the initial number of CFU and N is the number of CFU after 1 to 12 days of anaerobic incubation followed by 4 days of aerobic incubation. Filled diamonds, PM1482 (wild type); filled triangles, PM1864 (ΔponA3); filled circles, PM1536 (ΔponA2); multiplication signs, PM1865 (ΔponA2 ΔponA3). *, P value for statistical analysis of the ΔponA2 mutant compared to the wild type; **, P value for the comparison between the ΔponA2 ΔponA3 mutant and the ΔponA3 mutant.