In this study we demonstrated that the class A PBP PonA2 is essential for the survival of M. smegmatis under nonreplicating conditions. We also discovered a previously unrecognized class A PBP, PonA3, which is present in only a subset of environmental mycobacteria. Our results show that the PonA3 function is dispensable under the conditions that we used to identify phenotypes for the ΔponA2 mutant. The ponA3 gene is probably not expressed from the chromosome at a high level since we could detect the PonA3 protein in PBP assays only when the gene was expressed from a constitutive promoter on a multicopy plasmid. Under these conditions, the ponA3 gene was able to complement most (but not all) of the phenotypes of the ΔponA2 mutant. We postulate that PonA3 is a paralog of PonA2 that provides adaptive functions in the environmental saprophytic mycobacteria.
Our analysis revealed a proline-rich domain located at the C termini of all three class A PBPs and another PRR located at the N terminus of PonA1. These domains contain a PxxP core motif that is found primarily in proteins from eukaryotic organisms but have been found to be present in a large number of M. tuberculosis
). This motif is commonly embedded in polyproline tracts that constitute a helix with a hydrophobic surface for protein-protein interactions (24
). We performed comparative searches with the GenBank database and found that the class A PBPs of other actinobacteria belonging to the same family as Mycobacterium
, such as Nocardia farcinica
, Gordonia bronchialis
, and Rhodococcus erythropolis
, also have termini with similar proline-rich regions. This phylogenetic association suggests that there is a functional relationship between these domains and the cell envelope structure shared by these bacteria.
Proline-rich domains with PxxP motifs are found in many eukaryotic proteins and have been shown to interact with Src homology 3 (SH3) domains (24
). Proteins with SH3-like domains (SH3b) are present in bacteria and are involved in peptidoglycan degradation (33
), This suggests that the PRR domains of the PonA proteins might interact with PG disassembly proteins. We have examined the sequences of various mycobacterial proteins involved in PG metabolism (the hydrolase CwlM [12
], the peptidase RipA [21
], and the resuscitation promoting factor [Rpf] proteins [44
]) and found no apparent SH3b domains in any of them. In fact, there do not appear to be any mycobacterial proteins with recognizable SH3b domains. However, the mycobacterial SH3b-like sequences may have diverged significantly from the corresponding sequences of other bacteria because of the high G+C content of their genomes, so the possibility that SH3b-bearing proteins are present in mycobacteria cannot be excluded. We should also consider the possibility that the hydrophobic PRR domains might interact with each other. Thus, we propose that the proline-rich domains could be involved in modulating the interaction between the PBPs and proteins involved in PG turnover. Alternatively, since polyproline tracts can function as hydrogen bond acceptors (24
), these domains could allow the PBPs to interact with cell envelope precursors to coordinate assembly of the PG with assembly of other components of the mycolyl-arabinogalactan-peptidoglycan (mAGP) cell wall skeleton (35
). These functions do not have to be mutually exclusive.
The PonA2 and PonA3 proteins also share a PASTA domain, which was originally discovered in an analysis of the crystal structure of the PBP2x protein of Streptococcus pneumoniae
, and it is now known that single or multiple PASTA domains are present in a variety of proteins in Gram-positive bacteria (20
). The PASTA domain is thought to interact with the terminal d
-Ala of unlinked PG peptides (23
). This domain occurs four times in the C terminus of PknB, a mycobacterial serine/threonine kinase that is required for the phosphorylation of PbpA, resulting in movement of PbpA to the cell septum for division (11
). It has been proposed that the function of a PASTA domain is to guide proteins to the site of cell division where the concentration of PG pentapeptide precursors is highest. Alternatively, duplicated PASTA domains in the same protein may sense different types of PG peptides, allowing the protein to react to different modifications of the cell wall precursors (23
). Since PonA2 and PonA3 each have a single PASTA domain, we propose that this domain may allow these proteins to detect PG pentapeptide precursors as a mechanism to sense whether the cell is dividing. This would be consistent with results presented here that show that PonA2 is involved in survival of the cell under nonreplicating conditions and that PonA3 can substitute for PonA2 during stationary-phase adaptation. The idea that the PonA2 function is related to cell cycle progression is supported by the observation that the number of ponA2
transcripts decreases in M. tuberculosis
cells treated with antibiotics that dysregulate cell division (56
). It is unclear why PonA3 cannot rescue the anaerobic phenotype, but this finding might indicate that cessation of cell growth as a result of anaerobic conditions involves more complicated signals. Mutagenesis, deletion, and domain-swapping experiments with the PonA2 and PonA3 PRR and PASTA domains should provide additional information about the roles of these domains.
Our results show that PonA2 functions in adaptation of cultures to stationary phase, during which many of the cells do not replicate, and to anaerobic conditions, during which none of the cells replicate. These results are in agreement with previous work that identified an M. smegmatis ponA2
transposon mutant in a screen for mutants unable to survive starvation conditions (25
). In that study, the survival defect manifested over several months, as opposed to the few weeks observed in our study. The difference is probably the result of our mutant having a lysine auxotrophic phenotype, since we showed that a prototrophic derivative took approximately seven times longer to exhibit a survival defect in stationary phase than the auxotrophic strain. We surmise that the prototrophic strain probably experienced a different type of starvation, perhaps carbon limitation. The results of our comparison of the auxotrophic and prototrophic strains suggest that amino acid starvation plays a role in this adaptation, indicating that the stringent response potentially contributes to this phenomenon.
The mechanism of PonA2 in adaptation to stationary phase and anaerobiosis is not entirely clear. A recent report demonstrated that a Vibrio cholerae
PBP1A mutant quickly became spherical in stationary phase as a result of d
-amino acid efflux into the medium (30
). It was shown that PG metabolism is regulated by the endogenous production of specific d
-amino acids by a novel d
-amino acid racemase during stationary phase (30
). We do not know if the same phenomenon can occur in mycobacteria, but given the widespread alterations in the phenotype of the ΔponA2
mutant, the length of time required for the phenotype to occur, and the lack of any additional amino acid racemases in the M. smegmatis
genome, we think that d
-amino acid sensitivity is probably not a factor that contributes to the stationary-phase phenotype of this mutant. Other workers have proposed that PonA2 may be one of the ld
-transpeptidases responsible for the synthesis of novel DAP-DAP (3-3) PG cross-links that have been implicated in adaptation to stationary phase (17
). However, more recent work on a class of novel ld
-transpeptidases involved in PG assembly and modification that were first discovered in Enterococcus faecium
and are now known to be present in a variety of bacteria, including mycobacteria, has challenged this idea (31
). Determining the transpeptidase activity of the PonA2 protein and the cross-link composition of the PG of the ΔponA2
mutant should provide important information to help us understand how this PBP functions in PG assembly.
In addition to the stationary-phase survival defect of the ΔponA2 mutant, we also showed that the surface of the mutant is different than that of the wild type in stationary phase. Our data from the lysozyme aggregation experiments suggest that the net negative charge density of M. smegmatis cells increases in stationary phase but that this does not happen in the ΔponA2 mutant. To our knowledge, this is the first indication that the electrostatic nature of the surface of a mycobacterial species differs depending on the growth phase. The increase in the net negative charge density could result from the shedding of positively charged molecules, such as basic proteins, or from an increase in the expression of negatively charged glycolipids in the outer cell envelope as the wild-type cells transition to stationary phase. A decrease in the negative charge density of the ΔponA2 mutant could result from an increase in the number of positively charged molecules that might shield the surface or from shedding of the negatively charged species in the outer envelope as a result of the dysregulation of envelope assembly. The latter explanation is consistent with the substantial disorganization of the cell envelope structure indicated by the swollen cells seen in the TEM images of ΔponA2 cells taken from stationary-phase cultures.
The only phenotype that we examined that was not related to nonreplicative conditions was the antibiotic susceptibility of exponentially growing cultures. The loss of a PBP usually results in an increase in the susceptibility to β-lactam antibiotics. However, we found that the ΔponA2
mutant has decreased susceptibility to both penicillin- and cephalosporin-type β-lactams. This suggests that this mutant may somehow compensate for the loss of PonA2 by producing additional cross-links in the PG so that a higher concentration of antibiotics is required to affect the cell. Structural analysis of PG purified from the mutant should help us interpret the β-lactam antibiotic phenotype. The susceptibility of the mutant to hydrophilic antibiotics, such as isonizaid and ethambutol, is not changed, but its susceptibility to the hydrophobic antibiotic rifampin is increased, suggesting that loss of PonA2 may somehow affect the lipid-rich regions of the cell envelope. Our previously identified M. tuberculosis ponA2
transposon mutant displayed increased susceptibility to β-lactam antibiotics and no change in susceptibility to rifampin, suggesting that loss of PonA2 in M. tuberculosis
has different effects (14
). However, the M. tuberculosis
mutant was isolated from a library specifically screened for increased susceptibility to β-lactam antibiotics, so we cannot rule out the possibility that there is a suppressor mutation in this strain. We are currently reconstructing the M. tuberculosis ponA2
mutant by using allelic exchange to resolve this issue and to test the ability of the mutant to survive under stationary-phase and anaerobic conditions.