Characterization of serine hydroxymethyltransferase GlyA as a potential source of D-alanine in Chlamydia pneumoniae

Abstract

For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin.

Peptidoglycan in obligate intracellular bacteria

“…Whilst these are present in most of the organisms possessing classical peptidoglycan, their presence in intermediate peptidoglycan species is inconsistent. MetC has been shown to possess alternative alanine racemase activity in E. coli (Kang et al ., ), and GlyA has been shown to be the alanine racemase in Chlamydia pneumoniae (De Benedetti et al ., ), suggesting that the proteins used for racemase activity need not be strictly conserved in these pathways. It is also possible that Ddl, MurF and MurD may have different amino acid specificities in different organisms, and this can ultimately only be resolved by determining the structure of purified peptidoglycan from specific bacterial species.…”

Building peptidoglycan inside eukaryotic cells: A view from symbiotic and pathogenic bacteria

“…Another interesting feature of Chlamydiales is the conservation of the entire pathway of lipid II except for the Glu racemase (MurI) and with GlyA proposed to be the Ala racemase (De Benedetti et al, ; Jacquier, Viollier, et al, ). Until recently, there was no formal evidence of the presence of PG in intracellular Chlamydiae despite their susceptibility for beta‐lactams, becoming the presence of an intact lipid II route enigmatic (Henrichfreise et al, ).…”

Simkania negevensis, an Example of the Diversity of the Antimicrobial Susceptibility Pattern among Chlamydiales

“…It is not known whether the serine hydroxymethyltransferase GlyA encoded in the S. negevensis genome could compensate for the absence of Alr, as described for Chlamydiaceae (20). …”