A prototrophic suppressor of a D-glutamate auxotroph reveals a member of the periplasmic broad-spectrum racemase family (BsrF).

Unlabelled: Although bacterial peptidoglycan (PG) is highly conserved, some natural variations in PG biosynthesis and structure have evolved. Understanding the mechanisms and limits of such variation will inform our understanding of antibiotic resistance, innate immunity, and the evolution of bacteria. We have explored the constraints on PG evolution by blocking essential steps in PG biosynthesis in and then selecting mutants with restored prototrophy. Here, we attempted to select prototrophic suppressors of a D-glutamate auxotrophic mutant. No suppressors were isolated on unsupplemented lysogeny broth salts (LBS), despite plating >10 cells, nor were any suppressors generated through mutagenesis with ethyl methanesulfonate. A single suppressor was isolated on LBS supplemented with iso-D-gln, although the iso-D-gln subsequently appeared irrelevant. This suppressor has a genomic amplification formed by the creation of a novel junction that fuses to a gene encoding a putative road-pectrum acemase of , . An engineered allele lacking the putative secretion signal (ΔSS-) also suppressed D-glu auxotrophy, resulting in PG that was indistinguishable from the wild type. The ΔSS- allele similarly suppressed the D-alanine auxotrophy of an mutant and restored prototrophy to a double mutant auxotrophic for both D-ala and D-glu. The ΔSS- allele increased resistance to D-cycloserine but had no effect on sensitivity to PG-targeting antibiotics penicillin, ampicillin, or vancomycin. Our work helps define constraints on PG evolution and reveals a periplasmic broad-spectrum racemase in that can be co-opted for PG biosynthesis, with concomitant D-cycloserine resistance.

Importance: D-Amino acids are used and produced by organisms across all domains of life, but often, their origins and roles are not well understood. In bacteria, D-ala and D-glu are structural components of the canonical peptidoglycan cell wall and are generated by dedicated racemases Alr and MurI, respectively. The more recent discovery of additional bacterial racemases is broadening our view and deepening our understanding of D-amino acid metabolism. Here, while exploring alternative PG biosynthetic pathways in , we unexpectedly shed light on an unusual racemase, BsrF. Our results illustrate a novel mechanism for the evolution of antibiotic resistance and provide a new avenue for exploring the roles of non-canonical racemases and D-amino acids in bacteria.