Mutant <i>gltS</i> alleles enable a <i>Vibrio fischeri</i> D-glutamate auxotroph to grow with lower requirements for exogenous D-glutamate

D-glu is a key component of peptidoglycan (PG) and is essential for growth in most bacteria. To assess constraints on PG evolution and bacterial requirements for D-glu, we sought to artificially evolve PG biosynthesis, leading to either replacement of D-glu in the PG peptide or alternative pathways to D-glu incorporation. We previously found that suppression of D-glu auxotrophy in a <i>murI racD</i> mutant of <i>Vibrio fischeri</i> grown on lysogeny broth salts (LBS) medium was rare but could be accomplished by mutation of <i>bsrF</i>, with restoration of wild-type PG structure. Here, we selected nine additional prototrophic suppressors of the same <i>murI racD</i> mutant from 10¹⁰ colony-forming units (CFU) plated on LBS supplemented with ~2.7 mM D-gln. Each suppressor had a mutation in <i>gltS</i>, which encodes a putative sodium:glutamate symporter. Increased copy numbers of mutant <i>gltS</i> alleles enabled growth on unsupplemented LBS and resulted in PG containing D-glu. Examination of media components suggests that D-gln supplementation had inadvertently added ~14 μM D-glu, and that LBS itself contains ~1.4 μM D-glu. The mutations in <i>gltS</i> enabled growth with similarly low D-glu concentrations, but also increased sensitivity to homocysteic acid, suggesting more promiscuous transport. Surprisingly, we discovered that expression of mutant <i>gltS</i> in the auxotroph leads to incorporation of lysine into PG, in addition to canonical D-glu. When seawater is supplemented with D-glu, this <i>V. fischeri</i> mutant still colonized <i>Euprymna scolopes</i> and triggered PG-induced morphogenesis. Our results shed light on glutamate transport, highlight trade-offs in GltS structure and function, and reveal an unusual PG modification.IMPORTANCED-glu is an important building block in the peptidoglycan (PG) component of the bacterial cell wall, and its endogenous production is considered essential in most bacteria, even when grown in complex media. In <i>Vibrio fischeri</i>, <i>in trans</i> expression of mutant GltS symporters allows D-glu auxotrophic strains to grow on lysogeny broth salts (LBS) medium without exogenous D-glu, although there is a fitness trade-off of increased sensitivity to homocysteic acid. Our finding that LBS contains sufficient D-glu to support robust growth highlights the undervalued importance of D-amino acid transport and the ubiquity of D-amino acids. Moreover, the discovery of D-lysine in the PG peptide is an unusual PG modification that warrants further study.