Vibrio fischeri DarR Directs Responses to d -Aspartate and Represents a Group of Similar LysR-Type Transcriptional Regulators

Abstract

Mounting evidence suggests that d-amino acids play previously underappreciated roles in diverse organisms. In bacteria, even d-amino acids that are absent from canonical peptidoglycan (PG) may act as growth substrates, as signals, or in other functions. Given these proposed roles and the ubiquity of d-amino acids, the paucity of known d-amino-acid-responsive transcriptional control mechanisms in bacteria suggests that such regulation awaits discovery. We found that DarR, a LysR-type transcriptional regulator (LTTR), activates transcription in response to d-Asp. The d-Glu auxotrophy of a ::Tn mutant was suppressed, with the wild-type PG structure maintained, by a point mutation in This mutation resulted in the overexpression of an adjacent operon encoding a putative aspartate racemase, RacD, which compensated for the loss of the glutamate racemase encoded by Using transcriptional reporters, we found that wild-type DarR activated transcription in response to exogenous d-Asp but not upon the addition of l-Asp, l-Glu, or d-Glu. A DNA sequence typical of LTTR-binding sites was identified between and the divergently oriented operon, and scrambling this sequence eliminated activation of the reporter in response to d-Asp. In several proteobacteria, genes encoding LTTRs similar to DarR are linked to genes with predicted roles in d- and/or l-Asp metabolism. To test the functional similarities in another bacterium, and mutants were also generated in In and, growth on d-Asp required the presence of both and Our results suggest that multiple bacteria have the ability to sense and respond to d-Asp. d-Amino acids are prevalent in the environment and are generated by organisms from all domains of life. Although some biological roles for d-amino acids are understood, in other cases, their functions remain uncertain. Given the ubiquity of d-amino acids, it seems likely that bacteria will initiate transcriptional responses to them. Elucidating d-amino acid-responsive regulators along with the genes they control will help uncover bacterial uses of d-amino acids. Here, we report the discovery of DarR, a novel LTTR in that mediates a transcriptional response to environmental d-Asp and underpins the catabolism of d-Asp. DarR represents the founding member of a group of bacterial homologs that we hypothesize control aspects of aspartate metabolism in response to d-Asp and/or to d-Asp-containing peptides.

Double Auxotrophy to Improve the Safety of a Live Anti-Pseudomonas aeruginosa Vaccine

“…We have observed that the vaccine candidate PAO1 Δ murI is sporadically able to restore D-glutamate prototrophy when used at high concentrations. Similarly, the D-glutamate auxotrophy of a murI mutant of Vibrio fischeri was restored by overexpression of the aspartate racemase, RacD [ 14 ]. Likewise, an L-aspartate/glutamate racemase from E. coli containing only one cysteine residue in the active site, EcL-DER, showed threefold higher racemase activity with L-glutamate than L-aspartate [ 15 ].…”

Versatility and Complexity: Common and Uncommon Facets of LysR-Type Transcriptional Regulators

“…Moreover, the response can be highly specific. For example, two paralogs in Acinetobacter baylyi together regulate aspartate catabolism, yet one, DarR, responds to d-Asp and the other, AalR, to l-Asp (8,61). Identifying specific effectors is challenging, and in most cases, signals to which LTTRs respond remain unknown.…”

d -Fining DarR, a LysR-Type Transcriptional Regulator That Responds to d -Aspartate

“…1. This work was presented by R. Mark Jones at the ASM Conference on Vibrio in November 2017, in Chicago, IL, and is published in this issue (15).…”

“…In their current work, Jones et al (15) then conducted suppressor analysis, identifying spontaneous suppressors of the murI transposon insertions that were no longer D-glutamate auxotrophs. Three isolated suppressors all exhibited the same amino acid replacement (M202I) in a LysR-type transcriptional regulator (LTTR), which they named DarR (D-aspartate-responsive regulator).…”

“…Key results from Jones et al(15). DarR is a LysR-type transcription factor (LTTR) that regulates transcription of the VF_1546-racD operon in response to environmental D-aspartate.…”