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 LysRtype transcriptional regulator (LTTR), activates transcription in response to D-Asp. The D-Glu auxotrophy of a Vibrio fischeri murI::Tn mutant was suppressed, with the wildtype PG structure maintained, by a point mutation in darR. This darR 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 murI. Using transcriptional reporters, we found that wild-type DarR activated racD 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 darR and the divergently oriented racD 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, darR and racD mutants were also generated in Acinetobacter baylyi. In V. fischeri and A. baylyi, growth on D-Asp required the presence of both darR and racD. Our results suggest that multiple bacteria have the ability to sense and respond to D-Asp.