Regulation of D-alanyl-lipoteichoic acid biosynthesis in Streptococcus agalactiae involves a novel two-component regulatory system

The dlt operon of gram-positive bacteria comprises four genes (dltA, dltB, dltC, and dltD) that catalyze the incorporation of D-alanine residues, into the lipoteichoic acids (LTAs). In this work, we characterized the dlt oper on of Streptococcus, agalactiae, which, in addition to the dltA to dltD gen es, included two regulatory genes, designated dltR and dltS, located upstre am of dltA. The AM gene encodes a 224-amino-acid putative response regulato r belonging to the OmpR family of regulatory proteins. The dltS gene codes for a 395-amino-acid putative histidine kinase thought to be involved in th e sensing of environmental signals. The dlt operon of S. agalactiae is main ly transcribed from the P-dltR promoter, which directs synthesis of a 6.5-k b transcript encompassing dltR, dltS, dltA, dltB, dltC, and dltD, and from a weaker promoter, P-dltA, which is located in the 3' extremity of dltS. We demonstrate that P-dltR, but not P-dlA, is activated by DltR in the presen ce of DltS in D-Ala-deficient LTA mutants resulting from insertional inacti vation of the dltA gene, which encodes the cytoplasmic D-alanine-D-alanyl c arrier, ligase DltA. Expression of the dlt operon does not require DltR and DRS, since the basal activity of P-dltR, is high, being 20-fold that of th e constitutive promoter PaphA-3 which directs synthesis of the kanamycin re sistance gene aphA-3 in various gram-positive bacteria. We hypothesize that the role of DltR and DltS in the control of expression of the dlt operon i s to maintain the level of D-Ala esters in LTAs at a constant and appropria te value whatever the environmental conditions. The DltA(-) mutant displaye d the ability to form clumps in standing culture and exhibited an increased susceptibility to the cationic antimicrobial polypeptide colistin.