Two acid-inducible promoters from Lactococcus lactis require the cis-acting ACiD-box and the transcription regulator RcfB.

We previously characterized three Lactococcus lactis promoters, P170, P1 and P3, which are induced by low pH. Here, we identified a novel 14 bp regulatory DNA region centred at around -41.5 and composed of three tetranucleotide sequences, boxes A, C and D. Boxes A and C contribute to P1 activity, whereas box D and the position of boxes ACD (renamed ACiD-box) are essential to P1 activity and acid response. We also identified a trans -acting protein, RcfB, which is involved in P170 and P1 basal activity and is essential for their pH induction. The regulator belongs to the Crp-Fnr family of transcription regulators. Overexpression of rcfB resulted in increased beta-galactosidase activities and lantibiotic lacticin 481 production from P170- and P1-controlled genes, respectively, in acid condition. RcfB is thus probably activated when cells encounter an acid environment. rcfB is co-transcribed with genes encoding an universal stress-like protein and a multidrug transporter. RcfB plays a role in acid adaptation, as the survival rate of an rcfB mutant after a lethal acid challenge was 130-fold lower than that of the wild-type strain, when the bacteria were first grown in acidic medium. The groESL promoter includes a sequence resembling an ACiD-box and the chaperone GroEL production is partly RcfB dependent in acid condition. Our results suggest that the ACiD-box could be the DNA target site of RcfB.