Identification of a new locus in Listeria monocytogenes involved in cellobiose-dependent repression of hly expression

Expression of the PrfA-controlled virulence gene hly (encoding the pore-for ming cytolysin listeriolysin) is down-regulated by readily metabolized carb on sources in Listeria monocytogenes. We isolated a Tn917-insertional mutan t of L. monocytogenes (strain LO28), which expressed a hemolytic phenotype in the presence of cellobiose. Using hly fusions to luxAluxB genes, we show that hly expression was derepressed in the presence of cellobiose at the t ranscriptional level. Surprisingly, hly expression was still repressed by g lucose, as observed for the parental strain. Genetic analysis of the Tn917- flanking regions indicated that the transposon had inserted in a non-coding region located between two genes in opposite orientations. These two newly identified genes were designated orfA and mdrL. The insertion occurred imm ediately upstream of orfA, likely into its promoter region. Transcriptional analysis of orfA and mdrL revealed that Tn917 had abolished orfA expressio n whereas it had activated expression of mdrL. orfA encodes a putative prot ein of 176 amino acids homologous to YfiO of Bacillus subtilis (28% identit y), a protein of unknown function. mdrL codes for a putative protein of 398 amino acids homologous to Bmr and Bit of B. subtilis (21-24% identity), tw o members of the multidrug resistance efflux pump family. Our results indic ate that we have identified a new locus which plays a crucial role in the c ellobiose-dependent repression of hly expression. (C) 1999 Federation of Eu ropean Microbiological Societies. Published by Elsevier Science B.V. All ri ghts reserved.