Transcriptional activation of the citrate permease P gene of Lactococcus lactis biovar diacetylactis by an insertion sequence-like element present in plasmid pCIT264

Abstract

The lactococcal plasmid pCIT264 contains a cluster of three genes (citQ, citR and citP) involved in the transport of citrate in Lactococcus lactis biovar diacetylactis. The cit cluster contains a copy of a newly discovered insertion sequence (IS)-like element located between its promoter P1 and the first gene of the cluster. In this report, we show that this IS-like element can act as a mobile switch for the downstream genes, creating two new transcriptional promoters named P2 and P2'. The P2 promoter is recognized by the lactococcal RNA polymerase in vivo. This is a hybrid promoter composed of a -35 region reading outwards 12bp from the right end of the IS-like element, and a nucleotide sequence from the recipient plasmid, adjacent to the element, which provides an appropriately spaced -10 region. Transcription of the citQRP cluster from this promoter takes place during the exponential and stationary phases of growth in L. lactis. Promoter P2' is included in the IS-like element and is the only promoter responsible for expression of citP in E. coli. Thus, it appears that the introduction of this element into pCIT264 allows expression of the citQRP cluster in E. coli, and increases its levels of expression in L. lactis.