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The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501

Jolanta Kope^1,2

Marion Mägdefrau^1,

¹ Department for Environmental Microbiology, University of Technology Berlin, FR1-2, Franklinstrasse 28/29, D-10587 Berlin, Germany
² Institute for Chemistry, Karl-Franzens-Universität Graz, Heinrichstrasse 28, A-8010 Graz, Austria

Correspondence
Elisabeth Grohmann
elisabeth.grohmann{at}tu-berlin.de

The conjugative multiple antibiotic resistance plasmid pIP501 can be transferred and stably maintained in a variety of Gram-positive genera, including multicellular Streptomyces lividans, as well as in Gram-negative Escherichia coli. The 15 putative pIP501 transfer (tra) genes are organized in an operon-like structure terminating in a strong transcriptional terminator. This paper reports co-transcription of the pIP501 tra genes in exponentially growing Enterococcus faecalis JH2-2 cells, as shown by RT-PCR. The tra genes are expressed throughout the life cycle of Ent. faecalis, and the expression level is independent of the growth phase. Electrophoretic mobility shift assays indicated that the TraA relaxase, the first gene of the tra operon, binds to the tra promoter P_tra, which partially overlaps with the origin of transfer (oriT). DNase I footprinting experiments further delimited the TraA binding region and defined the nucleotides bound by TraA. -Galactosidase assays with P_tra–lacZ fusions proved P_tra promoter activity, which was strongly repressed when TraA was supplied in trans. Thus, it is concluded that the pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA.

Present address: Competence Center for Fluorescence Analysis, Josef-Engert-Strasse 9, D-93053 Regensburg, Germany.

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