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

Abstract

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. b-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.

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…For both pMV158 and pIP501, the oriT regions are compactly organized, and the promoter for the relaxase gene overlaps the nic site (92,163). Correspondingly, in the pIP501 system, relaxase binding to oriT autoregulates the expression of the relaxase and downstream transfer genes (163). Gram-negative plasmid pBBR1 has a nic region identical to that of pMV158, and reminiscent of pIP501, the binding of pBBR1 relaxase at oriT autoregulates downstream tra gene expression (255).…”

“…For both pMV158 and pIP501, the oriT regions are compactly organized, and the promoter for the relaxase gene overlaps the nic site (92,163). Correspondingly, in the pIP501 system, relaxase binding to oriT autoregulates the expression of the relaxase and downstream transfer genes (163).…”

“…Gram-negative plasmid pBBR1 has a nic region identical to that of pMV158, and reminiscent of pIP501, the binding of pBBR1 relaxase at oriT autoregulates downstream tra gene expression (255). It is postulated that this mode of regulation evolved for the feedback control of plasmid transfer rates to minimize the metabolic burden associated with conjugative DNA transfer (163).…”

“…Relaxases associated with grampositive T4SS have been classified into three phylogenetic groups (100,116), but the characterized relaxases catalyze nicking by the same transesterase mechanism as that described above for gram-negative enzymes. Characterized relaxases include MobA (staphylococcal pC221) (46,47), PcfG (E. faecalis plasmid pCF10) (58,59), MobM (streptococcal plasmid pMV158) (92,120), and TraA (S. agalactiae plasmid pIP501) (158,163,276). In vitro studies of the staphylococcal pC221 and E. faecalis pCF10 processing reactions established the importance of the accessory factors MobC and PcfF in coordinating the binding and catalytic activities of the relaxases MobA and PcfG at their respective oriT target sites (46,47,58).…”

Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501

“…Most knowledge about Gram-positive T4SS is based on similarity to their Gram-negative counterparts Abajy et al, 2007). However, much more information regarding proteins involved in the T4S processes is available for bacteria of Gramnegative origin Kurenbach et al, 2006;Wallden et al, 2010;Clewell, 2011). Only very recently has the first structural information on Gram-positive transfer proteins become available (Porter et al, 2012;Walldé n et al, 2012).…”