The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation

Abstract

Bacterial conjugation is the process by which a conjugative element (CE) is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer (oriT), where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis. We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1LS20 and aux2LS20, and which we show are essential for conjugation. Both Aux1LS20 and Aux2LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1LS20 and Aux2LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriTLS20, although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1LS20 and/or Aux2LS20 are located upstream of almost 400 relaxase genes of the RelLS20 family (MOBL) of relaxases. Thus, Aux1LS20 and Aux2LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

Novel regulatory mechanism of establishment genes of conjugative plasmids

“…Members of the superfamily of RHH transcription factors are involved in the regulation of diverse bacterial processes such as amino-acid biosynthesis, cell division, control of plasmid copy number and lytic cycle of bacteriophages (for review see, 46). Recently, we have shown that many auxiliary relaxosome proteins encoded by conjugative plasmids of G+ bacteria are also RHH type proteins (22). Here we show that Reg 576 too is a RHH type protein, thereby expanding even further the range of processes in which this family of proteins is involved.…”

“…Electrophoretic mobility shift assay (EMSA) assays were essentially performed as described before (22). In short, 120 ng of the polymerase chain reaction fragment was incubated for 20 min at RT in Binding Buffer (20 mM Tris HCl [pH 8.0], 1 mM EDTA, 5 mM MgCl 2 , 100 mM KCl, 10% (v/v) glycerol) without and with increasing amounts of purified His (6) Reg 576 in a total volume of 20 μl.…”

Enterococcal PcfF Is a Ribbon-Helix-Helix Protein That Recruits the Relaxase PcfG Through Binding and Bending of the oriT Sequence

“…In contrast to the common helix-turn-helix motif for DNA binding, RHH domains bind DNA via a small N-proximal β-sheet composed of one β-strand from each monomer (Schreiter and Drennan, 2007). So far, no accessory factors of G + origin have been structurally characterized, albeit one in silico analysis has indicated that many of them also contain RHH-binding domains (Miguel-Arribas et al, 2017).…”

A novel method for transforming the thermophilic bacterium Geobacillus kaustophilus

“…pLS20, a conjugative plasmid isolated from B. subtilis natto [ 19 ], can transfer itself to various B. subtilis -related Gram-positive bacteria, including Bacillus anthracis , Bacillus cereus , Bacillus licheniformis , Bacillus megaterium , Bacillus pumilus , and Bacillus thuringiensis [ 20 ]. pLS20cat, a derivative of pLS20 carrying a chloramphenicol resistance gene possesses the outstanding ability of rapid transfer: after simply mixing liquid cultures of donor and recipient cells the plasmid is efficiently transferred within 15 min [ 21 – 26 ]. In addition, pLS20cat can mobilize a co-resident plasmid if it contains a functional copy of the oriT of pLS20cat ( oriT LS20 ) [ 26 , 27 ].…”

“…pLS20cat, a derivative of pLS20 carrying a chloramphenicol resistance gene possesses the outstanding ability of rapid transfer: after simply mixing liquid cultures of donor and recipient cells the plasmid is efficiently transferred within 15 min [ 21 – 26 ]. In addition, pLS20cat can mobilize a co-resident plasmid if it contains a functional copy of the oriT of pLS20cat ( oriT LS20 ) [ 26 , 27 ]. Plasmid pLS20 or a derivative has been used to mobilize plasmids in various studies [ 20 , 28 – 30 ].…”