Abstract
In conjugative elements such as integrating conjugative elements (ICEs) or conjugative plasmids (CPs) transcription of DNA transfer genes is a prerequisite for cells to become transfer competent, i.e., capable of delivering plasmid DNA via bacterial conjugation into new host bacteria. In the large family of F-like plasmids belonging to the MobF12A group, transcription of DNA transfer genes is tightly controlled and dependent on the activation of a single promoter, designated PY. Plasmid encoded TraJ and chromosomally encoded ArcA proteins are known activators, whereas the nucleoid associated protein heat-stable nucleoid structuring (H-NS) silences the PY promoter. To better understand the role of these proteins in PY promoter activation, we performed in vitro DNA binding studies using purified H-NS, ArcA, and TraJR1 (TraJ encoded by the conjugative resistance plasmid R1). All proteins could bind to R1PY DNA with high affinities; however, only ArcA was found to be highly sequence specific. DNase I footprinting studies revealed three H-NS binding sites, confirmed the binding site for ArcA, and suggested that TraJ contacts a dyad symmetry DNA sequence located between −51 and −38 in the R1PY promoter region. Moreover, TraJR1 and ArcA supplied together changed the H-NS specific protection pattern suggesting that these proteins are able to replace H-NS from R1PY regions proximal to the transcription start site. Our findings were corroborated by PY-lacZ reporter fusions with a series of site specific R1PY promoter mutations. Sequential changes of some critical DNA bases in the TraJ binding site (jbs) from plasmid R1 to plasmid F led to a remarkable specificity switch: The PY promoter became activatable by F encoded TraJ whereas TraJR1 lost its activation function. The R1PY mutagenesis approach also confirmed the requirement for the host-encoded response-regulator ArcA and indicated that the sequence context, especially in the −35 region is critical for PY regulation and function.
Highlights
Horizontal gene transfer by conjugation is ubiquitous among microorganisms belonging to the kingdoms of bacteria and archaea
Expression of the tra operon is an absolute requirement for bacteria carrying conjugative plasmids (CPs) to develop transfer competence
We investigated the molecular basis of tra gene activation of the classical antibiotic resistance plasmid R1 which belongs to a large family of F-like plasmids, categorized as MOBF12A (Fernandez-Lopez et al, 2017)
Summary
Horizontal gene transfer by conjugation is ubiquitous among microorganisms belonging to the kingdoms of bacteria and archaea. If a transfer competent cell harboring a mobile genetic element contacts a recipient in a suitable environment, subsequent transfer of genetic information can decisively expand the metabolic, resistance, or virulence capabilities of the recipient bacterium. Single-stranded DNA is transported unidirectionally by means of a cell envelopespanning multi-protein transport complex [termed type IV secretion system (T4SS)]. The process of DNA transfer through the T4SS is ATP dependent and coupled to DNA replication. The transported DNA is usually guided by at least one protein and derived from an autonomously replicating conjugative plasmid (CP) or from an integrating conjugative element (ICE). Additional cargo genes integrated into the mobile DNA element can rapidly spread throughout bacterial populations
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