Transcriptional Organization of the Stability Module of Broad-Host-Range Plasmid RA3, from the IncU Group

Abstract

The broad-host-range conjugative plasmids have developed diverse adaptive mechanisms defining the range of their promiscuity. The BHR conjugative RA3 plasmid, the archetype of the IncU group, can transfer between, replicate and be maintained in representatives of Alpha-, Beta- and Gammaproteobacteria. Its stability module encompasses ten ORFs apparently organized into five operons, all transcribed in the same direction from several strong promoters that are tightly regulated either by autorepressors or by global plasmid-encoded regulators. In this paper, we demonstrate that owing to an efficient RNA polymerase read-through, the transcription from the first promoter, orf02p, may continue through the whole module. Moreover, an analysis of mRNA produced from the WT stability module and its deletion variants deprived of particular internal transcription initiation sites reveals that in fact each operon may be transcribed from any upstream promoter giving rise to multicistronic transcripts of variable length creating an additional level of gene expression control by transcript dosage adjustment. The gene expression patterns differ among various hosts indicating that promoter recognition, regulation and the RNAP read-through mechanisms are modulated in a species-specific manner. Importance The efficiently disseminating conjugative or mobilizable BHR plasmids play key roles in the horizontal spread of genetic information between closely related and phylogenetically distant species, which can be harmful from the medical, veterinary or industrial point of view. Understanding the mechanisms determining the plasmid's ability to function in diverse hosts is essential to help limit the spread of undesirable plasmid-encoded traits, e.g., antibiotic resistance. The range of plasmids' promiscuity depends on the adaptations of its transfer, replication and stability functions to the various hosts. IncU plasmids, with the archetype RA3, are considered to constitute a reservoir of antibiotic resistance genes in aquatic environments, however, the molecular mechanisms determining their adaptability to a broad range of hosts are rather poorly characterized. Here, we present the transcriptional organization of the stability module and show that gene transcript dosage effect is an important determinant of the RA3 stable maintenance in different hosts.

Unique Properties of the Alpha-Helical DNA-Binding Protein KfrA Encoded by the IncU Incompatibility Group Plasmid RA3 and Its Host-Dependent Role in Plasmid Maintenance

“…6A). It has been also shown that the kfrA operon of RA3 was vital for heterologous plasmid maintenance in E. coli and significantly important in other hosts (18). which was unable to bind to DNA and repress kfrAp.…”

“…1B ). Recently, we showed that the stability module is organized as a multicistronic operon with at least five differently regulated internal promoters of various strengths ( 18 ). Such an organization may provide, on the one hand, tuned expression of the region when required and, on the other, a species-dependent separation of the particular transcriptional units.…”

“…1C ), are localized upstream of the RA3 partition operon and preceded by internal promoters. The gene klcA , encoding a putative antirestriction protein ( 16 , 21 ), is expressed from orf02p and klcAp ( 18 ); both are under the control of the global repressor KorC, and orf02p is additionally under the control of KorB ( 22 ). kfrC and kfrA encode alpha-helical proteins that in R751 of the IncPβ group were postulated to support plasmid maintenance ( 23 ).…”

“…K d = [L] / [P] [LP], where L, P and LP represent molar concentrations of the proteins and the protein complex.Determination of catechol 2,3-dioxygenase activity (XylE)E. coli C600 carrying an appropriate pPT01 derivative was transformed with either the pGBT30 (tacp) expression vector or pESB5.58 (tacp-kfrA RA3 ). XylE activity(73) was assayed spectrophotometrically in cleared extracts of cells collected from logarithmically growing cultures without and with 0.5 mM IPTG, as described previously(18). One unit of the catechol 2,3-oxygenase activity is defined as the amount of enzyme needed to convert 1 μmole of catechol to 2-hydroxymuconic semialdehyde per mg of protein in one minute.Protein concentration was determined using the Bradford method(69).…”

“…One unit of the catechol 2,3-oxygenase activity is defined as the amount of enzyme needed to convert 1 μmole of catechol to 2-hydroxymuconic semialdehyde per mg of protein in one minute.Protein concentration was determined using the Bradford method(69). Experiments were performed in triplicate and the mean values with standard deviations are reported.Plasmid stability assaysThe stability of pESB36 or its derivative in E. coli EC1250, A. tumefaciens LBA1010 Rif r , P. aminovorans JCM 7685 Rif r , P. putida KT2442 Rif r or C. necator JMP228 Rif r was tested as described previously(18). Briefly, stationary-phase cultures grown under antibiotic selection were diluted10 5 -fold in fresh medium (without antibiotic) and cultivated for approximately 20 generations.…”