The first sequence of bacterial chromosome was published more than 10 years ago. Now, hundreds of bacterial genomes are available for comparative genomics. The vast genetic information as well as introduction of new bacterial strains to industrial usage require the suitable tools for rapid genetic manipulations with a wide variety of bacterial species. Broad-host-range plasmids provide a valuable tool for such work. Numerous broad-host-range shuttle vectors, promoter-probe vectors, expression vectors and other special-purpose vectors are available now. The majority of them are based on the replicons of the IncQ or IncP groups [see for example [1–3]].
Undoubtedly, RSF1010 can be considered as the most studied plasmid of this type. RSF1010 is a mobilizable, non-conjugative, IncQ group plasmid that has broad host range replication properties in most of the Gram- bacteria [4] and at least some of the Gram+ species [5]. RSF1010 can use many different transfer systems for its mobilization. This property enables it to be transferred to a wide variety of bacterial hosts and even to plants [6]. The nucleotide sequence of this plasmid has been determined [7] and the detailed functional study of all plasmid loci involved in replication and mobilization has been performed [see for review [4, 8, 9]]. Relatively small size (8684 bp), moderate copy-number (in E. coli, RSF1010 is present at a copy number of 12 per cell [10]), capability to replicate and stable maintenance in a broad range of bacterial hosts where RSF1010 could be easily transferred from the standard laboratory E. coli strains by conjugative mobilization, have made this plasmid an attractive molecular cloning vector for basic research.
On the other hand, RSF1010 was traditionally used in biotechnology. It is very suitable vector for gene amplification in bacterial producing strains because of its high stability in different hosts. Moreover, in E. coli it is compatible with other popular replicons such as pBR322, pSC101, etc. Due to this property, RSF1010 was used as a second and even third plasmid vector in the real industrial strains producing different metabolites. However, industrial usage of mobilizable plasmids is undesirable because of biosafety considerations and is restricted today by legislations of several leading countries described, for example, in "Guidelines for research involving recombinant DNA molecules" published by the NIH on the 7th of May, 1987 and in the European Council Directives 90/220/EEC, 98/81/EC and 90/219/EEC. That is why construction of the stably maintained mob- derivative of RSF1010 (the derivative carrying mutation(s) in mob locus, which decreases mobilization frequency significantly) could have important practical application. The modern advanced methods of bacterial transformation, especially the electro transformation technique, provide a simple way for introduction of plasmid DNA to a broad range of bacterial hosts, which is not dependent on conjugation and mobilization [see e.g. [11, 12] and for review [13, 14]]. Thus, in principle, mob- derivatives of RSF1010 could be used not only in E. coli, but also in other bacterial hosts supporting RSF1010 replication for that methods of chemical or electro transformation have been elaborated.
Previously, several mutations of RSF1010 have been selected and characterized that significantly decreased efficiency of the plasmid mobilization in the standard laboratory tests [15]. But these mutations inactivate only few from the total set of genetic loci of RSF1010 that participate in mobilization process, and so, from the formal point of view the corresponding plasmids could not be considered as non-mobilizable for non-restricted practical application in the industrial biotechnology. All known genetic loci involved in mobilization have to be eliminated from the officially safe RSF1010-derivatives.
At present, the following loci involved in the plasmid vegetative replication and its mobilization, have been identified in RSF1010 [16, 17] (see Fig. 1 as well):
1) Transcription of all corresponding genes is initiated from the promoters (P1 – P4) (see Fig. 1). It is under multiple regulatory controls, and the individual plasmid-encoded proteins are involved, as a rule, in several functions essential for plasmid replication, mobilization and regulation of transcription. P1 and P3 controlled by MobC and MobA provide transcription of mobA/repB, mobB, and, probably, (E-F-repA-repC)-operon. The P2-promoter is under the same auto-regulated control and provides transcription of mobC in the opposite direction. The P4 promoter providing transcription of (E-F-repA-repC)-operon, is under the auto-regulated control of the F-repressor, whose gene is located in the same operon [18]. Earlier it was shown [15] that deletion of the P4 promoter does not lead to the loss of the plasmid "availability": transcription of repB (repB') – (E-F-repA-repC)-operon from the P1/P3 promoters is sufficient for the plasmid replication;
2) oriV – the unique origin of vegetative DNA replication;
3) repA, repB (alternative designation that more precisely reflects the function of its protein product – mobA/repB), and repC – the genes for essential replication proteins. Hereat, repA and repC encode a helicase and an oriV binding protein, correspondingly, and the expression level of repC, in particular, regulates the extent of plasmid replication. The product of mobA/repB – RepB is a multifunctional protein where its C-terminal domain, RepB', exhibits DNA primase activity in the process of vegetative plasmid replication, whereas its N-terminal part, MobA, is essential for the plasmid mobilization, and as well as MobC, plays a role in transcription regulation of the plasmid genes;
4) oriT – the site of relaxation complex and origin of conjugative DNA transfer;
Activities of MobA, MobB and MobC providing the specific cleavage of plasmid DNA in oriT followed by mobilizable transfer, are essential for mobilization of RSF1010. MobC encoded by mobC, in cooperation with MobA are co-regulators of of plasmid gene transcription, participating, in particular, in auto-regulation of their own synthesis. The mobB gene encoding MobB, is located in the structural part of mobA/repB, but translation of the corresponding proteins is occurred in the different reading frames.
The auto-regulated expression of RSF1010 genes referred to above provides the control of plasmid copy number, its stable maintenance, vegetative replication and mobilization. The corresponding genetic elements are partially overlapped in a rather complicated fashion. All recently obtained RSF1010-based plasmids with partially deleted mob-genes possessed mobilization frequencies detectable even under laboratory conditions. Besides, most of the mutant plasmids had significantly increased copy number [15].
For construction of RSF1010 derivatives that could be officially considered as mob-, we have decided to substitute the plasmid fragment comprising mobC, oriT (overlapping with P1, P2 and P3) and 5'-terminal portion of mobA/repB encoding MobB and MobA, by an artificial DNA fragment that could provide efficient transcription and translation of repB. The earlier described [19] genetic element, P
lac
UV5
→lacI linked to sub-optimal SD sequence GGGGGG has been used initially for reproducing the auto-regulated expression pattern of the native mobA/repB gene.