Horizontal gene transfer (HGT) contributes greatly to the plasticity and evolution of prokaryotic and eukaryotic genomes. The main carriers of foreign DNA in HGT are mobile genetic elements (MGEs) that have extremely diverse genetic structures and properties. Various strategies are used for the maintenance and spread of MGEs, including (i) vegetative replication, (ii) transposition (and other types of recombination), and (iii) conjugal transfer. In many MGEs, all of these processes are dependent on rolling-circle replication (RCR). RCR is one of the most well characterized models of DNA replication. Although many studies have focused on describing its mechanism, the role of replication initiator proteins has only recently been subject to in-depth analysis, which indicates their involvement in multiple biological process associated with RCR. In this review, we present a general overview of RCR and its impact in HGT. We focus on the molecular characteristics of RCR initiator proteins belonging to the HUH and Rep_trans protein families. Despite analogous mechanisms of action these are distinct groups of proteins with different catalytic domain structures. This is the first review describing the multifunctional character of various types of RCR initiator proteins, including the latest discoveries in the field. Recent reports provide evidence that (i) proteins initiating vegetative replication (Rep) or mobilization for conjugal transfer (Mob) may also have integrase (Int) activity, (ii) some Mob proteins are capable of initiating vegetative replication (Rep activity), and (iii) some Rep proteins can act like Mob proteins to mobilize plasmid DNA for conjugal transfer. These findings have significant consequences for our understanding of the role of RCR, not only in DNA metabolism but also in the biology of many MGEs.

The segregation of newly replicated chromosomes in bacterial cells is a highly coordinated spatiotemporal process. In the majority of bacterial species, a tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target(s) parS sequence(s), facilitates the initial steps of chromosome partitioning. ParB nucleates around parS(s) located in the vicinity of newly replicated oriCs to form large nucleoprotein complexes, which are subsequently relocated by ParA to distal cellular compartments. In this review, we describe the role of ParB in various processes within bacterial cells, pointing out interspecies differences. We outline recent progress in understanding the ParB nucleoprotein complex formation and its role in DNA segregation, including ori positioning and anchoring, DNA condensation, and loading of the structural maintenance of chromosome (SMC) proteins. The auxiliary roles of ParBs in the control of chromosome replication initiation and cell division, as well as the regulation of gene expression, are discussed. Moreover, we catalog ParB interacting proteins. Overall, this work highlights how different bacterial species adapt the DNA partitioning ParAB-parS system to meet their specific requirements.

Klebsiella pneumoniae 287-w carries three small narrow host range (NHR) plasmids (pIGMS31, pIGMS32, and pIGRK), which could be maintained in several closely related species of Gammaproteobacteria, but not in Alphaproteobacteria. The plasmids contain different mobilization systems (MOB), whose activity in Escherichia coli was demonstrated in the presence of the helper transfer system originating from plasmid RK2. The MOBs of pIGMS31 and pIGMS32 are highly conserved in many bacterial plasmids (members of the MOB family), while the predicted MOB of pIGRK has a unique structure, encoding a protein similar to phage-related integrases. The MOBs of pIGMS31 and pIGMS32 enabled the transfer of heterologous replicons from E. coli into both gammaproteobacterial and alphaproteobacterial hosts, which suggests that these NHR plasmids contain broad host range MOB systems. Such plasmids therefore represent efficient carrier molecules, which may act as natural suicide vectors promoting the spread of diverse genetic information (including other types of mobile elements, e.g. resistance transposons) among evolutionarily distinct bacterial species. Thus, mobilizable NHR plasmids may play a much more important role in horizontal gene transfer than previously thought.

BackgroundGene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called “trivial overlapping”, there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome’s novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family.ResultsA single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR’ protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR’ have opposing functions – RepR is crucial for initiation of pIGRK replication, while RepR’ is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression.ConclusionsRegulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

Antibiotic resistance determinants are frequently associated with plasmids and other mobile genetic elements, which simplifies their horizontal transmission. Several groups of plasmids (including replicons of the IncL/M incompatibility group) were found to play an important role in the dissemination of resistance genes encoding β-lactamases. The IncL/M plasmids are large, broad host range, and self-transmissible replicons. We have identified and characterized two novel members of this group: pARM26 (isolated from bacteria inhabiting activated sludge from a wastewater treatment plant) and pIGT15 (originating from a clinical strain of Escherichia coli). This instigated a detailed comparative analysis of all available sequences of IncL/M plasmids encoding β-lactamases. The core genome of these plasmids is comprised of 20 genes with conserved synteny. Phylogenetic analyses of these core genes allowed clustering of the plasmids into four separate groups, which reflect their antibiotic resistance profiles. Examination of the biogeography of the IncL/M plasmids revealed that they are most frequently found in bacteria of the family Enterobacteriaceae originating from the Mediterranean region and Western Europe and that they are able to persist in various ecological niches even in the absence of direct antibiotic selection pressure.