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.

A group of proteic toxin-antitoxin (TA) cassettes whose representatives are widely distributed among bacterial genomes has been identified. These cassettes occur in chromosomes, plasmids, bacteriophages, and noncomposite transposons, as well as in the SXT conjugative element of Vibrio cholerae. The following four homologous loci were subjected to detailed comparative studies: (i) tad-ata from plasmid pAMI2 of Paracoccus aminophilus (the prototype of this group), (ii) gp49-gp48 from the linear bacteriophage N15 of Escherichia coli, (iii) s045-s044 from SXT, and (iv) Z3230-Z3231 from the genomic island of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. Functional analysis revealed that all but one of these loci (Z3230-Z3231) are able to stabilize heterologous replicons, although the host ranges varied. The TA cassettes analyzed have the following common features: (i) the toxins are encoded by the first gene of each operon; (ii) the antitoxins contain a predicted helix-turn-helix motif of the XRE family; and (iii) the cassettes have two promoters that are different strengths, one which is located upstream of the toxin gene and one which is located upstream of the antitoxin gene. All four toxins tested are functional in E. coli; overexpression of the toxins (in the absence of antitoxin) results in a bacteriostatic effect manifested by elongation of bacterial cells and growth arrest. The toxins have various effects on cell viability, which suggests that they may recognize different intracellular targets. Preliminary data suggest that different cellular proteases are involved in degradation of antitoxins encoded by the loci analyzed.Bacterial plasmids have modular structures, since it is possible to dissect them into several functional cassettes. The core region of the plasmid backbone is composed of a set of conserved modules, coding for replication and stability functions, which are crucial for plasmid maintenance. Comparative analyses of plasmid genomes have revealed that the same modules or closely related modules can be found in various combinations in different plasmids hosted by phylogenetically distinct bacteria. The plasticity of plasmid genomes is the result of horizontal gene transfer, as well as numerous recombinational events, which take place in diverse bacterial hosts. Recent advances in bacterial genomics have revealed that shuffling of modules is not limited to plasmids but is a much more common phenomenon, occurring in all bacterial mobile genetic elements. These elements may thus be considered combinations of exchangeable functional cassettes (78).Shuffling of modules, initially defined in plasmids, bacteriophages, and transposable elements, has resulted in (i) the generation of novel mobile elements, such as conjugative transposons (containing phage-related recombination modules plus plasmid-borne modules for conjugational transfer) (64), and (ii) the appearance of chimeric elements exhibiting new, unusual properties, including transposable bacteriophage Mu (containing a transposition mod...

The second replicator region of the native plasmid pTAVl of Paracoccus versutus has been identified thus proving the composite nature of this replicon. The minimal replicon designated pTAV320 (43 kb) was cloned and sequenced. pTAV320 encodes three putative proteins -RepA, RepB and RepC.This replicator region shows strong structural and functional similarity t o mpABC-type rep1 icons found in several Agrobacterium and Rhizobium plasmids. The origin of replication appears to be localized within the coding sequence of the repC gene. RepC was shown t o be essential for replication. RepA and RepB were necessary for stable maintenance of the plasmid, which implies a role in active partitioning. The presence of the complete sequence of pTAV320 (in its non-replicative form) could stabilize in cis pTAV202, a rninireplicon derived from the other pTAVl replicator region. Deletions introduced into the mpC gene abolished the 'stabilizing' activity of pTAV320, suggesting that the centromere-like sequence, necessary for partitioning, might be localized within this gene. The two replicator regions of pTAV1 (pTAV320 and pTAV202) expressed incompatibility towards the parental plasmid but were compatible in trans in P. wersutus cells. The pTAV320 replicon can be maintained in several Paracoccus, Agrobacterium, Rhizobium and Rhodohcter strains in addition t o P. versutus.

Paracoccus aminophilus JCM 7686 (Alphaproteobacteria) is a facultative, heterotrophic methylotroph capable of utilizing a wide range of C1 compounds as sole carbon and energy sources. Analysis of the JCM 7686 genome revealed the presence of genes involved in the oxidation of methanol, methylamine, dimethylamine, trimethylamine, N,N-dimethylformamide, and formamide, as well as the serine cycle, which appears to be the only C1 assimilatory pathway in this strain. Many of these genes are located in different extrachromosomal replicons and are not present in the genomes of most members of the genus Paracoccus, which strongly suggests that they have been horizontally acquired. When compared with Paracoccus denitrificans Pd1222 (type strain of the genus Paracoccus), P. aminophilus JCM 7686 has many additional methylotrophic capabilities (oxidation of dimethylamine, trimethylamine, N,N-dimethylformamide, the serine cycle), which are determined by the presence of three separate gene clusters. Interestingly, related clusters form compact methylotrophy islands within the genomes of Paracoccus sp. N5 and many marine bacteria of the Roseobacter clade.

Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B12 to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B12 was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B12 conjugates; namely the vitamin B12 azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B12 and PNA were tested, including a disulfide bond. We found that vitamin B12 transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF)3K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B12 as a carrier of PNA oligonucleotides into bacterial cells.