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We demonstrated that a single copy of insertion sequence ISPmel can mobilize adjacent segments of genomic DNA of Paracoccus methylutens DM12, which leads to the generation of diverse transposable elements of various size and DNA contents. All elements (named transposable modules [TMos]) contain ISPmel (placed at the 5' ends of the elements) and have variable 3'-end regions of between 0.5 and 5 kb. ISPmel was shown to encode an outwardly oriented promoter, which may activate the transcription of genes transposed within TMos in evolutionarily distinct hosts. TMos may therefore be considered to be natural systems enabling gene capture, expression, and spread. However, unless these elements have been inserted into a highly conserved genetic context to enable a precise definition of their termini, it is extremely difficult or even impossible to identify them in bacterial genomes by in silico sequence analysis. We showed that TMos are present in the chromosome and plasmids of strain DM12. Sequence analysis of plasmid pMTH1 (32 kb) revealed that four TMos, previously identified with a trap vector, pMEC1, comprise 87% of its genome. Repeated TMos within pMTH1 may stimulate other structural rearrangements resulting from homologous recombination between long repeat sequences. This illustrates that TMos may play a significant role in shaping the structure of natural plasmids, which consequently may have a great impact on the evolution of plasmid genomes.

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.

A novel shuttle entrapment vector, pMMB2, was used to identify a large transposable element, TnPpa1 (44.3 kb), of Paracoccus pantotrophus DSM 11072. TnPpa1 has a composite structure with divergently oriented copies of a cryptic transposon, Tn3434 (Tn3 family), located at both termini. The core region of the element contains a large set of putative genes, whose products show similarity to enzymes involved in central intermediary metabolism (e.g. tricarboxylic acid cycle or 2-methylcitrate cycle), transporters, transcriptional regulators and conserved proteins of unknown function. A 4.2 kb DNA segment of TnPpa1 is homologous to a region of chromosome cll of Rhodobacter sphaeroides 2.4.1, which exemplifies the mosaic structure of this element. TnPpa1 is bordered by 5 bp long directly repeated sequences and is located within a mega-sized replicon, pWKS5, in the DSM 11072 genome. Spontaneous inversion of the core region of TnPpa1 was detected in the host genome. Analysis of the distribution of TnPpa1 in three other strains of P. pantotrophus revealed that this element was present exclusively within DSM 11072, which suggests its relatively recent acquisition by lateral transfer. The identification of TnPpa1 (which may be considered a transposable genomic island) provides evidence for the transposition and lateral transfer of large DNA segments of chromosomal origin (carrying various housekeeping genes), which may have a great impact on the evolution of bacterial genomes.

Research has been conducted on Paracoccus pantotrophus DSM 11072 cryptic plasmid pWKS. The genetic structure of this plasmid has been investigated and functional modules responsible for its mobilization and replication have been identified and discussed.

Research has been conducted on Paracoccus versutus. The replicon of the bacterium's plasmid has been localized and its nucleoside sequence has been determined.

Research has been conducted on the repABC-type replicon of the composite Paracoccus versutus plasmid pTAV1. The localization of the repABC-type replicon's partitioning site which constitutes the functional partitioning system has been carried out and described.

Research was conducted to characterize the second replicator region of pTAV1 which is part of the mini-replicon pTAV320. An investigation of the possible interactions between both replication systems was performed using molecular analysis in the parental composite plasmid pTAV1. Results demonstrated that the two replicator regions of pTAV1 were incompatible with the parental plasmid but were compatible in trans in Paracoccus versutus cells.

The ability of the plasmid pAMI2 of Paracoccus aminophilus JCM 7686 to promote the growth of the N,N-dimethylformamide degradation-related genes is determined. The activation of these genes is shown to be completely dependent on the LuxR family regulator.

Three novel insertion sequences (ISs) that are ISPso1, ISPso2 and ISPso3 of the soil bacterium paracoccus solventivorans DSM 11592 were identified by transposition into entrapment vector pMEC1. Analysis of the distribution of P. solventivorans revealed that ISPso2- like elements is the most widely spread of the elements in nine species of the genus Paracoccus.