Genetic analysis of maintenance of pEC156, a naturally occurring Escherichia coli plasmid that carries genes of the EcoVIII restriction-modification system.

In the present study the role of the mechanisms responsible for maintenance of a natural plasmid pEC156, that carries genes of the EcoVIII restriction-modification system was investigated. Analysis of this plasmid's genetic content revealed the presence of genetic determinants suggesting two such mechanisms. The first of them relies on site specific recombination utilizing the Xer/cer molecular machinery, while the second involves a restriction-modification system as an addiction module. Our analysis indicated that three factors affect the maintenance of pEC156: (i) a cis-acting cer site involved in resolution of plasmid multimers, (ii) a gene coding for EcoVIII endonuclease, and (iii) plasmid copy number control. The lowest stability was observed with pEC156 derivatives deprived of the cer site. Decreased stability of pEC156 derivatives was also observed in E.coli strains deficient in genes coding for proteins involved in plasmid multimer resolution (XerC, XerD, ArgR and PepA). A similar effect, but to a much lesser extent was observed for the pEC156 derivative without a functional gene coding for EcoVIII endonuclease. Our results indicate that the presence of the cer site is more important for pEC156 stable maintenance than the presence of a functional gene coding for EcoVIII endonuclease. In our work we also tested maintenance of pEC156 possessing a ColE1-type replicon in bacteria belonging to Enterobacteriaceae family. We have found that pEC156 was most stably maintained in Enterobacter cloacae and Klebsiella oxytoca representing coli-type enterobacteria. We have found that in all enterobacteria tested pEC156 derivatives deficient in the cer site were significantly less stably maintained than cer(+) variants.

Molecular characterization of plasmid pMbo4.6 of Moraxella bovis ATCC 10900.

We report the characterization of a small cryptic plasmid unlike any previously described from Moraxella bovis ATCC 10900, a Gram-negative bacterium belonging to the family Moraxellaceae. The complete nucleotide sequence of the plasmid pMbo4.6 was determined. The plasmid was analyzed and found to be 4658 in size with a G+C content of 38.6 mol %. Computer analysis of the sequence data revealed four major open reading frames encoding putative proteins of 10.1 (ORF1), 64.2 (ORF2), 45.7 (ORF3), and 12.1 kDa (ORF4). ORF1 and ORF2 encode proteins that show a high level of amino acid sequence similarity (44 %) with some mobilization proteins. ORF3 encodes a protein showing a relatively high amino acid sequence similarity (about 40 %) with several plasmid replication initiator proteins. Upstream of ORF3, a 320-bp intergenic region, constituting the putative origin of replication that contained an AT-rich region followed by four direct repeats, was identified. This set of repeated sequences resembles iteron structures and plays an important role in the control of plasmid replication by providing a target site for the initiation of transcription and replication factors (IHF and RepA). Several palindromic sequences, inverted repeats, and hairpin-loop structures, which might confer regulatory effects on the replication of the plasmid, were also noted. ORF4 encodes an uncharacterized protein, conserved in bacteria, belonging to the DUF497 family. Sequence analysis and structural features indicate that pMbo4.6 replicates by a theta mechanism.

Unbalanced restriction impairs SOS-induced DNA repair effects.

The contribution of a type II restriction-modification system (R-M system) to genome integrity and cell viability was investigated. We established experimental conditions which enabled the achievement of hemimethylated and unmethylated states for the specific bases of the recognition sequences of the host's DNA. To achieve this, we constructed the MboII R-M system containing only one (i.e. M2.MboII) out of two functional MboII methyltransferases found in Moraxella bovis. Using the incomplete R-M system we were able to perturb the balance between methylation and restriction in an inducible manner. We demonstrate that upon the SOS-induced DNA repair in the mitomycin C treated cells, restriction significantly reduces cell viability. Similar results for the well-studied wild type EcoRI R-M system, expressed constitutively in Escherichia coli, were obtained. Our data provide further insights into the benefits and disadvantages of maintaining of a type II R-M system, highlighting its impact on host cell fitness.

M1.MboII and M2.MboII type IIS methyltransferases: different specificities, the same target.

Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction-modification (R-M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 5'-GAAGA-3'/3'-CTTCT-5'. M1.MboII modifies the last adenine in the recognition sequence 5'-GAAGA-3' to N(6)-methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 3'-CTTCT-5', yielding N(4)-methylcytosine, and moreover is able to methylate single-stranded DNA. The protein exists in solution as a monomer of molecular mass 30 000+/-1000 Da under denaturing conditions. Divalent cations (Ca(2+), Mg(2+), Mn(2+) and Zn(2+)) inhibit M2.MboII methylation activity. It was found that the isomethylomer M2.NcuI from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R-M system, consisting of two methyltransferases genes and the mboIIR gene, is the most stable and the least harmful to bacterial cells.