The DNA sequences of many small staphylococcal plasmids possess open reading frames with 70-80 sequence identity to that of the repD product of the chloramphenico1-resistance plasmid pC221. This thesis describes the results of studies of the REP D protein, required for initiation of replication of pC221 both in vivo and in vitro. REP D was expressed in Escherichia coli, using a purpose-built expression vector, as 10-15% of the total cellular protein. Purification yields 40-50mg of REP D from one litre of induced culture. The protein displays a molecular weight of 35 kDa on denaturing gel electrophoresis, in close agreement with that predicted from the DNA sequence, and 75 kDa by gel filtration of the native form, suggesting a dimeric quaternary structure. N-terminal amino acid sequence analysis of REP D revealed the absence of the presumed initiator methionine residue. In vitro REP D has both sequence-specific endonuclease and type-I topo-isomerase activities. The target is oriD, the replication origin of pC221, contained within the repD reading frame. Supercoiled pC221 and plasmids with related origin sequences (such as pT181) are relaxed by REP D in high ionic strength buffers, whereas relaxed, covalently closed plasmid pC221 only is nicked by REP D at a precise location in the (+) strand (oriD) under conditions of low ionic strength. Both reactions require Mg2+ but not ATP. Binding of REP D to oriD was demonstrated, using restriction fragments of pC221 and synthetic oligonucleotides. Competition experiments suggest two DNA binding sites. The first is involved in the recognition of the conserved cleavage site. The second site discriminates between such potential origins by binding only to a "specificity" sequence present within oriD. After nicking the relaxed origin DNA substrate REP D is found to be covalently attached to the resultant 5' end. Radiolabelling and hydrolysis revealed the linkage to be via a phosphodiester bond to a tyrosine residue. Peptide sequencing identified the tyrosine to be Tyr188 of REP D, conserved in the sequences of all six related REP proteins known. A mechanism for initiation of plasmid replication is suggested on the basis of these results. The nicking reaction is thought to trigger the initiation of replication of pC221, the free 3' hydroxyl group generated priming synthesis of the new (+) strand. The requirement in vitro for a relaxed substrate to demonstrate the absolute sequence specificity seen in vivo suggests the local topology of the origin region may have low superhelical density in vivo.

The 2 micron plasmid of the budding yeast Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number around 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-coded partitioning system and an amplification system to exhibit nearly chromosome-like stability in its host. Plasmid behavior under conditions that missegregate chromosomes suggest that the partitioning system couples plasmid segregation to chromosome segregation. However, the mechanism of this coupling has not been elucidated. A plausible model, consistent with current evidence, is the hitchhiking model, in which plasmid-chromosome tethering provides the basis for faithful plasmid partitioning. Testing this hypothesis unequivocally has been difficult, primarily because of the technical limitations posed by the small size of the budding yeast nucleus and poor resolution of chromosomes. As a result, cell biological assays based on fluorescence microscopy have had only modest success in addressing this problem. In the present study, I devised an experimental verification of the hitchhiking model using a single copy derivative of the 2 micron plasmid as a reporter. The rationale was to establish various conditions that force sister chromatids to co-segregate during mitosis in a bias-free manner or with a bias towards the daughter. The segregation patterns of plasmid sisters were followed under these conditions. The sum of the results from this analysis is accommodated by the hitchhiking model, with sister plasmids associating with sister chromatids in a one-to-one fashion. Episomes of mammalian viruses belonging to the gamma-herpes and papilloma families utilize a hitchhiking mechanism to persist in cells during the latent phase of their infection. Two of the viral partitioning systems have been reconstituted in S. cerevisiae. We wished to exploit these systems to characterize the efficiency of non-native chromosome tethering systems in promoting equal segregation of viral plasmids in S. cerevisiae. We find that the 2 micron plasmid partitioning system is considerably superior to the viral systems. This could be due to the higher efficiency of plasmid-chromosome association and/or due to the ability of plasmid sisters to tether to sister chromatids. / text

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Plasmid ColIb-P9 is a 93.2 kb self-transmissible plasmid, belonging to the I1 incompatibility group. Whilst much data had been gained concerning the molecular biology of conjugation mediated by this plasmid, a lack of information exsisted concerning the genetic organisation of the transfer genes. A physical map of the plasmid was constructed by detailed restriction analysis of DNA fragments sub-cloned from ColIb-P9. These fragments were also used to locate the positions of the transfer gene sog and the origin of transfer. Transposons Tn5 and Tnl723 were used to construct insertion mutants at defined points in ColIb-P9 and the effect of these on the expression of various transfer-related functions was studied. Using this technique, the probable location of the genes encoding the thick and thin sex pili were identified and also the site of the plasmid-encoded nuclease gene. The exact location of the entry exclusion gene was also determined. Complementation studies using the sub-cloned fragments of ColIb-P9 and a set of cosmid-clones generated from ColIbdrd-1 indicated that a positive regulator of the expression of the transfer genes exsisted and that this was composed of two genetically distinct elements. Studies involving wild type ColIb-P9 (drd+) indicated that this positive regulatory system is subject to negative control in cells containing the drd+ plasmid. The information gained from these studies was combined into a model of the organisation of the transfer genes of ColIb-P9. This defines at least three separate Tra regions, covering some 50 kb of the plasmid, with the origin of transfer located at one end of the transfer region.

BACKGROUND: Clostridium sordellii can cause severe infections in animals and humans, the latter associated with trauma, toxic shock and often-fatal gynaecological infections. Strains can produce two large clostridial cytotoxins (LCCs), TcsL and TcsH, related to those produced by Clostridium difficile, Clostridium novyi and Clostridium perfringens, but the genetic basis of toxin production remains uncharacterised. RESULTS: Phylogenetic analysis of the genome sequences of 44 strains isolated from human and animal infections in the UK, US and Australia placed the species into four clades. Although all strains originated from animal or clinical disease, only 5 strains contained LCC genes: 4 strains contain tcsL alone and one strain contains tcsL and tcsH. Four toxin-positive strains were found within one clade. Where present, tcsL and tcsH were localised in a pathogenicity locus, similar to but distinct from that present in C. difficile. In contrast to C. difficile, where the LCCs are chromosomally localised, the C. sordellii tcsL and tcsH genes are localised on plasmids. Our data suggest gain and loss of entire toxigenic plasmids in addition to horizontal transfer of the pathogenicity locus. A high quality, annotated sequence of ATCC9714 reveals many putative virulence factors including neuraminidase, phospholipase C and the cholesterol-dependent cytolysin sordellilysin that are highly conserved between all strains studied. CONCLUSIONS: Genome analysis of C. sordellii reveals that the LCCs, the major virulence factors, are localised on plasmids. Many strains do not contain the LCC genes; it is probable that in several of these cases the plasmid has been lost upon laboratory subculture. Our data are consistent with LCCs being the primary virulence factors in the majority of infections, but LCC-negative strains may precipitate certain categories of infection. A high quality genome sequence reveals putative virulence factors whose role in virulence can be investigated.

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