No description available.

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No description available.

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Stuttgart, Univ., Diss., 2003.

Berlin, Freie Universiẗat, Diss., 2004. / Dateiformat: zip, Dateien im PDF-Format.

Bielefeld, Universiẗat, Habil.-Schr., 1999.

A conjugation system in Staphylococcus aureus has been investigated and shown to be determined, at least in part, by genes carried on plasmids. Conjugation required cell-to-cell contact but not calcium ions. The frequency of conjugation depended on the recipient used and on the incubation conditions. Two conjugative plasmids were mapped by restriction enzyme analysis but experiments to clone the conjugation-determining region were unsuccessful although separate regions specifying gentamicin resistance, ethidium bromide resistance and cadmium resistance were cloned. The gentamicin resistance determinant was probably part of Tn4001. Deletion of various sized pieces of DNA from one of the plasmids resulted in reduction of its ability to specify conjugation but no specific part of this plasmid could be implicated in the process. Further experiments led to the conclusion that this particular plasmid (p8325-4) is probably not self-transmissible but transferred by a phage-mediated system. Strains of Staphylococcus aureus produced a pheromone-like substance that elicited a clumping response in Streptococcus faecalis but no evidence was found for the involvement of staphylococcal conjugative plasmids in this. The conjugative plasmid, p8325-2, mobilized a small plasmid (pT181) but not a chromosomal gene. Insertion of transposon Tn551 was used to produce mutants of the conjugative plasmid p8325-2. Some twenty-six mutants were studied and the position of Tn551 in them mapped. There were preferred regions of insertion for Tn551 and twenty out of the twenty-six mutants had altered ability to conjugate. One showed a significantly higher frequency of conjugation and the other nineteen, all with substantially lower frequencies of conjugation, were mapped to two well-separated regions of the plasmid. Similarity between the locations of these putative regions and those reported for some other conjugative plasmids from staphylococci is striking and suggests a common origin.

The obligate methylotroph, Methylophilus methylotrophus, used by ICI for its Single Cell Protein production, may represent a valuable alternative host organism to E. coli for the commercial production of heterologous gene products. The organism has the advantages of being safe, coupled with an ability to grow well, in large quantities, on a cheap carbon source. This thesis describes the construction, characterisation and analysis of a series of plasmid cloning vectors designed for use in M.methylotrophus. The vectors are based on the IncQ plasmid R300B and maintain a broad host range with an increased capacity for easy-to-use cloning sites mainly derived from the E.coli plasmids pBR322 and pBR328. All the vectors thus carry antibiotic- resistance genes containing restriction sites which could lead to insertional inactivation as a means of detecting recombinants. One plasmid in particular, pGSS33, has four antibiotic-resistance genes all of which contain at least one such restriction site. The first demonstration of expression of a eukaryotic coding sequence, murine dihydrofolate reductase (DHFR), in M.methylotrophus has been described. This has been followed by expression of E.coli B-galactosidase and synthetic human ?-1 interferon, all making use of pGSS vectors. The pGSS15-DHFR plasmid, pDHFR2.43, may turn out to be a valuable test plasmid for studying the stability of cloned eukaryotic coding sequences in both E.coli and M.methylotrophus; it is readily detected (trimethoprim-resistant) and can be grown with or without selection. A start has been made towards providing increased expression from vectors carrying strong E.coli promoters (lacUV5 and synthetic trp) which have been demonstrated to work well in M.methylotrophus. Broad host range cosmid vectors have been constructed which have the potential to be used for the production of gene-banks in Gram-negative organisms other than E.coli. Copy numbers of the vector plasmids have been determined in E.coli strains and several different methods of measurement reviewed in an attempt to find one suitable for use with M.methylotrophus. An encouraging lead has been identified in the search for a high copy number plasmid, which coupled with a strong promoter, could provide the basis for a very efficient batch production process. Thus with the availability of easy-to-use cloning vectors, convenient delivery systems and the accumulated evidence of strong E.coli promoters working efficiently in M.methylotrophus, this organism can seriously be considered as a safe alternative host to E.coli.