A study of gene expression in pseudomonas.
thesisposted on 19.11.2015, 09:07 by Rebecca Louise. Allen
An inherent problem in the study of the genetics of the interesting and potentially commercially useful properties of the pseudomonads is that of gene expression, since many of the genes encoding these properties are not well expressed in an E.coli background. The evidence available at the present time indicates that some Pseudomonas genes may possess different promoter sequences not recognised by E.coli RNA polymerase. An in vitro coupled transcription/translation system based on P.putida has been developed. A comparison of E.coli and broad host range plasmid DNA in this and the equivalent E. coli system showed that although cloned E. coli and vector polypeptides were synthesised in both systems, there was a difference in the polypeptide products directed by broad host range plasmid DNA in the two systems. In particular RSF1010 directed the synthesis of a 73kD polypeptide uniquely in the P.putida system. This was shown to be a polypeptide involved in mobilisation of the plasmid. A broad host rsmge proraoter-probe vector based on RSFlOlO was constructed and used for the shotgun cloning of P.putida promoters. A small subset of fragments which were active as promoters in P.putida but exhibited much lower activity in E.coli were isolated, sequenced and analysed with respect to concensus E. coli and nitrogen-regulated promoter sequences. These isolated DNA fragments may represent promoters which have sequences specifically recognised by Pseudomonas RNA polymerase. An analysis of published Pseudomonas chromosomally-encoded promoters revealed putative Pseudomonas-specific concensus regions.