Analysis of cloned genes for aromatic catabolism: The hydroxyphenylacetate genes of Escherichia coli and Klebsiella pneumoniae.
thesisposted on 19.11.2015, 09:08 by Tony. Fawcett
Cloned genes, from Escherichia coli C, for the catabolism of homoprotocatechuate were available. The organisation and expression of these genes was investigated by a number of molecular genetic techniques. The exact position of the 5' end of one of the genes, hpcG, was determined by a combination of Southern blotting and DNA sequencing and subsequently used as a marker to aid the positioning and illucidation of the direction of transcription of the other genes. The genes were found to be arranged in two blocks of structural genes which were both transcribed in the same direction and a separate regulatory gene. Northern hybridisation analysis of RNA from wild type cells and from cells containing cloned genes indicated the presence of three transcripts of 4.5, 2.7 and 1.6 kbp. A model for the position of the hpc genes, their regulation and number of transcriptional units is proposed. One of the catalytic proteins, CHMS dehydrogenase, was purified and some of its properties investigated; homology between the amino-terminal amino acid sequence of this enzyme and the equivalent protein from Klebsiella pneumoniae M5a1 was very high. An oligonucleotide derived from shared protein sequence of the CHMS dehydrogenases from E. coli C and K, pneumoniae M5a1 was used to investigate homology in related species by Southern hybridisation. E. coli strains B and W DNA contained a hybridising fragment when tested at high stringency, but Pseudomonas putida DNA did not hybridise under these conditions. The same oligonucleotide was used to screen a K. pneumoniae M5a1 genomic library by colony hybridisation and clones carrying hpc genes were succesfully isolated. Initial analysis of these clones suggest that little gross homology exsists between the hpc genes of E. coli C and K. pneumoniae M5a1.