Analysis of cloned aromatic catabolic genes from Klebsiella pneumaniae.
thesisposted on 19.11.2015, 09:07 by Neil David. Robson
The organisation of two aromatic catabolic pathways operational in Klebsiella pneumoniae has been investigated through the analysis of plasmid-based genomic clones. A single genomic clone encoding all of the activities required for the metabolism of 3-hydroxybenzoate was isolated on an 8.0Kbp SphI fragment via a cloning strategy determined by the analysis of an existing partial clone. The presence of a plasmid containing this fragment allowed strains of Escherichia coli to grow on 3- hydroxybenzoate as sole carbon and energy source. Sub-cloning experiments revealed that the genes involved in this pathway;mhbm encoding 3-hydroxybenzoate mono-oxygenase, mhbD encoding 2,5-dihydroxybenzoate dioxygenase, mhbl (maleylpyruvate isomerase), mhbH (fumarylpyruvate hydrolase) and the regulator gene mhbR, were arranged in the order mhbRDHMI. Preliminary analysis of the regulatory system has suggested that the expression of the mhb genes is under positive control. High level expression of the 2,5-dihydroxybenzoate dioxygenase encoded by mhbD was detected from several sub-clones. This allowed the development of a three-step purification procedure which resulted in a preparation suitable for N-terminal amino acid sequence determination. Full nucleotide sequence was obtained for the mhbD gene with assignment of the reading frame made on the basis of the protein's N-terminal sequence. Comparison of this sequence with existing databases failed to detect strong homologies with other dioxygenases. Two discrete genomic clones encoding genes involved in the catabolism of homoprotocatechuate (HPC); hpcB encoding HPC dioxygenase, hpcC (CHMS dehydrogenase), hpcD (CHM isomerase), hpcE (COHED decarboxylase), hpcF (HHDD isomerase), hpcG (OHED hydratase), hpcH (HHED aldolase) and hpcR (regulator gene), were subjected to restriction analysis and the gene organisation investigated by the sub-cloning. of specific fragments. The genes were found to be arranged in the order hpcR(EF)CBDGH where the precise relationship of the hpcE and hpcF genes was not defined. This gene order is identical to that determined for the corresponding system in Escherichia coli C.