Degradative Plasmids: Comparative Regulatory Sequences

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This proposal aims at studying the mode of regulation of catabolic and anabolic genes in Pseudomonas with a view to determining which nucleotide sequences are critical for the transcription of downstream gene(s). The systems to be studied comprise the structural and regulatory genetic regions of (i) the clc cluster, a group of plasmid-borne genes involved in the degradation of Chlorocatechol; (ii) the catBC operon, a tightly-linked cluster of two chromosomal genes involved in the degradation of catechol; and (iii) the alg cluster, a group of chromosomal genes involved in the biosynthesis of a polysaccharide alginate. We propose to delineate the critical nucleotides in the upstream regions of the clcABD, catBC and algD gene clusters by altering specific nucleotides at various locations by site directed mutagenesis and then examining the effects of such alterations on the expression of the downstream genes. In order to detect other critical nucleotides at other locations, nonspecific mutagenesis will be used to alter upstream nucleotides at random and the effects of such alterations will be noted. Since many regulatory genes controlling structural gene expression in a positive manner are known to interact at upstream sequences between 50 and 100 nucleotides upstream of the transcription initiation site, the products of algR, clcR and catR genes will be purified after hyperproduction and their interaction with the upstream sequences at -50 to -100 regions will be studied by footprinting and electrophoretic protein DNA binding assays. These experiments should provide us with a wealth of information on the nature of gene regulation in Pseudomonas, a bacterial species which is occasionally a human pathogen, in some cases an important plant pathogen, and in other cases is being exploited to an increasing degree for industrial purposes.