Analysis of the Promoter for the Pseudomonas Putida catBC Operon

Abstract

Pseudomonas putida is capable of dissimilating benzoate to succinate and acetyl Co-A via the β-ketoadipate pathway. Two of the essential enzymes involved in this process are muconate lactonizing enzyme I, which catalyzes the conversion of cis, cis-muconate to muconolactone, and muconolactone isomerase, which catalyzes the conversion of muconolactone to β-ketoadipate enol-lactone. These enzymes are encoded by the catB and catC genes, respectively. These tightly linked genes have been cloned from the chromosome of P. putida strain RB1 and their nucleotide sequences determined. Based upon the observation that a single mutation will affect the expression of both genes, catB and catC are believed to comprise an operon. Through the use of deletion clones and a promoter probe vector, a single promoter region has been located 5′ of catBC. Sequences located between -44 and -198 were determined to be essential for promoter activity. S1 nuclease and reverse transcriptase mapping were used to locate the transcription initiation site 63 base pairs upstream of the catB ATG initiation codon. Transcripts originating from this site were found only in RNA prepared from benzoate-grown cells. The nucleotide sequence of the catBC promoter was compared to the sequences of other prokaryotic promoters; however, a consensus sequence was not apparent. These results indicate that the catB and catC genes form an inducible operon which is regulated at the tr an scrip tional level and that nucleotide sequences upstream of −44 are required for promoter activity.