Expression and regulation of genes encoding quinoprotein alcohol dehydrogenases in Pseudomonas putida HK5

Abstract

Pseudomonas putida HK5 produces three different quinoprotein alcohol dehydrogenases, ADH-I (qedA), ADH-IIB (qbdBA) and ADH-IIG (qgdA). Gene organization of qedA, the gene for ADH-I, and other 10 genes in the cluster was related to the genome sequences of five other Pseudomonas strains. Insertion mutations in either qedA, exaE, or agmR eliminated ADH-I activity, although the mutants were still able to grow on ethanol but at a slower rate than that of the wild-type strain. In addition, roles of genes encoding two response regulators, exaE or agmR, located downstream of qedA were inferred from the properties of exaE- or agmR-disrupted mutants and the gene complementation results. Both exaE and agmR gene products were strictly necessary for qedA transcriptions, although there may be other sensing regulatory factor(s) involved. Mutant analysis demonstrated the tentative involvement of agmR, but not exaE, in the induction of ADH-IIB and ADH-IIG activities. The promoter activities of genes involving in alcohol oxidation were determined using a transcriptional lacZ fusion promoter-probe vector. Ethanol was the best inducer for the divergent promoters of qedA and qedC encoding ADH-I and a cytochrome c, respectively. Primary and secondary C3-, C4-alcohols and butyraldehyde specifically induced the divergent promoters of qbdBA and aldA encoding ADH-IIB and an NAD-aldehyde dehydrogenase, respectively. The qgdA promoter of ADH-IIG was well responded to S-(+)-1,2-propanediol induction. The hypothetical regulatory network scheme of three distinct alcohol dehydrogenases in the oxidation system of alcohol in P. putida HK5 was derived.