GENETIC AND FUNCTIONAL-ANALYSIS OF THE STYRENE CATABOLIC CLUSTER OF PSEUDOMONAS SP. STRAIN Y2

The chromosomal region of Pseudomonas sp, strain Y2 involved in the co nversion of styrene to phenylacetate (upper catabolic pathway) has bee n cloned and sequenced, Four catabolic genes, styABCD, and two regulat ory genes, stySR, were identified, This gene cluster when transferred to Escherichia coli W confers to this phenylacetate-degrading host the ability to grow on styrene as the sole carbon and energy source, Gene s styABCD are homologous to those encoding the styrene upper catabolic pathway in Pseudomonas fluorescens ST, Northern blot analyses have co nfirmed that genes styABCD constitute a transcription unit, The transc ription start site of the sty operon was mapped 33 nucleotides upstrea m of the styA translational start codon, The styS and styR genes, whic h form an independent transcriptional unit, are located upstream of th e styABCD operon, and their gene products show high similarity to memb ers of the superfamily of two-component signal transduction systems, T he styS gene product is homologous to histidine kinase proteins, where as the styR gene product exhibits similarity at its N-terminal domain with cluster 1 of receiver modules and at its C terminus with the LuxR /FixJ family 3 of DNA-binding domains, Expression of the catabolic ope ron decreased significantly in the absence of the stySR genes and was restored when the stySR genes were provided in trans in the presence o f styrene, suggesting that the stySR system behaves as a styrene-induc ible positive regulator of the styABCD operon, Finally, a gene encodin g a phenylacetyl-coenzyme A ligase that catalyzes the first step in th e phenylacetate catabolism (styrene lower catabolic pathway) has been identified upstream of the styS gene, This activity was found to be in duced in Pseudomonas sp. strain Y2 cells grown on styrene but not pres ent in cells grown on glycerol, These results strongly suggest that th e genes responsible for the complete mineralization of styrene are clu stered in the chromosome of Pseudomonas sp, strain Y2.