REVERSE TRANSCRIPTION-PCR ANALYSIS OF THE REGULATION OF ETHYLBENZENE DIOXYGENASE GENE-EXPRESSION IN PSEUDOMONAS-FLUORESCENS CA-4

Pseudomonas fluorescens strain CA-4 is a bioreactor isolate previously characterised by the presence of a side chain oxidation pathway for e thylbenzene breakdown. In this report a second pathway involving ethyl benzene ring dioxygenation has been identified in this strain. We exam ine here second substrate inhibition of the genes encoding the initial enzymes of this pathway, using reverse transcription (RT)-PCR. The ge nes of the ring-dioxygenation have been cloned and sequenced. They exh ibit near identity to the gene clusters encoding the aromatic ring dio xygenase enzymes of two previously described isopropyl degrading strai ns, Pseudomonas sp. strain JR1 and P. fluorescens IP01. This dioxygena se pathway appears to be the major pathway for ethylbenzene degradatio n in this strain. The expression of these genes appears to be affected by the presence of second carbon substrates. Using RT-PCR we demonstr ate that the negative effect of glutamate present in the growth medium together with ethylbenzene on the rate of ethylbenzene metabolism is mediated at the transcriptional level on the ethylbenzene dioxygenase genes. (C) 1998 Federation of European Microbiological Societies. Publ ished by Elsevier Science B.V. All rights reserved.