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 ethylbenzene breakdown. In this report a second pathway involving ethylbenzene ring dioxygenation has been identified in this strain. We examine here second substrate inhibition of the genes encoding the initial enzymes of this pathway, using reverse transcription (RT)-PCR. The genes of the ring-dioxygenation have been cloned and sequenced. They exhibit near identity to the gene clusters encoding the aromatic ring dioxygenase enzymes of two previously described isopropyl degrading strains, Pseudomonas sp. strain JR1 and P. fluorescens IP01. This dioxygenase pathway appears to be the major pathway for ethylbenzene degradation in this strain. The expression of these genes appears to be affected by the presence of second carbon substrates. Using RT-PCR we demonstrate 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.