Physiological Analysis of the Expression of the Styrene Degradation Gene Cluster in Pseudomonas fluorescens ST

Abstract

The effects of different carbon sources on expression of the styrene catabolism genes in Pseudomonas fluorescens ST were analyzed by using a promoter probe vector, pPR9TT, which contains transcription terminators upstream and downstream of the ␤-galactosidase reporter system. Expression of the promoter of the stySR operon, which codes for the styrene two-component regulatory system, was found to be constitutive and not subject to catabolite repression. This was confirmed by the results of an analysis of the stySR transcript in P. fluorescens ST cells grown on different carbon sources. The promoter of the operon of the upper pathway, designated PstyA, was induced by styrene and repressed to different extents by organic acids or carbohydrates. In particular, cells grown on succinate or lactate in the presence of styrene started to exhibit ␤-galactosidase activity during the mid-exponential growth phase, before the preferred carbon sources were depleted, indicating that there is a threshold succinate and lactate concentration which allows induction of styrene catabolic genes. In contrast, cells grown on glucose, acetate, or glutamate and styrene exhibited a diauxic growth curve, and ␤-galactosidase activity was detected only after the end of the exponential growth phase. In each experiment the reliability of the reporter system constructed was verified by comparing the ␤-galactosidase activity and the activity of the styrene monooxygenase encoded by the first gene of the styrene catabolic operon.Styrene is a chemical that is used extensively in the manufacturing of plastics and synthetic rubbers. This toxic compound is released into the environment mainly through factory wastewater, evaporation, and pyrolysis of polystyrene. Different routes for styrene catabolism in different microorganisms have been described (8,9,17,21,29,31). Recently, strains belonging to the genus Pseudomonas have been studied more extensively both at the physiological level (21-23) and the molecular level (2,17,24,30). In these strains the catabolic genes are organized in a cluster whose expression requires the presence of two genes, styS and styR, which are organized in an operon and code for a sensor kinase and a regulatory DNA binding protein, respectively. Two-component regulatory systems for genes involved in aromatic hydrocarbon degradation have been described previously only for toluene degradation in Pseudomonas putida F1 and Thauera sp. strain T1 (6, 15) and for degradation of biphenyls in Rhodococcus sp. strain M5 (14).In our laboratory, Pseudomonas fluorescens ST, which is able to grow on styrene as a sole carbon source, has been characterized, and both the regulatory genes (styS and styR) and the upper pathway genes (styA, styB, styC, and styD), which code for conversion of styrene into phenylacetic acid, have been sequenced (2,17,18). At the moment, our interest is focused on characterization of the regulatory system and, in particular, on the effects of different carbon sources on styrene-induced expression of the regulatory an...

Transcriptional regulation of styrene degradation in Pseudomonas putida CA-3 The GenBank accession number for the sequence determined in this work is AF257095.

“…Therefore, it appears that a transcriptional termination signal(s) exists between the paak and the styR genes which prevents readthrough transcription into the regulatory elements styS and styR. This conclusion is supported by recent work involving the promoter region of stySR from the styrene-degrading P. fluorescens ST (Santos et al, 2000). Similarly, it would appear that a transcriptional termination signal(s) exists between styR and styA, which prevents the progress of RNA polymerase into the upper pathway genes beginning with styA.…”

“…This is in contrast to the styrenedegrading strain Xanthobacter 124X, as growth of the bacterium on PAA results in detectable levels of activity from upper-pathway-associated enzymes styrene oxide isomerase and phenylacetaldehyde dehydrogenase (Hartmans et al, 1989). Furthermore, a recent study with P. fluorescens ST suggested that stySR transcription is constitutive regardless of the carbon source (Santos et al, 2000). Therefore, while a common route for styrene catabolism is observed in many of the bacterial species studied to date, it is clear that there are significant differences in how these degradative pathways are regulated.…”

Expansion of growth substrate range in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a ring-fission hydrolase CmtE and induce the tod catabolic operon, respectively

“…In this regard, both TodS and TodT proteins are known to be required for the chemotaxis to aromatic hydrocarbons in P. putida F1 (Parales et al, 2000), indicating that the catabolism and microbial behaviour are co-ordinately regulated, and the chemotaxis is probably controlled at the transcriptional level. Similar two-component signal transduction systems have been identified in the pathways of aerobic styrene degradation (O'Leary et al, 2002;Panke et al, 1999;Santos et al, 2000;Velasco et al, 1998), in other forms of aerobic toluene degradation (Mosqueda et al, 1999), and in anaerobic toluene degradation (Coschigano & Young, 1997;Leuthner & Heider, 1998). The sensor proteins found in anaerobic toluene degradation are shorter than TodS and contain two PAS domains and one histidine kinase domain.…”

Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

“…1). Catabolic repression on the expression of degradation pathways of aromatic compounds by small organic acid molecules has been reported (34,41,53). The mechanism of catabolic repression of the 2,4,6-TCP degradation pathway in JMP134 by glutamate is unknown, but can occur at the transcriptional level, as reported in Pseudomonas spp.…”