Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST

Abstract

The nucleotide sequence of the 4,377-bp chromosomal region of Pseudomonas fluorescens ST that codes for the oxidation of styrene to phenylacetic acid was determined. Four open reading frames, named styA, styB, styC, and styD, were identified in this region. Sequence analysis and biotransformation assays, performed with batch and continuous cultures, allowed us to identify the functions of the sequenced genes. styA and styB encode a styrene monooxygenase responsible for the transformation of styrene to epoxystyrene; styC codes for the second enzyme of the pathway, an epoxystyrene isomerase that converts epoxystyrene to phenylacetaldehyde; and the styD gene produces a phenylacetaldehyde dehydrogenase that oxidizes phenylacetaldehyde to phenylacetic acid. StyA, 415-amino-acids long, was found to be weakly homologous to p-hydroxybenzoate hydroxylase from both P. fluorescens and P. aeruginosa and to salicylate hydroxylase from P. putida, suggesting that it might be a flavin adenine dinucleotide-binding monooxygenase. StyB was found to be partially homologous to the carboxyterminal part of the 2,4-dichlorophenol-6-monooxygenase encoded by plasmid pJP4, while the styC product did not share significant homology with any known proteins. The fourth open reading frame, styD, could encode a protein of 502 amino acids and was strongly homologous to several eukaryotic and prokaryotic aldehyde dehydrogenases. The order of the genes corresponds to that of the catabolic steps. The previously suggested presence of the gene for epoxystyrene reductase, which directly converts epoxystyrene to 2-phenylethanol (A. M. Appl. Environ. Microbiol. 61:121-127, 1996), has not been confirmed by sequencing and by biotransformation assays performed in continuous cultures. A copy of the insertion sequence IS1162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.

Toward Biorecycling: Isolation of a Soil Bacterium That Grows on a Polyurethane Oligomer and Monomer

“…Considering the genomic potential and the substrate spectrum a degradation pathway for 2,4-TDA with candidate genes encoding the enzymes involved can be suggested (Figure 3). Although also a monooxygenation of an aromatic ring lacking hydroxyl groups has been reported in the case of styrene (Beltrametti et al, 1997), an initiation of the degradation of not yet activated aromatics by flavin monooxygenases is rather unlikely (Van Berkel et al, 2006). In contrast to that, hydroxylation of substituents at the aromatic ring, like the methyl group of toluene, is common (Assinder and Williams, FIGURE 3 | Proposed degradation pathway including extradiol cleavage of 4-aminocatechol for 2,4-TDA in the putative Pseudomonas sp.…”

Biosurfactants produced by Pseudomonas fluorescens and soft‐rotting of harvested florets of broccoli and cauliflower

“…2b) suggests that biosurfactant production might be highly regulated or that insertion sequences abolish its production. IS21-like insertion sequences affecting expression of specific genes are found in P. fluorescens isolates (Solinas et al, 1995;Beltrametti et al, 1997). Clearly, the 7·1-kb DNA fragment complementing the biosurfactant activity awaits nucleotide sequencing and translational analyses, from which the open reading frame encoding the type of surfactant produced could be identified and insights into the genetic organization with regard to the regulation of the locus could be obtained.…”

Genetic and Functional Analysis of the Styrene Catabolic Cluster of Pseudomonas sp. Strain Y2

“…Airborne emissions of styrene, even at low concentrations, often cause a problem because of their malodorous character and their toxic and carcinogenic effects (42). The removal of styrene from industrial waste gases could be accomplished by using styrene-degrading bacteria as biocatalysts; however, little is known concerning the microbial metabolism of styrene (6,42). Two main routes of aerobic styrene breakdown have been described: (i) oxidation of the vinyl side chain with the formation of phenylacetate and (ii) initial oxidation of the aromatic nucleus (42).…”

“…The only information about the organization of the styrene catabolic genes has been recently obtained for Pseudomonas fluorescens ST (6,25). This strain degrades styrene by oxidation of its lateral chain, and it has been shown that the upper pathway for the conversion of styrene to phenylacetate is encoded by four catabolic genes, styABCD (Fig.…”

“…1A). Although 2-phenylethanol accumulates in styrene-grown cells (25), genetic and biochemical analyses of the styABCD cluster have suggested that this compound is not an intermediate of the styrene catabolism in P. fluorescens ST (6). However, 2-phenylethanol appears to be an intermediate of styrene catabolism in Corynebacterium sp.…”