Dataset Information

Coregulation by phenylacetyl-coenzyme A-responsive PaaX integrates control of the upper and lower pathways for catabolism of styrene by Pseudomonas sp. strain Y2.

ABSTRACT: The P(styA) promoter of Pseudomonas sp. strain Y2 controls expression of the styABCD genes, which are required for the conversion of styrene to phenylacetate, which is further catabolized by the products of two paa gene clusters. Two PaaX repressor proteins (PaaX1 and PaaX2) regulate transcription of the paa gene clusters of this strain. In silico analysis of the P(styA) promoter region revealed a sequence located just within styA that is similar to the reported PaaX binding sites of Escherichia coli and the proposed PaaX binding sites of the paa genes of Pseudomonas species. Here we show that protein extracts from some Pseudomonas strains that have paaX genes, but not from a paaX mutant strain, can bind and retard the migration of a P(styA) specific probe. Purified maltose-binding protein (MBP)-PaaX1 fusion protein specifically binds the P(styA) promoter proximal PaaX site, and this binding is eliminated by the addition of phenylacetyl-coenzyme A. The sequence protected by MBP-PaaX1 binding was defined by DNase I footprinting. Moreover, MBP-PaaX1 represses transcription from the P(styA) promoter in a phenylacetyl-coenzyme A-dependent manner in vitro. Finally, the inactivation of both paaX gene copies of Pseudomonas sp. strain Y2 leads to a higher level of transcription from the P(styA) promoter, while heterologous expression of the PaaX1 in E. coli greatly decreases transcription from the P(styA) promoter. These findings reveal a control mechanism that integrates regulation of styrene catabolism by coordinating the expression of the styrene upper catabolic operon to that of the paa-encoded central pathway and support a role for PaaX as a major regulatory protein in the phenylacetyl-coenzyme A catabolon through its response to the levels of this central metabolite.

SUBMITTER: del Peso-Santos T

PROVIDER: S-EPMC1483010 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Coregulation by phenylacetyl-coenzyme A-responsive PaaX integrates control of the upper and lower pathways for catabolism of styrene by Pseudomonas sp. strain Y2.

del Peso-Santos Teresa T Bartolomé-Martín David D Fernández Cristina C Alonso Sergio S García José Luis JL Díaz Eduardo E Shingler Victoria V Perera Julián J

Journal of bacteriology 20060701 13

The P(styA) promoter of Pseudomonas sp. strain Y2 controls expression of the styABCD genes, which are required for the conversion of styrene to phenylacetate, which is further catabolized by the products of two paa gene clusters. Two PaaX repressor proteins (PaaX1 and PaaX2) regulate transcription of the paa gene clusters of this strain. In silico analysis of the P(styA) promoter region revealed a sequence located just within styA that is similar to the reported PaaX binding sites of Escherichia ...[more]

PMID: 16788190

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Project description:The chromosomal region of Pseudomonas sp. strain Y2 involved in the conversion of styrene to phenylacetate (upper catabolic pathway) has been cloned and sequenced. Four catabolic genes, styABCD, and two regulatory 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. Genes styABCD are homologous to those encoding the styrene upper catabolic pathway in Pseudomonas fluorescens ST. Northern blot analyses have confirmed that genes styABCD constitute a transcription unit. The transcription start site of the sty operon was mapped 33 nucleotides upstream of the styA translational start codon. The styS and styR genes, which form an independent transcriptional unit, are located upstream of the styABCD operon, and their gene products show high similarity to members of the superfamily of two-component signal transduction systems. The styS gene product is homologous to histidine kinase proteins, whereas 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 operon decreased significantly in the absence of the stySR genes and was restored when the stySR genes were provided in trans in the presence of styrene, suggesting that the stySR system behaves as a styrene-inducible positive regulator of the styABCD operon. Finally, a gene encoding a phenylacetyl-coenzyme A ligase that catalyzes the first step in the phenylacetate catabolism (styrene lower catabolic pathway) has been identified upstream of the styS gene. This activity was found to be induced in Pseudomonas sp. strain Y2 cells grown on styrene but not present in cells grown on glycerol. These results strongly suggest that the genes responsible for the complete mineralization of styrene are clustered in the chromosome of Pseudomonas sp. strain Y2.

Project description:During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl coenzyme A (PAA-CoA) by a ligase, PaaK, and then PAA-CoA is epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation through the tricarboxylic acid (TCA) cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under the control of the LuxIR-like quorum sensing (QS) system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than that of the wild-type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in levels of internal accumulation compared to the wild-type level. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in suppressing B. cenocepacia CepIR-activated virulence.IMPORTANCE The opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR which, upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.

Project description:Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for ?-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 10(5) ± 0.03 × 10(5) M(-1) s(-1)) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1?(2'-propanoate)-3a?-H-4?(3?-propanoate)-7a?-methylhexahydro-5-indanone (kcat/Km = 2.4 × 10(5) ± 0.1 × 10(5) M(-1) s(-1) and 3.2 × 10(5) ± 0.3 × 10(5) M(-1) s(-1), respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes.

Project description: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. Marconi, F. Beltrametti, G. Bestetti, F. Solinas, M. Ruzzi, E. Galli, and E. Zennaro, 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 ISI162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.

Dataset Information

Coregulation by phenylacetyl-coenzyme A-responsive PaaX integrates control of the upper and lower pathways for catabolism of styrene by Pseudomonas sp. strain Y2.

Publications

Coregulation by phenylacetyl-coenzyme A-responsive PaaX integrates control of the upper and lower pathways for catabolism of styrene by Pseudomonas sp. strain Y2.

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