Characterization and Role of
            <i>tbuX</i>
            in Utilization of Toluene by
            <i>Ralstonia pickettii</i>
            PKO1

Kahng, Hyung-Yeel; Byrne, Armando M.; Olsen, Ronald H.; Kukor, Jerome J.

doi:10.1128/jb.182.5.1232-1242.2000

Cited by 85 publications

(46 citation statements)

References 55 publications

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“…Interestingly, homologs apparently facilitating the diffusion of aromatic compounds have recently been identified. These include the TbuX toluene channel in Ralstonia picketii (313), the m-xylene channel XylN encoded by the TOL plasmid pWW0 of P. putida (324), the putative salicylate ester channel SalD of Acinetobacter (310), the putative p-cymene channel CymD of P. putida (189), and the putative cumene channel CumH of P. fluorescens (241).…”

Section: Specific Channelsmentioning

confidence: 99%

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Nikaido

2003

Microbiol Mol Biol Rev

3,780

3,800

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Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions

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Section: Specific Channelsmentioning

confidence: 99%

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Nikaido

2003

Microbiol Mol Biol Rev

3,780

3,800

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“…To learn about the transcriptional control of the tmo genes, we first sequenced the region upstream of the tmoABCDEF genes in pMC4 (2) by the dideoxy sequencing method with the ABI Prism dRhodamine terminator kit (Applied Biosystems). A 1,362-nucleotide-long open reading frame 27 bp upstream of tmoA was found, whose deduced amino acid sequence shared significant homology with outer membrane proteins encoded in different toluene degradation operons from a number of gram-negative bacteria that use toluene as the sole C source (i.e., 83% identity with TodX of Pseudomonas putida F1 [34] and DOT-T1E [23] and 48% identity with TbuX of Ralstonia pickettii [13]). The initial gene in the tmo cluster was called tmoX.…”

mentioning

confidence: 99%

Cross-Regulation between a Novel Two-Component Signal Transduction System for Catabolism of Toluene in Pseudomonas mendocina and the TodST System from Pseudomonas putida

et al. 2002

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The tmoABCDEF genes encode the toluene-4-monooxygenase from Pseudomonas mendocina KR1. Upstream from the tmoA gene an open reading frame, tmoX, encoding a protein 83% identical to TodX (todX being the initial gene in the todXFC1C2BADEGIH operon from Pseudomonas putida DOT-T1E) was found. The tmoX gene is also the initial gene in the tmoXABCDEF gene cluster. The transcription initiation point from the tmoX promoter was mapped, and the sequence upstream revealed striking identity with the promoter of the tod operon of P. putida. The tod operon is regulated by a two-component signal transduction system encoded by the todST genes. Two novel genes from P. mendocina KR1, tmoST, were rescued by complementation of a P. putida DOT-T1E todST knockout mutant, whose gene products shared about 85% identity with TodS-TodT. We show that transcription from P tmoX and P todX can be mediated by TmoS-TmoT or TodS-TodT, in the presence of toluene, revealing cross-regulation between these two catabolic pathways.The first step in the metabolism of toluene in Pseudomonas mendocina KR1 is carried out by toluene-4-monooxygenase (T4MO), encoded by the tmoABCDEF gene cluster (38, 39), which is responsible for the oxidation of toluene to p-cresol (35,36). Two other independently regulated gene clusters are known to be required for the metabolism of toluene in P. mendocina KR1 (37). One encodes p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase, which catalyze the successive oxidation of the methyl group of p-cresol to the corresponding acid, p-hydroxybenzoate; the other encodes phydroxybenzoate hydroxylase for forming protocatechuate, which is channeled to the ␤-ketoadipate route through an ortho-cleavage pathway (12). Although T4MO activity is known to be inducible by toluene, chlorinated solvents, and alkanes (6,19), no information regarding the regulators involved in the expression of the tmo genes was available when this work was envisaged. To learn about the transcriptional control of the tmo genes, we first sequenced the region upstream of the tmoABCDEF genes in pMC4 (2) by the dideoxy sequencing method with the ABI Prism dRhodamine terminator kit (Applied Biosystems). A 1,362-nucleotide-long open reading frame 27 bp upstream of tmoA was found, whose deduced amino acid sequence shared significant homology with outer membrane proteins encoded in different toluene degradation operons from a number of gram-negative bacteria that use toluene as the sole C source (i.e., 83% identity with TodX of Pseudomonas putida F1 [34] and and 48% identity with TbuX of Ralstonia pickettii [13]). The initial gene in the tmo cluster was called tmoX. Identification of the transcription initiation point of the tmoX promoter and time course induction in response to different effectors.To analyze the expression of the tmo genes, total RNA was isolated from P. mendocina KR1 grown on citrate in the presence and in the absence of toluene or pcresol, the substrate and the hydroxylated product of the T4MO, respectively. A 23-mer oligonucleotide (...

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“…strain Y2 (37), CumH from Pseudomonas fluorescens IP01 (8), IpbH from P. putida RE204 (GenBank accession no. AF006691), CymD from P. putida F1 (4), TodX from P. putida F1 (38), TbuX from Ralstonia picketti PKO1 (17), and PhlX from Ralstonia eutropha JMP134 (GenBank accession no. AF06589) ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

The TOL Plasmid pWW0 xylN Gene Product from Pseudomonas putida Is Involved in m- Xylene Uptake

Kasai¹,

Inoue²,

Harayama³

2001

J Bacteriol

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The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli. To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type and xylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylN mutant TOL plasmid was not. The apparent K s value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.TOL plasmids in Pseudomonas putida encode the metabolic pathways for the degradation of toluene, xylenes, and their alcohol and carboxylate derivatives (1). The TOL degradation pathways generally consist of two parts: an upper pathway that converts toluene and xylenes to their carboxylic acid derivatives (11) and a lower (or meta-cleavage) pathway that transforms the carboxylic acids to the precursors of Krebs cycle intermediates (12). Genetic studies have shown that the genes for these catabolic enzymes are organized into two operons, one encoding enzymes for the upper pathway (the upper operon) and the other encoding enzymes for the meta-cleavage pathway (the meta operon) (6, 9, 10, 23).The upper operon of TOL plasmid pWW0 contains seven genes in the order xylU-xylW-xylC-xylM-xylA-xylB-xylN in a region of about 8 kb (11,40). The xylU and xylW genes are not required for toluene and xylene catabolism (40). The xylC gene encodes benzaldehyde dehydrogenase (14,21), and the xylM and xylA genes encode subunits of xylene monooxygenase (31,33,34), while the xylB gene encodes benzyl alcohol dehydrogenase (32). The xylN gene, the last gene of the upper operon, synthesizes a 52-kDa protein which is processed to a 47-kDa polypeptide (11); the physiological role of the xylN gene product has remained unknown. In this study, we investigate the function of the xylN gene product. MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. pWW0-161 is a Tn401 insertion derivative of TOL plasmid pWW0 and confers resistance to ampicillin on Escherichia coli hosts (6). The Tn401 insertion occurs outside the catabolic genes in pWW0-161. In this study, an xylN::Km r mutant of pWW0-161 was isola...

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Characterization and Role of tbuX in Utilization of Toluene by Ralstonia pickettii PKO1

Cited by 85 publications

References 55 publications

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Cross-Regulation between a Novel Two-Component Signal Transduction System for Catabolism of Toluene in Pseudomonas mendocina and the TodST System from Pseudomonas putida

The TOL Plasmid pWW0 xylN Gene Product from Pseudomonas putida Is Involved in m- Xylene Uptake

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