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

Biotechnology Center for Agriculture and the Environment, Rutgers University, New Brunswick, New Jersey 08901-8520, USA.
Journal of Bacteriology (Impact Factor: 2.81). 04/2000; 182(5):1232-42. DOI: 10.1128/JB.182.5.1232-1242.2000
Source: PubMed


The tbu regulon of Ralstonia pickettii PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the tbu pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated tbuX. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in Escherichia coli. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in R. pickettii PKO1. In addition, the expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.

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Available from: Ronald H Olsen, Oct 06, 2015
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    • "The plates were incubated at 30°C for 3 days. Ctl refers to clone containing pBBR1MCS2, and pTcpY refers to the clones containing constitutively expressed tcpY Biodegradation a b-barrel outer membrane protein because of its structural similarity to characterized b-barrel outer membrane proteins, including FadL, TodX and TbuX (Wang et al. 1995; Kahng et al. 2000; van den Berg 2005). The toxicity and uptake studies support that TcpY facilitates the transport of polychlorophenols across the outer membrane of gram negative bacterium C. necator. "
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    ABSTRACT: The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a beta-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds.
    Biodegradation 11/2009; 21(3):431-9. DOI:10.1007/s10532-009-9313-8 · 2.34 Impact Factor
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    • "The role of phenol as an inducer for TpMO is unclear in the literature. Early work indicates phenol is not an inducer for TpMO (Leahy et al ., 1997; Olsen et al ., 1994) whereas later works show it is an inducer, albeit a poor one compared with toluene and benzene (Kahng et al ., 2000). Therefore phenol is listed both as an inducer and a non-inducer in Table I. "
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    ABSTRACT: We have recently found that toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1 (encoded by tbuA1UBVA2C) performs successive hydroxylations of benzene (Appl. Environ. Microbiol. 70: 3814, 2004) as well as hydroxylates toluene to a mixture of 90% p-cresol and 10% m-cresol which are then further oxidized to 100% 4-methylcatechol (J. Bacteriol. 186: 3117, 2004) whereas it was thought previously that TpMO forms 100% m-cresol and is not capable of successive hydroxylations. Here we propose a modification of the degradation pathway originally described by Olsen et al. (J. Bacteriol. 176: 3749, 1994) that now relies primarily on TpMO for conversion of toluene to 4-methylcatechol (instead of m-cresol) since both m-cresol and p-cresol are shown here to be good substrates for Escherichia coli expressing TpMO (Vmax/Km=0.046, 0.036, and 0.055 mL min−1 mg−1 protein for the oxidation of toluene, m-cresol, and p-cresol, respectively). In light of the broader activity of TpMO, phenol hydroxylase (encoded by tbuD) appears to facilitate conversion of any m-cresol or p-cresol formed from toluene oxidation by TpMO to 4-methylcatechol; hence, the cell has a redundant method for making this important intermediate 4-methylcatechol. Further, it is suggested that the physiological relevance of the 10% m-cresol formed from toluene oxidation by TpMO is needed for induction of the meta cleavage operon tbuWEFGKIHJ to enable full metabolism of toluene since p-cresol (and o-cresol) do not induce the meta-cleavage pathway. Therefore both the successive hydroxylation of toluene by TpMO and the product distribution are of physiological relevance to the cell.
    Biocatalysis and Biotransformation 07/2009; 22(4):283-289. DOI:10.1080/10242420400012008 · 0.69 Impact Factor
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    • "In addition, Alcaligenes, Brevibacterium, Nocardia, Bacillus, Bordetella, Arthrobacter, Bradyrhizobium, Acidovorax , Agrobacterium, Aquaspirillum, Variovorax and Stenotrophomonas were also detected as BTEX degraders in soil (Hendrickx et al. 2006a, b). From sewage and fresh water, the BTEX degraders were reported as Ralstonia (Kahng et al. 2000), Microbacterium (Cavalca et al. 2004), Mycrobacterium (Cavalca et al. 2004), Azoarcus (Cavalca et al. 2004; Mohamed et al. 2001), Thauera (Yoshifumi et al. 2004), Burkholderia (Johnson and Olsen 1997) and "
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    ABSTRACT: BTX (benzene, toluene and xylene) degrading bacteria were isolated from Pacific Ocean sediment and nearshore surface water. In the seawater near a ferry dock, degrading bacteria of a relatively wide diversity were detected, including species of Pseudomonas, Rhodococcus, Exiguobacterium and Bacillus; while species of Bacillus only have been detected from the deep-sea sediment. Most of the isolates showed degradation to more than one compound. Generally better growth was obtained with p-xylene and ethylbenzene than with the other two. All the bacteria could tolerate and grow with the compounds at 5-20% (v/v). Both benzene and toluene degradation related genes had been successfully PCR cloned from the isolates of nearshore water, the detected benzene dioxygenase gene was identical among all the species and close to its soil counterpart. However, they were not detected in all the isolates from deep sea. Results in this report suggested that BTX degrading bacteria widely spread in marine environments and they might be of potentials in biotreatment of BTEX in saline environments.
    Extremophiles 06/2008; 12(3):335-42. DOI:10.1007/s00792-007-0136-4 · 2.31 Impact Factor
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