Cometabolism ofMethyl
 tertiary
 Butyl Ether and Gaseous
 n
 -Alkanes by
 Pseudomonas mendocina
 KR-1 Grown on C
 5
 toC
 8
 n
 -Alkanes

Smith, Christy A.; O’Reilly, Kirk T.; Hyman, Michael R.

doi:10.1128/aem.69.12.7385-7394.2003

Cited by 58 publications

(58 citation statements)

References 29 publications

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“…M. petroleiphilum PM1 is the best characterized of the few bacterial pure cultures reported to grow on and completely degrade MTBE and its daughter product, TBA (20,30,36,65). The genetic basis for MTBE and TBA conversion is not known, although different classes of monooxygenases have been proposed to play a role in metabolism or cometabolism of these compounds (30,36,51,68,74), including P-450 monooxygenase and alkane monooxygenase (hydroxylase) systems, the latter of which was shown to play a role in cometabolic degradation of MTBE by P. putida GPo1 (69) and possibly also by Pseudomonas mendocina KR-1 (70). A known inducer of alkane hydroxylase, dicyclopropylketone, was also shown to induce MTBE conversion to TBA in GPo1 (69).…”

Section: Resultsmentioning

confidence: 99%

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

et al. 2007

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Methylibium petroleiphilum PM1 is a methylotroph distinguished by its ability to completely metabolize the fuel oxygenate methyl tert-butyl ether (MTBE). Strain PM1 also degrades aromatic (benzene, toluene, and xylene) and straight-chain (C 5 to C 12 ) hydrocarbons present in petroleum products. Whole-genome analysis of PM1 revealed an ϳ4-Mb circular chromosome and an ϳ600-kb megaplasmid, containing 3,831 and 646 genes, respectively. Aromatic hydrocarbon and alkane degradation, metal resistance, and methylotrophy are encoded on the chromosome. The megaplasmid contains an unusual t-RNA island, numerous insertion sequences, and large repeated elements, including a 40-kb region also present on the chromosome and a 29-kb tandem repeat encoding phosphonate transport and cobalamin biosynthesis. The megaplasmid also codes for alkane degradation and was shown to play an essential role in MTBE degradation through plasmid-curing experiments. Discrepancies between the insertion sequence element distribution patterns, the distributions of best BLASTP hits among major phylogenetic groups, and the G؉C contents of the chromosome (69.2%) and plasmid (66%), together with comparative genome hybridization experiments, suggest that the plasmid was recently acquired and apparently carries the genetic information responsible for PM1's ability to degrade MTBE. Comparative genomic hybridization analysis with two PM1-like MTBE-degrading environmental isolates (ϳ99% identical 16S rRNA gene sequences) showed that the plasmid was highly conserved (ca. 99% identical), whereas the chromosomes were too diverse to conduct resequencing analysis. PM1's genome sequence provides a foundation for investigating MTBE biodegradation and exploring the genetic regulation of multiple biodegradation pathways in M. petroleiphilum and other MTBE-degrading beta-proteobacteria.Methylibium petroleiphilum strain PM1, which belongs to a newly described genus and species (57), is a motile bacterium belonging to the Comamonadaceae family of the Betaproteobacteria and is an important member of subsurface microbial communities in many gasoline-contaminated aquifers. Furthermore, PM1 is a methylotroph that can grow aerobically on the fuel oxygenate methyl tert-butyl ether (MTBE) and oxidize it completely to carbon dioxide (9, 34). MTBE is a suspected carcinogen that has contaminated drinking water wells throughout the United States due to the preponderance of underground leaking storage tanks, the widespread usage of MTBE, and its recalcitrance and mobility in groundwater. PM1 can also oxidize aromatic hydrocarbons (toluene, benzene, oxylene, and phenol) (20) and n-alkanes (C 5 to C 12 ) (57; K. Hristova, unpublished data) and has been used in two bioaugmentation field trials in gasoline-contaminated aquifers in California (67) and Montana (18,73). In contaminated sites amended with oxygen, in situ MTBE degradation was observed and corresponded to increases in native populations of Methylibium sp. (ϳ99% similarity to PM1, based on the 16S rRNA gene) (38,67,82). PM1-l...

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Section: Resultsmentioning

confidence: 99%

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

et al. 2007

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“…The cells were grown in batch culture on C-limiting amounts of n-octane (10 l [0.04%, vol/vol, liquid phase]) in the presence or absence of gaseous n-alkanes (10 ml [ϳ7.5%, vol/vol, gas phase]). Concentrations of n-alkanes were determined by gas chromatography, as described previously (8). The figure shows the time course for n-octane consumption for uninoculated abiotic control cultures containing (छ) n-octane and inoculated cultures containing (}) n-octane, (ᮀ) n-octane plus methane, (⌬) n-octane plus ethane, (E) n-octane plus propane, and (ƒ) n-octane plus n-butane.…”

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“…Time course of n-octane and gaseous n-alkane consumption by P. putida GPo1. Cultures of P. putida GPo1 were grown in sealed glass serum vials (160 ml) containing mineral salts medium (25 ml) (8). The cells were grown in batch culture on C-limiting amounts of n-octane (10 l [0.04%, vol/vol, liquid phase]) in the presence or absence of gaseous n-alkanes (10 ml [ϳ7.5%, vol/vol, gas phase]).…”

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Propane and n -Butane Oxidation by Pseudomonas putida GPo1

Johnson

Hyman

2006

Appl Environ Microbiol

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Propane and n-butane inhibit methyl tertiary butyl ether oxidation by n-alkane-grown Pseudomonas putida GPo1. Here we demonstrate that these gases are oxidized by this strain and support cell growth. Both gases induced alkane hydroxylase activity and appear to be oxidized by the same enzyme system used for the oxidation of n-octane.The n-alkane-oxidizing activity of Pseudomonas putida GPo1 is a model for the bacterial oxidation of n-alkanes other than methane (11). In this strain, n-alkane oxidation is initiated by alkane hydroxylase and the n-alkane growth substrate range of the bacterium (C 5 to C 13 ) matches the n-alkane substrate range of the native hydroxylase (10-12). We recently demonstrated that alkane hydroxylase in strain GPo1 oxidizes the gasoline oxygenate MTBE to tertiary butyl alcohol (TBA) (9). Propane and n-butane inhibited this activity, and these effects appeared to be due to a competitive interaction between hydroxylase substrates. However, no evidence for oxidation of these gases was presented, and neither gas is currently recognized as an alkane hydroxylase substrate.In this study we first examined whether strain GPo1 oxidized gaseous n-alkanes (C 1 to C 4 ) during growth on a model nalkane, n-octane. No consumption of either methane or ethane occurred during growth on n-octane, while both propane (ϳ20%) and n-butane (ϳ60%) were consumed under the same conditions (Fig. 1). In abiotic incubations, Յ20% of noctane ( Fig. 1) and Յ3% of gaseous n-alkanes were lost over the same time period (not shown). In a separate experiment, we determined the final culture densities (optical densities at 600 nm [OD 600 ]) and protein contents of cultures grown on C-limiting n-octane with gaseous n-alkanes. After exhaustion of n-octane (5 days), the average culture density and protein concentration for three separate cultures were 0.22 and 46 g/ml, respectively, for cultures grown on n-octane alone. No increase in culture density was observed with methane or ethane, while the average (n ϭ 3) final culture densities and protein concentrations for cultures containing propane were 0.43 and 62 g/ml or for cultures containing n-butane were 1.1 and 148 g/ml, respectively.Propane and n-butane were also examined as potential independent n-alkane growth substrates. Cultures were established with propane, n-butane, n-pentane, or n-octane added individually as sole substrates. The time courses of n-pentane and n-octane consumption were almost identical, and changes in culture density closely reflected n-alkane consumption (Fig. 2). In contrast, no detectable n-alkane consumption or increases in culture density were initially observed in n-butanecontaining cultures. However, after ϳ4 days, n-butane consumption was initiated and accompanied by a corresponding increase in culture density. No discernible effect on this lag phase or final culture density was observed when cultures were supplemented with fourfold-higher (ϳ0.7% [vol/vol] gas phase) concentrations of the main hydrocarbon contaminant (isobutane) present in our ...

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“…Little is known about the organisms, pathways, or intermediates involved in anaerobic MTBE oxidation. Under aerobic conditions, MTBE biodegradation occurs either through growth-related metabolism (9,11,22) or through cometabolic processes catalyzed by organisms previously grown on various hydrocarbons (4,6,10,12,17,24,25,30). Growthrelated metabolism of MTBE by pure bacterial cultures can lead to substantial mineralization of this compound.…”

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Oxidation of Methyl tert -Butyl Ether by Alkane Hydroxylase in Dicyclopropylketone-Induced and n -Octane-Grown Pseudomonas putida GPo1

Smith

Hyman

2004

Appl Environ Microbiol

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The alkane hydroxylase enzyme system in Pseudomonas putida GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl tert-butyl ether (MTBE). We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity. Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of tert-butyl alcohol (TBA). Cells grown in the presence of DCPK also oxidized tert-amyl methyl ether but did not appear to oxidize either TBA, ethyl tert-butyl ether, or tert-amyl alcohol. Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches. First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK. Second, no TBA production from MTBE was observed in DCPK-treated cells of P. putida GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid. Third, all n-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells. Fourth, two non-growth-supporting n-alkanes (propane and n-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process. Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE. Sixth, MTBE-oxidizing activity was also observed in n-octane-grown cells. Kinetic studies with strain GPo1 grown on n-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in n-octane-grown cells because of the unusually high K s value (20 to 40 mM) for MTBE.

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Cometabolism ofMethyl tertiary Butyl Ether and Gaseous n -Alkanes by Pseudomonas mendocina KR-1 Grown on C ₅ toC ₈ n -Alkanes

Cited by 58 publications

References 29 publications

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

Propane and n -Butane Oxidation by Pseudomonas putida GPo1

Oxidation of Methyl tert -Butyl Ether by Alkane Hydroxylase in Dicyclopropylketone-Induced and n -Octane-Grown Pseudomonas putida GPo1

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Cometabolism ofMethyl tertiary Butyl Ether and Gaseous n -Alkanes by Pseudomonas mendocina KR-1 Grown on C 5 toC 8 n -Alkanes

Cited by 58 publications

References 29 publications

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

Propane and n -Butane Oxidation by Pseudomonas putida GPo1

Oxidation of Methyl tert -Butyl Ether by Alkane Hydroxylase in Dicyclopropylketone-Induced and n -Octane-Grown Pseudomonas putida GPo1

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Cometabolism ofMethyl tertiary Butyl Ether and Gaseous n -Alkanes by Pseudomonas mendocina KR-1 Grown on C ₅ toC ₈ n -Alkanes