Involvement of BmoR and BmoG in n-alkane metabolism in ‘Pseudomonas butanovora’

ABSTRACT Butane monooxygenases of butane-grown Pseudomonas butanovora, Mycobacterium vaccae JOB5, and an environmental isolate, CF8, were compared at the physiological level. The presence of butane monooxygenases in these bacteria was indicated by the following results. (i) O2 was required for butane degradation. (ii) 1-Butanol was produced during butane degradation. (iii) Acetylene inhibited both butane oxidation and 1-butanol production. The responses to the known monooxygenase inactivator, ethylene, and inhibitor, allyl thiourea (ATU), discriminated butane degradation among the three bacteria. Ethylene irreversibly inactivated butane oxidation by P. butanovora but not by M. vaccae or CF8. In contrast, butane oxidation by only CF8 was strongly inhibited by ATU. In all three strains of butane-grown bacteria, specific polypeptides were labeled in the presence of [14C]acetylene. The [14C]acetylene labeling patterns were different among the three bacteria. Exposure of lactate-grown CF8 and P. butanovora and glucose-grownM. vaccae to butane induced butane oxidation activity as well as the specific acetylene-binding polypeptides. Ammonia was oxidized by all three bacteria. P. butanovora oxidized ammonia to hydroxylamine, while CF8 and M. vaccae produced nitrite. All three bacteria oxidized ethylene to ethylene oxide. Methane oxidation was not detected by any of the bacteria. The results indicate the presence of three distinct butane monooxygenases in butane-grown P. butanovora, M. vaccae, and CF8.

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Site-Directed Amino Acid Substitutions in the Hydroxylase α Subunit of Butane Monooxygenase from Pseudomonas butanovora: Implications for Substrates Knocking at the Gate

Journal of Bacteriology ◽

10.1128/jb.00280-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4962-4969 ◽

Cited By ~ 21

Author(s):

Kimberly H. Halsey ◽

Luis A. Sayavedra-Soto ◽

Peter J. Bottomley ◽

Daniel J. Arp

Keyword(s):

Amino Acid ◽

Methane Oxidation ◽

In Vivo Studies ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Butanol Production ◽

Α Subunit ◽

Wide Range ◽

Pseudomonas Butanovora ◽

Butane Monooxygenase

ABSTRACT Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase α subunit of butane monooxygenase (BMOH-α) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-α were altered to the corresponding residues from the α subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to 1-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 μM methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 μM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-α.

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Product Repression of Alkane Monooxygenase Expression in Pseudomonas butanovora

Journal of Bacteriology ◽

10.1128/jb.188.7.2586-2592.2006 ◽

2006 ◽

Vol 188 (7) ◽

pp. 2586-2592 ◽

Cited By ~ 17

Author(s):

D. M. Doughty ◽

L. A. Sayavedra-Soto ◽

D. J. Arp ◽

P. J. Bottomley

Keyword(s):

Chain Length ◽

Growth Medium ◽

Transcriptional Activity ◽

Propane Oxidation ◽

Regulatory Mechanisms ◽

Reporter Strain ◽

Pseudomonas Butanovora ◽

Alkane Monooxygenase ◽

Butane Monooxygenase

ABSTRACT Physiological and regulatory mechanisms that allow the alkane-oxidizing bacterium Pseudomonas butanovora to consume C2 to C8 alkane substrates via butane monooxygenase (BMO) were examined. Striking differences were observed in response to even- versus odd-chain-length alkanes. Propionate, the downstream product of propane oxidation and of the oxidation of other odd-chain-length alkanes following β-oxidation, was a potent repressor of BMO expression. The transcriptional activity of the BMO promoter was reduced with as little as 10 μM propionate, even in the presence of appropriate inducers. Propionate accumulated stoichiometrically when 1-propanol and propionaldehyde were added to butane- and ethane-grown cells, indicating that propionate catabolism was inactive during growth on even-chain-length alkanes. In contrast, propionate consumption was induced (about 80 nmol propionate consumed · min−1 · mg protein−1) following growth on the odd-chain-length alkanes, propane and pentane. The induction of propionate consumption could be brought on by the addition of propionate or pentanoate to the growth medium. In a reporter strain of P. butanovora in which the BMO promoter controls β-galactosidase expression, only even-chain-length alcohols (C2 to C8) induced β-galactosidase following growth on acetate or butyrate. In contrast, both even- and odd-chain-length alcohols (C3 to C7) were able to induce β-galactosidase following the induction of propionate consumption by propionate or pentanoate.

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Molecular analysis of the soluble butane monooxygenase from ‘Pseudomonas butanovora’ a aThe GenBank accession number for the bmoXYBZDC sequence is AY093933.

Microbiology ◽

10.1099/00221287-148-11-3617 ◽

2002 ◽

Vol 148 (11) ◽

pp. 3617-3629 ◽

Cited By ~ 69

Author(s):

Miriam K. Sluis ◽

Luis A. Sayavedra-Soto ◽

Daniel J. Arp

Keyword(s):

Molecular Analysis ◽

Accession Number ◽

Pseudomonas Butanovora ◽

Butane Monooxygenase

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Butane metabolism by butane-grown ‘Pseudomonas butanovora’

Microbiology ◽

10.1099/13500872-145-5-1173 ◽

1999 ◽

Vol 145 (5) ◽

pp. 1173-1180 ◽

Cited By ~ 56

Author(s):

Daniel J. Arp

Keyword(s):

Pseudomonas Butanovora

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Evidence for Modified Mechanisms of Chloroethene Oxidation in Pseudomonas butanovora Mutants Containing Single Amino Acid Substitutions in the Hydroxylase α-Subunit of Butane Monooxygenase

Journal of Bacteriology ◽

10.1128/jb.00189-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5068-5074 ◽

Cited By ~ 7

Author(s):

Kimberly H. Halsey ◽

David M. Doughty ◽

Luis A. Sayavedra-Soto ◽

Peter J. Bottomley ◽

Daniel J. Arp

Keyword(s):

Amino Acid ◽

Mutant Strain ◽

Parent Compound ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Α Subunit ◽

Mutant Strains ◽

Pseudomonas Butanovora ◽

Butane Monooxygenase ◽

The Impact

ABSTRACT The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the α-subunit of butane monooxygenase hydroxylase (BMOH-α) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O2 uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-α mutants might allow insights into the catalytic mechanism of BMO to be obtained.

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Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism by Pseudomonas butanovora

Journal of Bacteriology ◽

10.1128/jb.184.7.1916-1924.2002 ◽

2002 ◽

Vol 184 (7) ◽

pp. 1916-1924 ◽

Cited By ~ 34

Author(s):

Alisa S. Vangnai ◽

Daniel J. Arp ◽

Luis A. Sayavedra-Soto

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Alcohol Dehydrogenase ◽

Primary Alcohol ◽

Leader Sequence ◽

Butane Oxidation ◽

Oxidation Activity ◽

Alcohol Dehydrogenases ◽

Cell Extracts ◽

Pseudomonas Butanovora

ABSTRACT The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P. butanovora. The P. butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of the P. butanovora strain with bdh disrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD+-independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P. butanovora.

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