scholarly journals Evidence for Involvement of Copper Ions and Redox State in Regulation of Butane Monooxygenase in Pseudomonas butanovora

2008 ◽  
Vol 190 (8) ◽  
pp. 2933-2938 ◽  
Author(s):  
D. M. Doughty ◽  
E. G. Kurth ◽  
L. A. Sayavedra-Soto ◽  
D. J. Arp ◽  
P. J. Bottomley
Keyword(s):  
Phosphate Buffer ◽  
Copper Ions ◽  
Galactosidase Activity ◽  
Reporter Strain ◽  
Mineral Salts ◽  
Anoxic Conditions ◽  
Repressive Effect ◽  
Pseudomonas Butanovora ◽  
Reporter Strains ◽  
Butane Monooxygenase

ABSTRACT Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C2-C9 alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of β-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O2 when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH4Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O2 (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O2, CuSO4 (0.5 μM) repressed 1-butanol-dependent induction of β-galactosidase activity. Under oxic conditions (20% O2 [vol/vol]), significantly higher concentrations of CuSO4 (2 μM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu2+ reducing agent Na ascorbate (100 μM) and CuSO4 (0.5 μM) repressed the induction of β-galactosidase activity under oxic conditions to the same extent that 0.5 μM CuSO4 alone repressed it under anoxic conditions. Under oxic conditions, 2 μM CuSO4 repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO4 repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

scholarly journals Product Repression of Alkane Monooxygenase Expression in Pseudomonas butanovora

2006 ◽  
Vol 188 (7) ◽  
pp. 2586-2592 ◽  
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.

scholarly journals Effects of Dichloroethene Isomers on the Induction and Activity of Butane Monooxygenase in the Alkane-Oxidizing Bacterium “Pseudomonas butanovora”

2005 ◽  
Vol 71 (10) ◽  
pp. 6054-6059 ◽  
Author(s):  
D. M. Doughty ◽  
L. A. Sayavedra-Soto ◽  
D. J. Arp ◽  
P. J. Bottomley
Keyword(s):  
Gene Expression ◽  
Lactic Acid ◽  
Organic Acids ◽  
The Other ◽  
Reporter Strain ◽  
Negative Effects ◽  
Intact Cells ◽  
Pseudomonas Butanovora ◽  
Butane Monooxygenase

ABSTRACT We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium “Pseudomonas butanovora.” Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of ∼20 nmol mg protein−1 of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein−1. Oxidation of similar amounts of each DCE isomer (∼20 nmol mg protein−1) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown “P. butanovora” did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.

scholarly journals Influence of pH and Matric Potential on Germination of Cephalosporium gramineum Conidia

Plant Disease ◽  
1998 ◽  
Vol 82 (9) ◽  
pp. 975-978 ◽  
Author(s):  
Cynthia A. Blank ◽  
Timothy D. Murray
Keyword(s):  
Soil Moisture ◽  
Soil Ph ◽  
Significant Influence ◽  
Phosphate Buffer ◽  
Mineral Salts ◽  
Matric Potential ◽  
High Soil Moisture ◽  
Soil Fungistasis ◽  
Cephalosporium Gramineum ◽  
Influence Of Ph

Germination of Cephalosporium gramineum conidia in soil was up to twofold greater at -0.064 MPa than at -0.037 and -0.007 MPa when incubated at 5°C for 2 days. Soil pH from 4.7 to 7.5 did not have a significant influence on germination of conidia and the interaction between soil pH and matric potential on germination was not significant. Soil fungistasis, which was previously observed for conidia of C. gramineum, was not observed in these studies. Germination of conidia on mineral salts agar containing phosphate buffer was significantly less at pH 4.5 than at 5.5, 6.5, or 7.5 at 5°C in one of two experiments; however, pH had no influence on germination at 10 or 20°C in two experiments. Although Cephalosporium stripe is more severe under conditions of high soil moisture and low soil pH, increased germination of conidia in response to these factors does not explain the observed increase in disease.

scholarly journals Propionate inactivation of butane monooxygenase activity in ‘Pseudomonas butanovora’: biochemical and physiological implications

Microbiology ◽  
2007 ◽  
Vol 153 (11) ◽  
pp. 3722-3729 ◽  
Author(s):  
D. M. Doughty ◽  
K. H. Halsey ◽  
C. J. Vieville ◽  
L. A. Sayavedra-Soto ◽  
D. J. Arp ◽  
...  
Keyword(s):  
Monooxygenase Activity ◽  
Pseudomonas Butanovora ◽  
Butane Monooxygenase

scholarly journals The Formation of Polycomplexes of Poly(Methyl Vinyl Ether-Co-Maleic Anhydride) and Bovine Serum Albumin in the Presence of Copper Ions

2014 ◽  
Vol 16 (3) ◽  
pp. 97-105 ◽  
Author(s):  
Mesut Karahan ◽  
Zeynep Mustafaeva ◽  
Cemal Özeroğlu
Keyword(s):  
Bovine Serum Albumin ◽  
Maleic Anhydride ◽  
Phosphate Buffer ◽  
Bovine Serum ◽  
Copper Ions ◽  
Phosphate Buffer Solution ◽  
Buffer Solution ◽  
Methyl Vinyl ◽  
Polymer Metal ◽  
Methyl Vinyl Ether

Abstract The binary and ternary complex formations of poly(methyl vinyl ether-co-maleic anhydride) (PMVEMA) with copper ions and with bovine serum albumin (BSA) in the presence of copper ions in phosphate buffer solution at pH = 7 were examined by the techniques of UV-visible, fluorescence, dynamic light scattering, atomic force microscopy measurements. In the formation of binary complexes of PMVEMA-Cu(II), the addition of copper ions to the solution of PMVEMA in phosphate buffer solution at pH = 7 forms homogeneous solutions when the molar ratio of Cu(II)/MVEMA is 0.5. Then the formations of ternary complexes of PMVEMA-Cu(II)-BSA were examined. Study analysis revealed that the toxicities of polymer-metal and polymer-metal-protein mixture solutions depend on the nature and ratio of components in mixtures.

scholarly journals Chromogenic Escherichia coli reporter strain for screening DNA damaging agents

AMB Express ◽  
2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Josué Daniel Mora-Garduño ◽  
Jessica Tamayo-Nuñez ◽  
Felipe Padilla-Vaca ◽  
Fátima Berenice Ramírez-Montiel ◽  
Ángeles Rangel-Serrano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Water Samples ◽  
Membrane Integrity ◽  
Analytical Techniques ◽  
Bacterial Cells ◽  
Reporter Strain ◽  
Dna Damaging Agents ◽  
Reporter Strains ◽  
High Concentrations

AbstractThe presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

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

2006 ◽  
Vol 188 (13) ◽  
pp. 4962-4969 ◽  
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.

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

2007 ◽  
Vol 189 (14) ◽  
pp. 5068-5074 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document