scholarly journals Diversity in Butane Monooxygenases among Butane-Grown Bacteria

1999 ◽  
Vol 65 (10) ◽  
pp. 4586-4593 ◽  
Author(s):  
Natsuko Hamamura ◽  
Ryan T. Storfa ◽  
Lewis Semprini ◽  
Daniel J. Arp
Keyword(s):  
Ethylene Oxide ◽  
Methane Oxidation ◽  
Environmental Isolate ◽  
Butane Oxidation ◽  
Butanol Production ◽  
Oxidation Activity ◽  
Physiological Level ◽  
Mycobacterium Vaccae ◽  
Pseudomonas Butanovora ◽  
Allyl Thiourea

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.

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 Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism by Pseudomonas butanovora

2002 ◽  
Vol 184 (7) ◽  
pp. 1916-1924 ◽  
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.

scholarly journals Anaerobic oxidation of methane in grassland soils used for cattle husbandry

2012 ◽  
Vol 9 (10) ◽  
pp. 3891-3899 ◽  
Author(s):  
A. Bannert ◽  
C. Bogen ◽  
J. Esperschütz ◽  
A. Koubová ◽  
F. Buegger ◽  
...  
Keyword(s):  
Methane Oxidation ◽  
Enrichment Culture ◽  
Anaerobic Oxidation Of Methane ◽  
Anaerobic Oxidation ◽  
Incubation Experiment ◽  
Nutrient Source ◽  
Oxidation Activity ◽  
Oxidation Of Methane ◽  
Cattle Husbandry

Abstract. While the importance of anaerobic methane oxidation has been reported for marine ecosystems, the role of this process in soils is still questionable. Grasslands used as pastures for cattle overwintering show an increase in anaerobic soil micro-sites caused by animal treading and excrement deposition. Therefore, anaerobic potential methane oxidation activity of severely impacted soil from a cattle winter pasture was investigated in an incubation experiment under anaerobic conditions using 13C-labelled methane. We were able to detect a high microbial activity utilizing CH4 as nutrient source shown by the respiration of 13CO2. Measurements of possible terminal electron acceptors for anaerobic oxidation of methane were carried out. Soil sulfate concentrations were too low to explain the oxidation of the amount of methane added, but enough nitrate and iron(III) were detected. However, only nitrate was consumed during the experiment. 13C-PLFA analyses clearly showed the utilization of CH4 as nutrient source mainly by organisms harbouring 16:1ω7 PLFAs. These lipids were also found as most 13C-enriched fatty acids by Raghoebarsing et al. (2006) after addition of 13CH4 to an enrichment culture coupling denitrification of nitrate to anaerobic oxidation of methane. This might be an indication for anaerobic oxidation of methane by relatives of "Candidatus Methylomirabilis oxyfera" in the investigated grassland soil under the conditions of the incubation experiment.

scholarly journals Abundance and Activity of Methanotrophic Bacteria in Littoral and Profundal Sediments of Lake Constance (Germany)

2008 ◽  
Vol 75 (1) ◽  
pp. 119-126 ◽  
Author(s):  
M. Rahalkar ◽  
J. Deutzmann ◽  
B. Schink ◽  
I. Bussmann
Keyword(s):  
Methane Oxidation ◽  
Lake Constance ◽  
Type I ◽  
Sediment Depth ◽  
Oxidation Activity ◽  
Oxidation Rates ◽  
Methane Oxidizing Bacteria ◽  
Littoral Sediment ◽  
Sediment Layers

ABSTRACT The abundances and activities of aerobic methane-oxidizing bacteria (MOB) were compared in depth profiles of littoral and profundal sediments of Lake Constance, Germany. Abundances were determined by quantitative PCR (qPCR) targeting the pmoA gene and by fluorescence in situ hybridization (FISH), and data were compared to methane oxidation rates calculated from high-resolution concentration profiles. qPCR using type I MOB-specific pmoA primers indicated that type I MOB represented a major proportion in both sediments at all depths. FISH indicated that in both sediments, type I MOB outnumbered type II MOB at least fourfold. Results obtained with both techniques indicated that in the littoral sediment, the highest numbers of methanotrophs were found at a depth of 2 to 3 cm, corresponding to the zone of highest methane oxidation activity, although no oxygen could be detected in this zone. In the profundal sediment, highest methane oxidation activities were found at a depth of 1 to 2 cm, while MOB abundance decreased gradually with sediment depth. In both sediments, MOB were also present at high numbers in deeper sediment layers where no methane oxidation activity could be observed.

scholarly journals Methanotrophic activity and diversity in different <i>Sphagnum magellanicum</i> dominated habitats in the southernmost peat bogs of Patagonia

2012 ◽  
Vol 9 (1) ◽  
pp. 47-55 ◽  
Author(s):  
N. Kip ◽  
C. Fritz ◽  
E. S. Langelaan ◽  
Y. Pan ◽  
L. Bodrossy ◽  
...  
Keyword(s):  
Methane Oxidation ◽  
Northern Hemisphere ◽  
Water Levels ◽  
Peat Bogs ◽  
Oxidation Activity ◽  
Moss Species ◽  
Sphagnum Mosses ◽  
Methane Concentrations ◽  
Sphagnum Magellanicum

Abstract. Sphagnum peatlands are important ecosystems in the methane cycle. Methanotrophs living inside the dead hyaline cells or on the Sphagnum mosses are able to act as a methane filter and thereby reduce methane emissions. We investigated in situ methane concentrations and the corresponding activity and diversity of methanotrophs in different Sphagnum dominated bog microhabitats. In contrast to the Northern Hemisphere peat ecosystems the temperate South American peat bogs are dominated by one moss species; Sphagnum magellanicum. This permitted a species-independent comparison of the different bog microhabitats. Potential methane oxidizing activity was found in all Sphagnum mosses sampled and a positive correlation was found between activity and in situ methane concentrations. Substantial methane oxidation activity (23 μmol CH4 gDW−1 day−1) was found in pool mosses and could be correlated with higher in situ methane concentrations (>35 μmol CH4 l−1 pore water). Little methanotrophic activity (<0.5 μmol CH4 gDW−1 day−1) was observed in living Sphagnum mosses from lawns and hummocks. Methane oxidation activity was relatively high (>4 μmol CH4 gDW−1 day−1) in Sphagnum litter at depths around the water levels and rich in methane. The total bacterial community was studied using 16S rRNA gene sequencing and the methanotrophic communities were studied using a pmoA microarray and a complementary pmoA clone library. The methanotrophic diversity was similar in the different habitats of this study and comparable to the methanotrophic diversity found in peat mosses from the Northern Hemisphere. The pmoA microarray data indicated that both alpha- and gammaproteobacterial methanotrophs were present in all Sphagnum mosses, even in those mosses with a low initial methane oxidation activity. Prolonged incubation of Sphagnum mosses from lawn and hummock with methane revealed that the methanotrophic community present was viable and showed an increased activity within 15 days. The high abundance of methanotrophic Methylocystis species in the most active mosses suggests that these might be responsible for the bulk of methane oxidation.

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