Purification, Characterization, and Overexpression of Flavin Reductase Involved in Dibenzothiophene Desulfurization byRhodococcus erythropolis D-1

ABSTRACT The dibenzothiophene (DBT)-desulfurizing bacterium,Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the Km values for NADH and FMN were 208 and 10.8 μM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35°C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80°C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705–1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD ofR. erythropolis IGTS8, and the enzyme was overexpressed inEscherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.

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Biochemical Characterization of StyAB from Pseudomonas sp. Strain VLB120 as a Two-Component Flavin-Diffusible Monooxygenase

Journal of Bacteriology ◽

10.1128/jb.186.16.5292-5302.2004 ◽

2004 ◽

Vol 186 (16) ◽

pp. 5292-5302 ◽

Cited By ~ 146

Author(s):

Katja Otto ◽

Karin Hofstetter ◽

Martina Röthlisberger ◽

Bernard Witholt ◽

Andreas Schmid

Keyword(s):

Biochemical Characterization ◽

Specific Activity ◽

Styrene Oxide ◽

Sole Source ◽

Nadh Oxidation ◽

Flavin Mononucleotide ◽

Wild Type ◽

Oxidation Activity ◽

Epoxidation Reaction ◽

Sequential Binding

ABSTRACT Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of C═C double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg−1 (k cat = 1.6 s−1) and Km values for styrene of 0.45 ± 0.05 mM (wild type) and 0.38 ± 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg−1 (k cat [NADH] = 60 s−1). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.

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Megaplasmid pRme2011a of Sinorhizobium meliloti Is Not Required for Viability

Journal of Bacteriology ◽

10.1128/jb.182.12.3582-3586.2000 ◽

2000 ◽

Vol 182 (12) ◽

pp. 3582-3586 ◽

Cited By ~ 71

Author(s):

Ivan J. Oresnik ◽

Shu-Lin Liu ◽

Christopher K. Yost ◽

Michael F. Hynes

Keyword(s):

Gel Electrophoresis ◽

Type Strain ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Sole Source ◽

Pulsed Field Gel Electrophoresis ◽

Selection Strategy ◽

Wild Type ◽

Defined Media

ABSTRACT We report the curing of the 1,360-kb megaplasmid pRme2011a fromSinorhizobium meliloti strain Rm2011. With a positive selection strategy that utilized Tn5B12-S containing thesacB gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.

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Functional Dissection of Yeast Hir1p, a WD Repeat–Containing Transcriptional Corepressor

Genetics ◽

10.1093/genetics/148.2.657 ◽

1998 ◽

Vol 148 (2) ◽

pp. 657-667

Author(s):

Heshani DeSilva ◽

Kenneth Lee ◽

Mary Ann Osley

Keyword(s):

Wild Type Strain ◽

Protein Complexes ◽

Histone Gene ◽

Gene Products ◽

Wild Type ◽

Transcriptional Corepressor ◽

C Terminus ◽

Terminal Amino ◽

Wd Repeats

Abstract The HIR1 gene product is required to repress transcription of three of the four histone gene loci in Saccharomyces cerevisiae, and like its counterpart, the HIR2 protein, it functions as a transcriptional corepressor. Although Hir1p and Hir2p are physically associated in yeast, Hir1p is able to function independently of Hir2p when it is artificially recruited to the histone HTA1 promoter. A deletion analysis of HIR1 has revealed two separate repression domains: one in its N terminus, where seven copies of the β-transducin or WD40 motif reside, and the second in the remaining C-terminal amino acids. Overexpression of the WD repeats in a hir1Δ strain complemented its Hir− phenotype, while overexpression of the C terminus in a wild-type strain caused both Hir− and Spt− phenotypes. The Hir1p C terminus physically interacted in vivo with Hir2p, and both Hir1p repression domains interacted with full-length Hir1p. It was additionally found that the Hir1p WD repeats functionally interacted with the SPT4, SPT5, and SPT6 gene products, suggesting that these repeats may direct Hir1p to different protein complexes.

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Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.7.1811 ◽

1998 ◽

Vol 42 (7) ◽

pp. 1811-1814 ◽

Cited By ~ 45

Author(s):

Leonardo K. Basco ◽

Rachida Tahar ◽

Pascal Ringwald

Keyword(s):

Dihydrofolate Reductase ◽

Point Mutations ◽

Gene Mutations ◽

Adult Patients ◽

Wild Type ◽

Dihydropteroate Synthase ◽

Reductase Gene ◽

Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

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Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan

Applied and Environmental Microbiology ◽

10.1128/aem.68.12.6237-6245.2002 ◽

2002 ◽

Vol 68 (12) ◽

pp. 6237-6245 ◽

Cited By ~ 36

Author(s):

Tara D. Sutherland ◽

Irene Horne ◽

Robyn J. Russell ◽

John G. Oakeshott

Keyword(s):

Shuttle Vector ◽

Open Reading Frame ◽

Cosmid Library ◽

Flavin Mononucleotide ◽

Flavin Reductase ◽

Reading Frame ◽

Reductase Gene ◽

Degradation Activity ◽

Layer Chromatography

ABSTRACT The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.

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A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

Journal of Bacteriology ◽

10.1128/jb.00261-10 ◽

2010 ◽

Vol 192 (18) ◽

pp. 4776-4785 ◽

Cited By ~ 29

Author(s):

Rabeb Dhouib ◽

Françoise Laval ◽

Frédéric Carrière ◽

Mamadou Daffé ◽

Stéphane Canaan

Keyword(s):

Mycobacterium Smegmatis ◽

Wild Type Strain ◽

Specific Activity ◽

Cell Interaction ◽

Monoacylglycerol Lipase ◽

Homologous Proteins ◽

Dependent Activity ◽

Hydrolysis Of

ABSTRACT MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.

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Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1805 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1805-1812 ◽

Cited By ~ 58

Author(s):

J Zhu ◽

R H Schiestl

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Aberrations ◽

Topoisomerase I ◽

Wild Type Strain ◽

Hot Spot ◽

Illegitimate Recombination ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae

Chromosome aberrations may cause cancer and many heritable diseases. Topoisomerase I has been suspected of causing chromosome aberrations by mediating illegitimate recombination. The effects of deletion and of overexpression of the topoisomerase I gene on illegitimate recombination in the yeast Saccharomyces cerevisiae have been studied. Yeast transformations were carried out with DNA fragments that did not have any homology to the genomic DNA. The frequency of illegitimate integration was 6- to 12-fold increased in a strain overexpressing topoisomerase I compared with that in isogenic control strains. Hot spot sequences [(G/C)(A/T)T] for illegitimate integration target sites accounted for the majority of the additional events after overexpression of topoisomerase I. These hot spot sequences correspond to sequences previously identified in vitro as topoisomerase I preferred cleavage sequences in other organisms. Furthermore, such hot spot sequences were found in 44% of the integration events present in the TOP1 wild-type strain and at a significantly lower frequency in the top1delta strain. Our results provide in vivo evidence that a general eukaryotic topoisomerase I enzyme nicks DNA and ligates nonhomologous ends, leading to illegitimate recombination.

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In VivoEfficacy of Human Simulated Regimens of Carbapenems and Comparator Agents against NDM-1-Producing Enterobacteriaceae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01946-13 ◽

2013 ◽

Vol 58 (3) ◽

pp. 1671-1677 ◽

Cited By ~ 23

Author(s):

Dora E. Wiskirchen ◽

Patrice Nordmann ◽

Jared L. Crandon ◽

David P. Nicolau

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Similar Efficacy ◽

Infection Model ◽

High Dose ◽

Wild Type ◽

Prolonged Infusion ◽

Content Type

ABSTRACTDoripenem and ertapenem have demonstrated efficacy against several NDM-1-producing isolatesin vivo, despite having high MICs. In this study, we sought to further characterize the efficacy profiles of humanized regimens of standard (500 mg given every 8 h) and high-dose, prolonged infusion of doripenem (2 g given every 8 h, 4-h infusion) and 1 g of ertapenem given intravenously every 24 h and the comparator regimens of ceftazidime at 2 g given every 8 h (2-h infusion), levofloxacin at 500 mg every 24 h, and aztreonam at 2 g every 6 h (1-h infusion) against a wider range of isolates in a murine thigh infection model. An isogenic wild-type strain and NDM-1-producingKlebsiella pneumoniaeand eight clinical NDM-1-producing members of the familyEnterobacteriaceaewere tested in immunocompetent- and neutropenic-mouse models. The wild-type strain was susceptible to all of the agents, while the isogenic NDM-1-producing strain was resistant to ceftazidime, doripenem, and ertapenem. Clinical NDM-1-producing strains were resistant to nearly all five of the agents (two were susceptible to levofloxacin). In immunocompetent mice, all of the agents produced ≥1-log10CFU reductions of the isogenic wild-type and NDM-1-producing strains after 24 h. Minimal efficacy of ceftazidime, aztreonam, and levofloxacin against the clinical NDM-1-producing strains was observed. However, despitein vitroresistance, ≥1-log10CFU reductions of six of eight clinical strains were achieved with high-dose, prolonged infusion of doripenem and ertapenem. Slight enhancements of doripenem activity over the standard doses were obtained with high-dose, prolonged infusion for three of the four isolates tested. Similar efficacy observations were noted in neutropenic mice. These data suggest that carbapenems are a viable treatment option for infections caused by NDM-1-producingEnterobacteriaceae.

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CO-Dependent H2Production by Genetically Engineered Thermococcus onnurineus NA1

Applied and Environmental Microbiology ◽

10.1128/aem.03298-12 ◽

2013 ◽

Vol 79 (6) ◽

pp. 2048-2053 ◽

Cited By ~ 57

Author(s):

Min-Sik Kim ◽

Seung Seob Bae ◽

Yun Jae Kim ◽

Tae Wan Kim ◽

Jae Kyu Lim ◽

...

Keyword(s):

Gene Cluster ◽

Type Strain ◽

Wild Type Strain ◽

Specific Activity ◽

Steel Production ◽

Transcriptional Analysis ◽

Bioreactor Culture ◽

Wild Type ◽

Steel Mill ◽

Engineered Strain

ABSTRACTHydrogenogenic CO oxidation (CO + H2O → CO2+ H2) has the potential for H2production as a clean renewable fuel.Thermococcus onnurineusNA1, which grows on CO and produces H2, has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H2, the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na+/H+antiporter and a 1.8-fold-higher specific activity for CO-dependent H2production than did the wild-type strain. The H2production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H2production rate of the engineered strain was severalfold higher than those of any other CO-dependent H2-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H2production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.

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Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2801 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2801-2808 ◽

Cited By ~ 9

Author(s):

D T Mooney ◽

D B Pilgrim ◽

E T Young

Keyword(s):

Point Mutations ◽

Wild Type ◽

Urea Treatment ◽

Amino Terminal ◽

In Vitro Processing ◽

The Matrix ◽

Terminal Amino ◽

Processing Protease

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.

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