scholarly journals Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

2004 ◽  
Vol 186 (5) ◽  
pp. 1229-1238 ◽  
Author(s):  
Trygve Brautaset ◽  
Øyvind M. Jakobsen ◽  
Michael C. Flickinger ◽  
Svein Valla ◽  
Trond E. Ellingsen
Keyword(s):  
Carbon Source ◽  
Shuttle Vector ◽  
Genetic Manipulations ◽  
Optimum Growth Temperature ◽  
Bacillus Methanolicus ◽  
Dehydrogenase Gene ◽  
Fructose 1,6 Bisphosphatase ◽  
Ribulose Monophosphate Pathway ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50°C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

scholarly journals Characterization of D-Arabitol as Newly Discovered Carbon Source of Bacillus methanolicus

2019 ◽  
Vol 10 ◽  
Author(s):  
Marina Gil López ◽  
Marta Irla ◽  
Luciana F. Brito ◽  
Volker F. Wendisch
Keyword(s):  
Carbon Source ◽  
Bacillus Methanolicus

scholarly journals Gluconeogenic fructose-1,6-bisphosphatase from the mature sporocarps of common aquatic ferns: partial purification and basic characterization of this enzyme from Marsilea minuta (Polypodiopsida)

2020 ◽  
Vol 77 (5) ◽  
pp. 386-397
Author(s):  
S.S. Ghosh ◽  
◽  
M. Das ◽  
S. Basu ◽  
J. Adhikari ◽  
...  
Keyword(s):  
Partial Purification ◽  
Light Illumination ◽  
Azolla Pinnata ◽  
Fructose 1,6 Bisphosphatase ◽  
Salvinia Natans ◽  
Km Value ◽  
Gluconeogenic Enzyme ◽  
Ph Range ◽  
Fructose 1,6 Bisphosphate

The present communication reports substantial activity of gluconeogenic fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) in three common heterosporous aquatic ferns (Marsilea minuta, Salvinia natans, and Azolla pinnata) and also describes a protocol for its partial purification from mature sporocarps of Marsilea minuta. The cytosolic FBPase, obtained from Marsilea minuta, Salvinia natans, and Azolla pinnata was recognized as gluconeogenic enzyme due to its drastic catabolic inactivation in presence of externally administered glucose and its insensitivity towards photosynthetic light illumination. Cytosolic gluconeogenic FBPase was partially purified from mature sporocarps of Marsilea minuta to about 22-fold over homogenate following low-speed centrifugation (11, 400 × g), 30–80% ammonium sulfate fractionation followed by subsequent chromatography using matrices like CM-Cellulose, Sephadex G-200, and Ultrogel AcA 34. The profile of partially purified FBPase in PAGE under non-denaturing condition was recorded. The enzyme activity increased linearly with respect to protein concentration to about 100 µg and with respect to time up to 75 minutes. Temperature optimum was found at 35 °C. The effect of substrate concentration and kinetic analyses for FBPase were carried out using D-fructose-1,6-bisphosphate (D-FBP, the substrate) in the range of 0.0 to 1.0 mM at an interval of 0.1 mM concentration. The Km value for D-FBP of FBPase was 0.06129 mM and Vmax was 4525 nmole Pi released (mg)-1 protein h-1 as determined by nonlinear regression kinetics using Prism 8 software (Graph Pad). The enzyme was functional in a constricted pH range of 7.0 to 8.0, giving maxima at pH 7.5. This cytosolic enzyme was significantly stimulated by Mg2+ and strongly inhibited by Hg2+, Cu2+ and Zn2+.

scholarly journals Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from Pyrococcus furiosus

2002 ◽  
Vol 184 (12) ◽  
pp. 3401-3405 ◽  
Author(s):  
Corné H. Verhees ◽  
Jasper Akerboom ◽  
Emile Schiltz ◽  
Willem M. de Vos ◽  
John van der Oost
Keyword(s):  
Biochemical Characterization ◽  
Pyrococcus Furiosus ◽  
Distinct Type ◽  
Sequence Motifs ◽  
Conserved Sequence ◽  
New Family ◽  
Fructose 1,6 Bisphosphatase ◽  
Conserved Sequence Motifs ◽  
Fructose 1,6 Bisphosphate

ABSTRACT The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a Km of 0.32 mM and a V max of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li+ (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

Morphological Characterization of Mycobacteriophage R1

1974 ◽  
Vol 32 ◽  
pp. 254-255
Author(s):  
B. L. Soloff ◽  
T. A. Rado
Keyword(s):  
Carbon Source ◽  
Stationary Phase ◽  
Growth Phase ◽  
Minimal Medium ◽  
Morphological Characterization ◽  
Logarithmic Growth Phase ◽  
Complete Lysis ◽  
Lysogenic Culture ◽  
Logarithmic Growth

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

Cloning and characterization of an aldehyde dehydrogenase gene GmALDH3-1 from soybean

2010 ◽  
Vol 32 (5) ◽  
pp. 492-497 ◽  
Author(s):  
Fang HUANG ◽  
Ying-Jun CHI ◽  
Hui HE ◽  
De-Yue YU
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Dehydrogenase Gene

scholarly journals Organization and characterization of the rat class 3 aldehyde dehydrogenase gene

1993 ◽  
Vol 268 (17) ◽  
pp. 12530-12536 ◽  
Author(s):  
D.C. Asman ◽  
K. Takimoto ◽  
H.C. Pitot ◽  
T.J. Dunn ◽  
R. Lindahl
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Dehydrogenase Gene

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

2002 ◽  
Vol 68 (12) ◽  
pp. 6237-6245 ◽  
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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