scholarly journals Characterization of Glycine SarcosineN-Methyltransferase and Sarcosine DimethylglycineN-Methyltransferase

2001 ◽  
Vol 67 (5) ◽  
pp. 2044-2050 ◽  
Author(s):  
Antti Nyyssölä ◽  
Tapani Reinikainen ◽  
Matti Leisola
Keyword(s):  
Escherichia Coli ◽  
Osmotic Pressure ◽  
Glycine Betaine ◽  
The Other ◽  
Substrate Specificities ◽  
Methyl Group ◽  
High Substrate ◽  
Ectothiorhodospira Halochloris ◽  
High Concentrations

ABSTRACT Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosineN-methyltransferase (GSMT) and sarcosine dimethylglycineN-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed inEscherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent Km andV max values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction productS-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.

scholarly journals Characterization of the Two Mycobacterium tuberculosis recA Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 6005-6015 ◽  
Author(s):  
Krishna K. Gopaul ◽  
Patricia C. Brooks ◽  
Jean-François Prost ◽  
Elaine O. Davis
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Mycobacterium Tuberculosis ◽  
Promoter Activity ◽  
The Other ◽  
Expression Level ◽  
Promoter Elements ◽  
Kinetics Of

ABSTRACT The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli σ70 −35 elements but located much closer to the −10 element is important for optimal expression of P1, whereas the sequence at the −35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.

scholarly journals Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

2005 ◽  
Vol 71 (11) ◽  
pp. 7224-7228 ◽  
Author(s):  
Tina Lütke-Eversloh ◽  
Gregory Stephanopoulos
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Molecular Level ◽  
Feedback Inhibition ◽  
Feedback Regulation ◽  
The Other ◽  
Chorismate Mutase ◽  
Amino Acid Substitutions ◽  
Prephenate Dehydrogenase

ABSTRACT In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA fbr mutants were selected by virtue of their resistance toward m-fluoro-d,l-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrAWT, but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrAfbr mutants were similar to those of the TyrAWT protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA fbr contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.

scholarly journals Characterization of the protein gp1 from bacteriophage φIN93

2011 ◽  
Vol 48 (1) ◽  
pp. 47-56 ◽  
Author(s):  
Isao Matsushita ◽  
Hideshi Yanase
Keyword(s):  
Escherichia Coli ◽  
Recombinant Plasmid ◽  
Shuttle Vector ◽  
Thermus Thermophilus ◽  
The Other ◽  
Replication Protein

Characterization of the protein gp1 from bacteriophage φIN93The protein gp1, encoded by theORF1gene in the genome of theThermusphage φIN93, is similar to the replication protein encoded byrepAin theThermus sp.plasmid. To confirm that gp1 functions as a replication protein, we constructed the recombinant plasmid pIA1, by insertingORF1and the kanamycin acetyltransferase (kat) gene into pBluescriptII SK(+), which enabled replication of pIA1 inThermus thermophilusHB27. By contrast, plasmid pIA1-del, which contained only a part ofORF1(the other part deleted usingKpnI), was not replicated. These results show that gp1 functions as a replication protein and that pIA1 can be used as a shuttle vector betweenThermus thermophilusHB27 andEscherichia coli.

scholarly journals Characterization of the Catalytic Activities of the PhoQ Histidine Protein Kinase of Salmonella entericaSerovar Typhimurium

2001 ◽  
Vol 183 (5) ◽  
pp. 1787-1791 ◽  
Author(s):  
Martin Montagne ◽  
Alexandre Martel ◽  
Hervé Le Moual
Keyword(s):  
Escherichia Coli ◽  
Phosphatase Activity ◽  
Divalent Cations ◽  
Histidine Protein Kinase ◽  
Catalytic Activities ◽  
Serovar Typhimurium ◽  
High Concentrations ◽  
Kinetics Of ◽  
Autokinase Activity

ABSTRACT Studies of Escherichia coli membranes that were highly enriched in the Salmonella enterica serovar Typhimurium PhoQ protein showed that the presence of ATP and divalent cations such as Mg2+, Mn2+, Ca2+, or Ba2+ resulted in PhoQ autophosphorylation. However, when Mg2+ or Mn2+was present at concentrations higher than 0.1 mM, the kinetics of PhoQ autophosphorylation were strongly biphasic, with a rapid autophosphorylation phase followed by a slower dephosphorylation phase. A fusion protein lacking the sensory and transmembrane domains retained the autokinase activity but could not be dephosphosphorylated when Mg2+ or Mn2+ was present at high concentrations. The instability of purified [32P]phospho-PhoP in the presence of PhoQ-containing membranes indicated that PhoQ also possesses a phosphatase activity. The PhoQ phosphatase activity was stimulated by increasing the Mg2+ concentration. These data are consistent with a model in which Mg2+ binding to the sensory domain of PhoQ coordinately regulates autokinase and phosphatase activities.

Ochrobactrum tritici strain 5bvl1 — characterization of a Cr(VI)-resistant and Cr(VI)-reducing strain

2004 ◽  
Vol 50 (9) ◽  
pp. 697-703 ◽  
Author(s):  
Rita Branco ◽  
M Carmen Alpoim ◽  
Paula V Morais
Keyword(s):  
Heavy Metal ◽  
Growth Rate ◽  
Type Strain ◽  
Treatment Plant ◽  
The Other ◽  
Reducing Ability ◽  
High Concentrations ◽  
Ph Range ◽  
Ochrobactrum Tritici

Bacterial strain 5bvl1, isolated from a chromium-contaminated wastewater treatment plant and identified as Ochrobactrum tritici, was resistant to a broad range of antibiotics, to Cr(VI), Ni(II), Co(II), Cd(II), and Zn(II), and was able to grow in the presence of 5% NaCl and within the pH range 4–10. Characterization showed that strain 5bvl1 could be considered a halotolerant and alkalitolerant microorganism resistant to high concentrations of Cr(VI). This strain was able to grow aerobically in up to 10 mmol·L–1 Cr(VI). Cr(VI) resistance was independent of sulphate concentration. Under aerobic conditions strain 5bvl1 was also able to reduce high Cr(VI) concentrations (up to 1.7 mmol·L–1). Increasing concentrations of Cr(VI) in the medium lowered the growth rate of strain 5bv11 but the reduction in growth rate could not be directly correlated with the amount of Cr(VI) reduced. Unlike the type strain, which was only able to reduce Cr(VI), strain 5bvl1 was resistant to Cr(VI) and able to reduce it. Moreover, in strain 5bvl1, the rate and extent of Cr(VI)-reduction were higher than in the other strains of the genus Ochrobactrum. Ochrobactrum strain 5bvl1 resists high Cr(VI) concentrations and has a high Cr(VI)-reducing ability, making it a valuable tool in bioremediation.Key words: Ochrobactrum, Cr(VI) resistance, Cr(VI)-reduction, heavy metal, bioremediation.

scholarly journals Selection, mapping, and characterization of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway.

1986 ◽  
Vol 165 (3) ◽  
pp. 856-863 ◽  
Author(s):  
O B Styrvold ◽  
P Falkenberg ◽  
B Landfald ◽  
M W Eshoo ◽  
T Bjørnsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Glycine Betaine

Colicinogeny among Escherichia coli Serotypes, Including O157:H7, Representing Four Closely Related Diarrheagenic Clones

1998 ◽  
Vol 61 (11) ◽  
pp. 1431-1438 ◽  
Author(s):  
SHELTON E. MURINDA ◽  
SHU-MIN LIU ◽  
ROBERT F. ROBERTS ◽  
RICHARD A. WILSON
Keyword(s):  
Escherichia Coli ◽  
Toxin Production ◽  
Coli Strain ◽  
The Other ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Reference Set ◽  
Almost All ◽  
Luria Agar

Twenty-seven diarrheagenic Escherichia coli (DEC) strains from five closely related, genetically distinct clones (DEC 3, 4, 8, 9, and 10), representing serotypes commonly associated with Shiga-like toxin production, i.e., 015:H−, 026:(H11, H−), 0111:(H8, H11, H−), and O157:H7, were evaluated for colicinogeny on Luria agar or Luria agar containing 0.25 μg/ml mitomycin C to induce colicin production. Ten (37%) of the DEC strains tested were colicinogenic. One of 11 serotype O157:H7 strains, DEC strain 4E, produced a colicin identified as Col D. DEC strains 8B, 9D, and 10B produced Col E1, whereas DEC strain 10A produced Col E2. DEC strains 8A, 8E, 10C, 10E, and 10F produced “untypable” colicins that killed almost all Pugsley Colicin Reference Set strains and the other DEC strains tested. To aid with further characterization of the colicins, plasmids extracted from each colicin-producing (Col+) DEC strain were used to transform E. coli strain DH5α. All Col+ DH5α transformants contained one plasmid ranging in size from 1.3 to 10 kb. Some transformants were stable colicin producers whereas others were unstable. The inhibitory activity and colicin sensitivity and insensitivity profiles of the Col+ transformants were similar to those of the corresponding Col+ donor DEC strains. It appears that the untypable colicins are novel and, thus, warrant further study. Colicin production by some of the DEC strains evaluated partly explains why they were insensitive to standard colicins in a previous study.

Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems

1989 ◽  
Vol 3 (8) ◽  
pp. 1025-1038 ◽  
Author(s):  
D. A. Stirling ◽  
C. S. J. Hulton ◽  
L. Waddell ◽  
S. F. Park ◽  
G. S. A. B. Stewart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Molecular Characterization ◽  
Glycine Betaine ◽  
Transport Systems

scholarly journals Characterization of Fungal FAD-Dependent AA3_2 Glucose Oxidoreductases from Hitherto Unexplored Phylogenetic Clades

2021 ◽  
Vol 7 (10) ◽  
pp. 873
Author(s):  
Sudarma Dita Wijayanti ◽  
Leander Sützl ◽  
Adèle Duval ◽  
Dietmar Haltrich
Keyword(s):  
Biochemical Characterization ◽  
Glucose Dehydrogenase ◽  
Catalytic Efficiency ◽  
Ustilago Maydis ◽  
The Other ◽  
Trichoderma Virens ◽  
Substrate Specificities ◽  
Anomeric Carbon ◽  
Substrate Spectrum

The CAZy auxiliary activity family 3 (AA3) comprises FAD-dependent enzymes belonging to the superfamily of glucose-methanol-choline (GMC) oxidoreductases. Glucose oxidase (GOx; EC 1.1.3.4) and glucose dehydrogenase (GDH; EC 1.1.5.9) are part of subfamily AA3_2 and catalyze the oxidation of β-D-glucose at its anomeric carbon to D-glucono-1,5-lactone. Recent phylogenetic analysis showed that AA3_2 glucose oxidoreductases can be grouped into four major clades, GOx I and GDH I–III, and in minor clades such as GOx II or distinct subclades. This wide sequence space of AA3_2 glucose oxidoreductases has, however, not been studied in detail, with mainly members of GOx I and GDH I studied biochemically or structurally. Here, we report the biochemical characterization of four fungal glucose oxidoreductases from distinct, hitherto unexplored clades or subclades. The enzyme from Aureobasidium subglaciale, belonging to the minor GOx II clade, showed a typical preference for oxygen and glucose, confirming the correct annotation of this clade. The other three enzymes exhibited strict dehydrogenase activity with different substrate specificities. GDH II from Trichoderma virens showed an almost six-fold higher catalytic efficiency for maltose compared to glucose. The preferred substrate for the two GDH III enzymes from Rhizoctonia solani and Ustilago maydis was gentiobiose, a β(1→6) disaccharide, as judged from the catalytic efficiency. Overall, the newly studied AA3_2 glucose oxidoreductases showed a much broader substrate spectrum than the archetypal GOx from Aspergillus niger, which belongs to clade GOx I.

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