scholarly journals Glutamyl-tRNA reductase activity in Bacillus subtilis is dependent on the hemA gene product

1992 ◽  
Vol 281 (3) ◽  
pp. 843-850 ◽  
Author(s):  
I Schröder ◽  
L Hederstedt ◽  
C G Kannangara ◽  
S P Gough
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Molecular Mass ◽  
Gene Product ◽  
Reductase Activity ◽  
Gel Filtration ◽  
Wild Type ◽  
C5 Pathway ◽  
Hema Gene

The Bacillus subtilis hemAXCDBL operon encodes enzymes for the synthesis of 5-aminolaevuline acid via the C5 pathway (hemA and hemL) and uroporphyrinogen III (hemB, hemC and hemD). B. subtilis HemA protein (molecular mass 50 kDa) was overexpressed in hemA mutant of both Escherichia coli and B. subtilis. A mutant B. subtilis HemA protein with a Cys to Tyr change at position 105 was also overexpressed. Both wild-type and mutant HemA proteins migrated as oligomers (molecular mass greater than or equal to 230 kDa) on gel-filtration columns. All column fractions containing wild-type HemA protein had glutamyl-tRNA reductase activity. No glutamyl-tRNA reductase activity was found with the mutant HemA protein. It is concluded that the B. subtilis hemA gene product is identical to, or part of, the glutamyl-tRNA reductase of the C5 pathway.

Involvement of the cgtA gene function in stimulation of DNA repair in Escherichia coli and Vibrio harveyi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1763-1770 ◽  
Author(s):  
Ryszard Zielke ◽  
Aleksandra Sikora ◽  
Rafał Dutkiewicz ◽  
Grzegorz Wegrzyn ◽  
Agata Czyż
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Repair ◽  
Gene Product ◽  
Uv Irradiation ◽  
Vibrio Harveyi ◽  
Bacterial Species ◽  
Wild Type ◽  
E Coli ◽  
Stimulation Of

CgtA is a member of the Obg/Gtp1 subfamily of small GTP-binding proteins. CgtA homologues have been found in various prokaryotic and eukaryotic organisms, ranging from bacteria to humans. Nevertheless, despite the fact that cgtA is an essential gene in most bacterial species, its function in the regulation of cellular processes is largely unknown. Here it has been demonstrated that in two bacterial species, Escherichia coli and Vibrio harveyi, the cgtA gene product enhances survival of cells after UV irradiation. Expression of the cgtA gene was found to be enhanced after UV irradiation of both E. coli and V. harveyi. Moderate overexpression of cgtA resulted in higher UV resistance of E. coli wild-type and dnaQ strains, but not in uvrA, uvrB, umuC and recA mutant hosts. Overexpression of the E. coli recA gene in the V. harveyi cgtA mutant, which is very sensitive to UV light, restored the level of survival of UV-irradiated cells to the levels observed for wild-type bacteria. Moreover, the basal level of the RecA protein was lower in a temperature-sensitive cgtA mutant of E. coli than in the cgtA + strain, and contrary to wild-type bacteria, no significant increase in recA gene expression was observed after UV irradiation of this cgtA mutant. Finally, stimulation of uvrB gene transcription under these conditions was impaired in the V. harveyi cgtA mutant. All these results strongly suggest that the cgtA gene product is involved in DNA repair processes, most probably by stimulation of recA gene expression and resultant activation of RecA-dependent DNA repair pathways.

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli

1979 ◽  
Vol 57 (6) ◽  
pp. 813-821 ◽  
Author(s):  
Peter Dickie ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sodium Cholate ◽  
Defined Medium ◽  
Exchange Chromatography ◽  
Fumarate Reductase ◽  
Molar Ratios ◽  
Membrane Bound

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic–lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme, whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, present in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Sephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.

scholarly journals Gonococcal MinD Affects Cell Division inNeisseria gonorrhoeae and Escherichia coli and Exhibits a Novel Self-Interaction

2001 ◽  
Vol 183 (21) ◽  
pp. 6253-6264 ◽  
Author(s):  
Jason Szeto ◽  
Sandra Ramirez-Arcos ◽  
Claude Raymond ◽  
Leslie D. Hicks ◽  
Cyril M. Kay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Morphological Changes ◽  
Shuttle Vector ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
E Coli ◽  
Two Hybrid

ABSTRACT The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae(MinDNg) in this mutant. Hence, MinDNg is required for maintaining proper cell division and growth in N. gonorrhoeae. Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinDNg is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinCNg and MinDNg from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinCNg or MinDNg alone did not display noticeable morphological changes. These studies suggest that MinDNg is involved in inhibiting gonococcal cell division, likely in conjunction with MinCNg. The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinDNg in wild-type Escherichia coli led to cell filamentation, while overexpression in an E. coli minD mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinDNg revealed a novel MinDNgself-interaction. We have also shown by yeast two-hybrid analysis that MinD from E. coli interacts with itself and with MinDNg. These results indicate that MinDNg is required for maintaining proper cell division and growth in N. gonorrhoeae and suggests that the self-interaction of MinD may be important for cell division site selection across species.

scholarly journals Substitution of an Alanine Residue for Glycine 146 in TMP Kinase from Escherichia coli Is Responsible for Bacterial Hypersensitivity to Bromodeoxyuridine

1998 ◽  
Vol 180 (16) ◽  
pp. 4291-4293 ◽  
Author(s):  
Lise Tourneux ◽  
Nadia Bucurenci ◽  
Ioan Lascu ◽  
Hiroshi Sakamoto ◽  
Gilbert Briand ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Mutant Strain ◽  
Wild Type ◽  
E Coli ◽  
Alanine Residue

ABSTRACT The wild-type TMP kinases from Escherichia coli and from a strain hypersensitive to 5-bromo-2′-deoxyuridine were characterized comparatively. The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase. Plasmids carrying the wild-type tmk gene from Escherichia coli orBacillus subtilis, but not the defective tmkgene, restored the resistance to bromodeoxyuridine of an E. coli mutant strain.

scholarly journals Purification and properties of a 1,3-1,4-β-d-glucanase (lichenase, 1,3-1,4-β-d-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli

1988 ◽  
Vol 255 (3) ◽  
pp. 833-841 ◽  
Author(s):  
J D Erfle ◽  
R M Teather ◽  
P J Wood ◽  
J E Irvin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Glucanase Activity ◽  
Ph Optimum ◽  
Beta Glucanase ◽  
Purification And Properties ◽  
Hydrolysis Of

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.

ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

2015 ◽  
Vol 25 (6) ◽  
pp. 394-402 ◽  
Author(s):  
Taylor L. Fischer ◽  
Robert J. White ◽  
Katherine F.K. Mares ◽  
Devin E. Molnau ◽  
Justin J. Donato
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Wild Type ◽  
E Coli ◽  
Enoyl Acp Reductase ◽  
Selection For

<b><i>Background/Aims:</i></b> We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in <i>Escherichia coli</i>. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. <b><i>Methods:</i></b> ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into <i>E. coli</i>, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. <b><i>Results:</i></b> Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into <i>E. coli</i>, whereas the mutant remained susceptible to triclosan<i>. </i>Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. <b><i>Conclusion:</i></b> ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

Sequencing reveals similarity of the wild-type div+ gene of Bacillus subtilis to the Escherichia coli secA gene

Gene ◽  
1991 ◽  
Vol 98 (1) ◽  
pp. 101-105 ◽  
Author(s):  
Sadaie Yoshito ◽  
Takamatsu Hiromu ◽  
Nakamura Kouji ◽  
Yamane Kunio
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Wild Type ◽  
Seca Gene

scholarly journals Genetic Analysis of an Incomplete mutSGene from Pseudomonas putida

2000 ◽  
Vol 182 (18) ◽  
pp. 5278-5279 ◽  
Author(s):  
Yasurou Kurusu ◽  
Tomoaki Narita ◽  
Makoto Suzuki ◽  
Taeko Watanabe
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Genetic Analysis ◽  
Pseudomonas Putida ◽  
Type Strain ◽  
Mutation Frequency ◽  
Wild Type Strain ◽  
Wild Type

ABSTRACT We genetically characterized the Pseudomonas putida mutS gene and found that it encodes a smaller MutS protein than do the genes of other bacteria. This gene is able to function in themutS mutants of Escherichia coli andBacillus subtilis. A P. putida mutS mutant has a mutation frequency 1,000-fold greater than that of the wild-type strain.

scholarly journals The yvyD Gene of Bacillus subtilis Is under Dual Control of ςB and ςH

1998 ◽  
Vol 180 (24) ◽  
pp. 6674-6680 ◽  
Author(s):  
Kathrin Drzewiecki ◽  
Christine Eymann ◽  
Gerhard Mittenhuber ◽  
Michael Hecker
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Gene Product ◽  
Stress Proteins ◽  
Stringent Response ◽  
Primer Extension ◽  
Wild Type ◽  
Computer Aided ◽  
Protein Gels ◽  
Amino Acid Depletion

ABSTRACT During a search by computer-aided inspection of two-dimensional (2D) protein gels for ςB-dependent general stress proteins exhibiting atypical induction profiles, a protein initially called Hst23 was identified as a product of the yvyD gene of Bacillus subtilis. In addition to the typical ςB-dependent, stress- and starvation-inducible pattern,yvyD is also induced in response to amino acid depletion. By primer extension of RNA isolated from the wild-type strain and appropriate mutants carrying mutations in the sigB and/orspo0H gene, two promoters were mapped upstream of theyvyD gene. The ςB-dependent promoter drives expression of yvyD under stress conditions and after glucose starvation, whereas a ςH-dependent promoter is responsible for yvyD transcription following amino acid limitation. Analysis of Northern blots revealed that yvyDis transcribed monocistronically and confirmed the conclusions drawn from the primer extension experiments. The analysis of the protein synthesis pattern in amino acid-starved wild-type and relAmutant cells showed that the YvyD protein is not synthesized in therelA mutant background. It was concluded that the stringent response plays a role in the activation of ςH. TheyvyD gene product is homologous to a protein which might modify the activity of ς54 in gram-negative bacteria. The expression of a ςL-dependent (ςL is the equivalent of ς54 in B. subtilis)levD-lacZ fusion is upregulated twofold in ayvyD mutant. This indicates that the yvyD gene product, being a member of both the ςB and ςH regulons, might negatively regulate the activity of the ςL regulon. We conclude that (i) systematic, computer-aided analysis of 2D protein gels is appropriate for the identification of genes regulated by multiple transcription factors and that (ii) YvyD might form a junction between the ςB and ςH regulons on one side and the ςL regulon on the other.

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