Characterization of Escherichia coli men Mutants Defective in Conversion of o -Succinylbenzoate to 1,4-Dihydroxy-2-Naphthoate

1982 ◽  
Vol 152 (3) ◽  
pp. 1132-1137
Author(s):  
Duncan J. Shaw ◽  
John R. Guest ◽  
Rangaswamy Meganathan ◽  
Ronald Bentley
Keyword(s):  
Escherichia Coli ◽  
Vitamin K ◽  
Gene Order ◽  
Cell Free Extract ◽  
E Coli ◽  
Mycobacterium Phlei ◽  
Form Part ◽  
A Cell

Four independent menaquinone (vitamin K 2 )-deficient mutants of Escherichia coli , blocked in the conversion of o -succinylbenzoate (OSB) to 1,4-dihydroxy-2-naphthoate (DHNA), were found to represent two distinct classes. Enzymatic complementation was observed when a cell-free extract of one mutant was mixed with extracts of any of the remaining three mutants. The missing enzymes in the two classes were identified by in vitro complementation with preparations of OSB-coenzyme A (CoA) synthetase or DHNA synthase isolated from Mycobacterium phlei . Mutants lacking DHNA synthase (and therefore complementing with M. phlei DHNA synthase) were designated menB , and the mutant lacking OSB-CoA synthetase (and therefore complementing with M. phlei OSB-CoA synthetase) was designated menE . The menB mutants produced only the spirodilactone form of OSB when extracts were incubated with [2,3- 14 C 2 ]OSB, ATP, and CoA; the OSB was unchanged on incubation with an extract from the menE mutant under these conditions. Experiments with strains lysogenized by a λ men transducing phage (λG68) and transduction studies with phage P1 indicated that the menB and menE genes form part of a cluster of four genes, controlling the early steps in menaquinone biosynthesis, located at 48.5 min in the E. coli linkage map. Evidence was obtained for the clockwise gene order gyrA ....-B-D, where the asterisk denotes the uncertain position of menE relative to menC and menB . The transducing phage (λG68) contained functional menB, menC , and menE genes, but only part of the menD gene, and it was designated λ menC B(D) .

Disassembly of the Drosophila nuclear lamina in a homologous cell-free system

1995 ◽  
Vol 108 (5) ◽  
pp. 2027-2035 ◽  
Author(s):  
N. Maus ◽  
N. Stuurman ◽  
P.A. Fisher
Keyword(s):  
Nuclear Lamina ◽  
Meiotic Metaphase ◽  
Two Dimensional ◽  
Cell Free Extract ◽  
Free System ◽  
Nuclear Lamin ◽  
A Cell

Stage 14 Drosophila oocytes are arrested in first meiotic metaphase. A cell-free extract of these oocytes catalyzes apparent disassembly of purified Drosophila nuclei as well as of nuclear lamin polymers formed in vitro from isolated interphase lamins. Biochemically, the oocyte extract catalyzes lamin solubilization and phosphorylation as well as characteristic changes in one- and two-dimensional gel mobility. A previously unidentified soluble lamin isoform is easily seen after in vitro disassembly. This isoform is detectable but present only in very small quantities in vivo and is apparently derived specifically from one of the two interphase lamin isoforms. Cell-free nuclear lamina disassembly is ATP-dependent and addition of calcium to extracts blocks disassembly as judged both morphologically and biochemically. This system will allow enzymological characterization of cell-free lamina disassembly as well as molecular analysis of specific Drosophila mutants.

scholarly journals Biotin synthase from Escherichia coli: isolation of an enzyme-generated intermediate and stoichiometry of S-adenosylmethionine use

1998 ◽  
Vol 330 (3) ◽  
pp. 1079-1085 ◽  
Author(s):  
M. Nicholas SHAW ◽  
M. Olwen BIRCH ◽  
Andreas TINSCHERT ◽  
Veronika VENETZ ◽  
Rüdiger DIETRICH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hplc Analysis ◽  
Cell Protein ◽  
Ph Optimum ◽  
Biotin Synthase ◽  
Reaction Velocity ◽  
E Coli ◽  
Soluble Cell ◽  
A Cell

A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1% of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8.5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in vitro was estimated to be 0.95 h-1, and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with 14C, and therefore derived from the [14C]dethiobiotin substrate; (2) it was present only in reaction mixtures containing biotin synthase; (3) it was not derived from [14C]biotin; (4) 35S from [35S]cystine was incorporated into the intermediate during the reaction; (5) its synthesis was dependent on the presence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the reaction mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversion to biotin was S-adenosylmethionine-dependent. During the reaction S-adenosylmethionine was cleaved to methionine and presumably 5ʹ-deoxyadenosine. Observation of the intermediate allowed us to perform experiments to determine the stoichiometry of S-adenosylmethionine use. We propose that two molecules of S-adenosylmethionine are used to synthesize one molecule of biotin, i.e. one from dethiobiotin to the intermediate, and a second from the intermediate to biotin.

scholarly journals Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

2005 ◽  
Vol 389 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Rajesh K. Soni ◽  
Parul Mehra ◽  
Gauranga Mukhopadhyay ◽  
Suman Kumar Dhar
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
E Coli ◽  
Dnab Helicase ◽  
H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

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.

scholarly journals ISOLATION AND CHARACTERIZATION OF dnaX AND dnaY TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI

Genetics ◽  
1979 ◽  
Vol 92 (4) ◽  
pp. 1041-1059
Author(s):  
Joan M Henson ◽  
Herman Chu ◽  
Carleen A Irwin ◽  
James R Walker
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
E Coli ◽  
Temperature Sensitive Mutants ◽  
Isolation And Characterization ◽  
Ts Mutations

ABSTRACT Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage PI, dnaX and Y have been mapped at minutes 10.4-10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dnaXts36 and YtslO are recessive to wild-type alleles present on episomes. F13 carries both dnaX  + and Y  +; the shorter F210 carries dnaY  +, but not X  +. Lambda transducing phages that carry dnaX  + or Y  + have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the CLARKE and CARBON (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the λ transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5.—The dnaXts36 mutant, after a shift to 42°, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to M°, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42° and was partially in-hibited even at 30°.—When the dnaYtslO mutant was shifted to 42°, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44°, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42° or by "preincubation" of cells at 42° before toluene treatment.— The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.

scholarly journals Purification and characterization of a diptericin homologue from Sarcophaga peregrina (flesh fly)

1992 ◽  
Vol 287 (2) ◽  
pp. 573-578 ◽  
Author(s):  
M Ishikawa ◽  
T Kubo ◽  
S Natori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Bactericidal Activity ◽  
Fat Body ◽  
Amino Acid Sequencing ◽  
Purification And Characterization ◽  
E Coli ◽  
Escherichia Coli Cells

A protein with a molecular mass of 8 kDa was found to be synthesized specifically when the fat-body from injured Sarcophaga peregrina larvae was cultured in vitro. This protein was purified from the haemolymph of the injured larvae to near-homogeneity. Partial amino acid sequencing revealed that this protein is a diptericin homologue. It showed bactericidal activity on growing, but not resting Escherichia coli cells. E. coli cells become elongated on treatment with this protein.

scholarly journals Characterization of Clinical Escherichia coli Strains Producing a Novel Shiga Toxin 2 Subtype in Sweden and Denmark

2021 ◽  
Vol 9 (11) ◽  
pp. 2374
Author(s):  
Xiangning Bai ◽  
Flemming Scheutz ◽  
Henrik Mellström Dahlgren ◽  
Ingela Hedenström ◽  
Cecilia Jernberg
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Epidemiological Surveillance ◽  
E Coli ◽  
Shiga Toxin 2 ◽  
Life Threatening ◽  
Macrolide Resistance Gene

Shiga toxin (Stx) is the key virulence factor in the Shiga Toxin-Producing Escherichia coli (STEC), which can cause diarrhea and hemorrhagic colitis with potential life-threatening complications. There are two major types of Stx: Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in E. coli, varying in sequences, toxicity and host specificity. Here, we report a novel Stx2 subtype (designated Stx2m) from three clinical E. coli strains isolated from diarrheal patients and asymptomatic carriers in Sweden and Denmark. The Stx2m toxin was functional and exhibited cytotoxicity in vitro. The two Swedish Stx2m-producing strains belonged to the same serotype O148:H39 and Multilocus Sequencing Typing (MLST) Sequence Type (ST) 5825, while the Danish strain belonged to the O96:H19 serotype and ST99 type. Whole-genome sequencing (WGS) data analysis revealed that the three Stx2m-producing strains harbored additional virulence genes and the macrolide resistance gene mdf (A). Our findings expand the pool of Stx2 subtypes and highlight the clinical significance of emerging STEC variants. Given the clinical relevance of the Stx2m-producing strains, we propose to include Stx2m in epidemiological surveillance of STEC infections and clinical diagnosis.

scholarly journals Distinct stress responses to pyocyanin by planktonic and sessile Staphylococcus aureus UFPEDA 02 and Escherichia coli UFPEDA 224 / Respostas distintas ao estresse causado pela piocianina em células planctônicas e sésseis de Staphylococcus aureus UFPEDA 02 e Escherichia coli UFPEDA 224

2021 ◽  
Vol 7 (10) ◽  
pp. 98074-98088
Author(s):  
Bianca Teixeira Morais De Oliveira ◽  
Kaíque Yago Gervazio De Lima ◽  
Ray Ravilly Alves Arruda ◽  
Ulrich Vasconcelos
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Stress Responses ◽  
Cell Concentration ◽  
The Other ◽  
Planktonic Cells ◽  
E Coli ◽  
Highly Sensitive ◽  
A Cell

Antimicrobial activity of pyocyanin against competing organisms of Pseudomonas aeruginosa is related to the oxidative stress that the compound promotes in susceptible cells. The objective of this work was to produce, extract and verify the activity of pyocyanin in planktonic and sessile forms from clinical strains, Staphylococcus aureus UFPEDA 02 and Escherichia coli UFPEDA 224. About 600 µg/mL of pyocyanin were obtained. The planktonic cells were highly sensitive. The MIC determined for S. aureus UFPEDA 02 and E. coli UFPEDA 224 were 18.75 and 37.5 µg/mL, respectively. The pyocyanin demonstrated biocidal effect against S. aureus UFPEDA 02. On the other hand, pyocyanin was not active in either sessile strain. The presence of the pigment allowed a greater adherence of the strains, forming more robust biofilms compared to the control. S. aureus UFPEDA 02 and E. coli UFPEDA 224 presented moderate and high hydrophobicity, respectively. Glass and dolomite surfaces were tested in the in vitro biofilm test. Both strains formed the biofilm better on the dolomite surface, obtaining a cell concentration (MPN/cm2) in the order of 3 log units after 48h of incubation.

scholarly journals Identification and Characterization of a Novel ABC Iron Transport System, fit, in Escherichia coli

2006 ◽  
Vol 74 (12) ◽  
pp. 6949-6956 ◽  
Author(s):  
Zhiming Ouyang ◽  
Richard Isaacson
Keyword(s):  
Escherichia Coli ◽  
Growth Promotion ◽  
Hypothetical Protein ◽  
Bidirectional Promoter ◽  
Intracellular Iron ◽  
E Coli ◽  
K 12 ◽  
Identification And Characterization

ABSTRACT A putative ABC transporter, fit, with significant homology to several bacterial iron transporters was identified in Escherichia coli. The E. coli fit system consists of six genes designated fitA, -B, -C, -D, -E, and -R. Based on DNA sequence analysis, fit encodes an outer membrane protein (FitA), a periplasmic binding protein (FitE), two permease proteins (FitC and -D), an ATPase (FitB), and a hypothetical protein (FitR). Introduction of the E. coli fit system into E. coli strain K-12 increased intracellular iron content and transformed bacteria were more sensitive to streptonigrin, which suggested that fit transports iron in E. coli. Expression of fit was studied using a lacZ reporter assay. A functional, bidirectional promoter was identified in the intergenic region between genes fitA and fitB. The expression of the E. coli fit system was found to be induced by iron limitation and repressed when Fe2+ was added to minimal medium. Several fit mutants were created in E. coli using an in vitro transposon mutagenesis strategy. Mutations in fit did not affect bacterial growth in iron-restricted media. Using a growth promotion test, it was found that fit was not able to transport enterobactin, ferrichrome, transferrin, and lactoferrin in E. coli.

scholarly journals The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase

2020 ◽  
Vol 295 (18) ◽  
pp. 6225-6235 ◽  
Author(s):  
Alexander C. Anderson ◽  
Alysha J. N. Burnett ◽  
Lana Hiscock ◽  
Kenneth E. Maly ◽  
Joel T. Weadge
Keyword(s):  
Escherichia Coli ◽  
Catalytic Mechanism ◽  
Cellulose Synthase ◽  
Gram Negative Bacteria ◽  
Membrane Phospholipids ◽  
Colistin Resistance ◽  
E Coli ◽  
Terminal Domain

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

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