scholarly journals Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase

2000 ◽  
Vol 66 (2) ◽  
pp. 481-486 ◽  
Author(s):  
Luying Xun ◽  
Erik R. Sandvik
Keyword(s):  
Escherichia Coli ◽  
Flavin Adenine Dinucleotide ◽  
Database Search ◽  
Gene Products ◽  
Flavin Reductase ◽  
Large Component ◽  
E Coli ◽  
Sequence Similarities

ABSTRACT 4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) ofEscherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH2)-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH2 in vitro, HpaB was able to use FADH2 and O2 for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH2 for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH2-utilizing monooxygenase. FADH2 generated by Fre was rapidly oxidized by O2 to form H2O2 in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH2 and transitorily protected it from rapid autoxidation by O2. When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O2 for FADH2 utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH2 produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH2 was autoxidized by O2, causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH2-utilizing monooxygenase that uses FADH2as a substrate rather than as a cofactor.

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 Gene Products Required for De Novo Synthesis of Polysialic Acid in Escherichia coli K1

2006 ◽  
Vol 188 (5) ◽  
pp. 1786-1797 ◽  
Author(s):  
Ekaterina N. Andreishcheva ◽  
Willie F. Vann
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
De Novo ◽  
Polysialic Acid ◽  
Neonatal Meningitis ◽  
Gene Products ◽  
De Novo Synthesis ◽  
E Coli ◽  
Tract Infections

ABSTRACT Escherichia coli K1 is responsible for 80% of E. coli neonatal meningitis and is a common pathogen in urinary tract infections. Bacteria of this serotype are encapsulated with the α(2-8)-polysialic acid NeuNAc(α2-8), common to several bacterial pathogens. The gene cluster encoding the pathway for synthesis of this polymer is organized into three regions: (i) kpsSCUDEF, (ii) neuDBACES, and (iii) kpsMT. The K1 polysialyltransferase, NeuS, cannot synthesize polysialic acid de novo without other products of the gene cluster. Membranes isolated from strains having the entire K1 gene cluster can synthesize polysialic acid de novo. We designed a series of plasmid constructs containing fragments of regions 1 and 2 in two compatible vectors to determine the minimum number of gene products required for de novo synthesis of the polysialic acid from CMP-NeuNAc in K1 E. coli. We measured the ability of the various combinations of region 1 and 2 fragments to restore polysialyltransferase activity in vitro in the absence of exogenously added polysaccharide acceptor. The products of region 2 genes neuDBACES alone were not sufficient to support de novo synthesis of polysialic acid in vitro. Only membrane fractions harboring NeuES and KpsCS could form sialic polymer in the absence of exogenous acceptor at the concentrations formed by wild-type E. coli K1 membranes. Membrane fractions harboring NeuES and KpsC together could form small quantities of the sialic polymer de novo.

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 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 Characterization of the fomA andfomB Gene Products from Streptomyces wedmorensis, Which Confer Fosfomycin Resistance on Escherichia coli

2000 ◽  
Vol 44 (3) ◽  
pp. 647-650 ◽  
Author(s):  
Seiji Kobayashi ◽  
Tomohisa Kuzuyama ◽  
Haruo Seto
Keyword(s):  
Escherichia Coli ◽  
Resistance Mechanism ◽  
Biosynthetic Gene Cluster ◽  
Magnesium Ion ◽  
Gene Products ◽  
Biosynthetic Gene ◽  
E Coli ◽  
Fosfomycin Resistance ◽  
High Level

ABSTRACT Together, the fomA and fomB genes in the fosfomycin biosynthetic gene cluster of Streptomyces wedmorensis confer high-level fosfomycin resistance onEscherichia coli. To elucidate their functions, thefomA and fomB genes were overexpressed inE. coli and the gene products were characterized. The recombinant FomA protein converted fosfomycin to fosfomycin monophosphate, which was inactive on E. coli, in the presence of a magnesium ion and ATP. On the other hand, the recombinant FomB protein did not inactivate fosfomycin. However, a reaction mixture containing FomA and FomB proteins converted fosfomycin to fosfomycin monophosphate and fosfomycin diphosphate in the presence of ATP and a magnesium ion, indicating that FomA and FomB catalyzed phosphorylations of fosfomycin and fosfomycin monophosphate, respectively. These results suggest that the self-resistance mechanism of the fosfomycin-producing organism S. wedmorensis is mono- and diphosphorylation of the phosphonate function of fosfomycin catalyzed by FomA and FomB.

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.

scholarly journals Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli

1990 ◽  
Vol 270 (2) ◽  
pp. 357-361 ◽  
Author(s):  
A E I Proudfoot ◽  
D Fattah ◽  
E H Kawashima ◽  
A Bernard ◽  
P T Wingfield
Keyword(s):  
Escherichia Coli ◽  
Inducible Promoter ◽  
Cellular Protein ◽  
Biological Assays ◽  
Interleukin 5 ◽  
E Coli ◽  
Gene Coding ◽  
High Level

The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three ‘in vitro’ biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines.

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