scholarly journals Treponema pallidum 3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete

2001 ◽  
Vol 183 (16) ◽  
pp. 4702-4708 ◽  
Author(s):  
Stéphane Benoit ◽  
James E. Posey ◽  
Matthew R. Chenoweth ◽  
Frank C. Gherardini
Keyword(s):  
Treponema Pallidum ◽  
Maximum Growth ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Phosphoglycerate Mutase ◽  
Heat Sensitivity ◽  
Dependent Manner ◽  
Gene Encoding ◽  
E Coli ◽  
Key Enzyme

ABSTRACT In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP inT. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn2+ while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25°C, retained only 50% activity after incubation for 20 min at 34°C or 10 min at 37°C, and was completely inactive after 10 min at 42°C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42°C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum.

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1421-1431 ◽  
Author(s):  
Patrice Bruscella ◽  
Laure Cassagnaud ◽  
Jeanine Ratouchniak ◽  
Gaël Brasseur ◽  
Elisabeth Lojou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acidithiobacillus Ferrooxidans ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Tat System ◽  
Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

scholarly journals Reconstitution of Acetosyringone-Mediated Agrobacterium tumefaciens Virulence Gene Expression in the Heterologous Host Escherichia coli

2001 ◽  
Vol 183 (12) ◽  
pp. 3704-3711 ◽  
Author(s):  
Scott M. Lohrke ◽  
Hongjiang Yang ◽  
Shouguang Jin
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Virulence Gene Expression ◽  
E Coli ◽  
Glucose Binding

ABSTRACT The ability to utilize Escherichia coli as a heterologous system in which to study the regulation ofAgrobacterium tumefaciens virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the rpoA gene encoding the alpha subunit of RNA polymerase is required for the expression of lacZ gene under the control of virB promoter (virBp::lacZ) in E. colicontaining a constitutively active virG gene [virG(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from E. coli and the C-terminal 89 residues from A. tumefaciens was able to significantly express virBp::lacZ in E. coli in a VirG(Con)-dependent manner. Utilization oflac promoter-driven virA and virGin combination with the A. tumefaciens rpoA construct resulted in significant inducer-mediated expression of thevirBp::lacZ fusion, and the level ofvirBp::lacZ expression was positively correlated to the copy number of the rpoA construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in A. tumefaciens. Furthermore, the effect of sugars on virgene expression was observed only in the presence of thechvE gene, suggesting that the glucose-binding protein ofE. coli, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in A. tumefaciens strain A348 from which the virA clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of thevir genes in E. coli is less than what is observed in A. tumefaciens, suggesting that additional gene(s) from A. tumefaciens may be required for the full expression of virulence genes in E. coli.

scholarly journals Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene (pdc) and Comparison to Bacterial Homologues

2002 ◽  
Vol 68 (6) ◽  
pp. 2869-2876 ◽  
Author(s):  
Krishnan Chandra Raj ◽  
Lee A. Talarico ◽  
Lonnie O. Ingram ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Codon Usage ◽  
Specific Activity ◽  
Pyruvate Decarboxylase ◽  
Biochemical Properties ◽  
Kinetic Properties ◽  
E Coli ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Zymobacter Palmae

ABSTRACT Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial pdc genes have been previously described and expressed as active recombinant proteins. The pdc gene from Zymomonas mobilis has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial pdc gene from Zymobacter palmae. The pattern of codon usage for this gene appears quite similar to that for Escherichia coli genes. In E. coli recombinants, the Z. palmae PDC represented approximately 1/3 of the soluble protein. Biochemical and kinetic properties of the Z. palmae enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the Z. palmae enzyme exhibited the highest specific activity (130 U mg of protein−1) and the lowest Km for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.

scholarly journals Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

1998 ◽  
Vol 180 (22) ◽  
pp. 5997-6004 ◽  
Author(s):  
Karl Matussek ◽  
Patrick Moritz ◽  
Nina Brunner ◽  
Christoph Eckerskorn ◽  
Reinhard Hensel
Keyword(s):  
Substrate Affinity ◽  
Kinetic Properties ◽  
Gene Encoding ◽  
Enzyme Preparations ◽  
E Coli ◽  
Gene Coding ◽  
Key Enzyme ◽  
Methanothermus Fervidus ◽  
Synthetic Reaction ◽  
Sequencing And Expression

ABSTRACT Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the V max of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher V max of the enzyme from M. fervidus in the synthesizing direction.

scholarly journals Curcumin Oxidation Is Required for Inhibition of Helicobacter pylori Growth, Translocation and Phosphorylation of Cag A

2021 ◽  
Vol 11 ◽  
Author(s):  
Ashwini Kumar Ray ◽  
Paula B. Luis ◽  
Surabhi Kirti Mishra ◽  
Daniel P. Barry ◽  
Mohammad Asim ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Growth Inhibition ◽  
Mrna Expression ◽  
Host Protein ◽  
Dependent Manner ◽  
Gene Encoding ◽  
E Coli ◽  
H Pylori

Curcumin is a potential natural remedy for preventing Helicobacter pylori-associated gastric inflammation and cancer. Here, we analyzed the effect of a phospholipid formulation of curcumin on H. pylori growth, translocation and phosphorylation of the virulence factor CagA and host protein kinase Src in vitro and in an in vivo mouse model of H. pylori infection. Growth of H. pylori was inhibited dose-dependently by curcumin in vitro. H. pylori was unable to metabolically reduce curcumin, whereas two enterobacteria, E. coli and Citrobacter rodentium, which efficiently reduced curcumin to the tetra- and hexahydro metabolites, evaded growth inhibition. Oxidative metabolism of curcumin was required for the growth inhibition of H. pylori and the translocation and phosphorylation of CagA and cSrc, since acetal- and diacetal-curcumin that do not undergo oxidative transformation were ineffective. Curcumin attenuated mRNA expression of the H. pylori virulence genes cagE and cagF in a dose-dependent manner and inhibited translocation and phosphorylation of CagA in gastric epithelial cells. H. pylori strains isolated from dietary curcumin-treated mice showed attenuated ability to induce cSrc phosphorylation and the mRNA expression of the gene encoding for IL-8, suggesting long-lasting effects of curcumin on the virulence of H. pylori. Our work provides mechanistic evidence that encourages testing of curcumin as a dietary approach to inhibit the virulence of CagA.

scholarly journals Functional Interaction between Apolipophorins and Complement Regulate the Mosquito Immune Response to Systemic Infections

2016 ◽  
Vol 8 (3) ◽  
pp. 314-326 ◽  
Author(s):  
Layla Kamareddine ◽  
Johnny Nakhleh ◽  
Mike A. Osta
Keyword(s):  
Immune Response ◽  
Fungal Infections ◽  
Negative Regulator ◽  
Rnai Phenotype ◽  
Immune Gene ◽  
Dependent Manner ◽  
Gene Encoding ◽  
E Coli ◽  
Systemic Infections ◽  
Kinase Pathway

The complement-like protein thioester-containing protein 1 (TEP1) is the hallmark effector molecule against Plasmodium ookinetes in the malaria vector Anopheles gambiae. We have previously shown that the knockdown of the noncatalytic clip domain serine protease CLIPA2 increased TEP1-mediated killing rendering mosquitoes more resistant to Plasmodium, bacterial and fungal infections. Here, CLIPA2 coimmunoprecipitation from the hemolymph of Beauveria bassiana-infected mosquitoes followed by mass spectrometry and functional genetic analysis led to the identification of the Apolipophorin-II/I gene, encoding the two lipid carrier proteins Apo-I and II, as a novel negative regulator of TEP1-mediated immune response during mosquito systemic infections. Apo-II/I exhibits a similar RNAi phenotype as CLIPA2 in mosquito bioassays characterized by increased resistance to B. bassiana and Escherichia coli infections. We provide evidence that this enhanced resistance to systemic infections is TEP1 dependent. Interestingly, silencing Apo-II/I but not CLIPA2 upregulated the expression of TEP1 following systemic infections with E. coli and B. bassiana in a c-Jun N-terminal kinase pathway-dependent manner. Our results suggest that mosquito Apo-II/I plays an important immune regulatory role during systemic infections and provide novel insight into the functional interplay between lipid metabolism and immune gene regulation.

Leishmanolysin (gp63 metallopeptidase)-like activity extracellularly released byHerpetomonas samuelpessoai

Parasitology ◽  
2005 ◽  
Vol 132 (1) ◽  
pp. 37-47 ◽  
Author(s):  
C. G. R. ELIAS ◽  
F. M. PEREIRA ◽  
B. A. SILVA ◽  
C. S. ALVIANO ◽  
R. M. A. SOARES ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Protein Profile ◽  
Immunoblot Analysis ◽  
Secretory Protein ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Dependent Manner ◽  
Sds Page ◽  
Dose Dependent ◽  
Anti Gp63

In previous studies, we showed thatHerpetomonas samuelpessoaiproduced a large amount of a surface-located metallopeptidase that presented similar biochemical properties to that of gp63 fromLeishmaniaspp., which is a well-known virulence factor expressed by these digenetic parasites. The present study aims to identify the proteolytic activity released by livingH. samuelpessoaicells. In this context, the parasites were incubated in phosphate buffer up to 4 h, and the supernatants were obtained by centrifugation and filtration steps and were then applied on SDS–PAGE to determine the secretory protein profile and on gelatin-SDS–PAGE to identify the proteolytic activity. The results demonstrated thatH. samuelpessoaisecreted at least 12 polypeptides and an extracellular peptidase of 66 kDa. This enzyme had its activity diminished by 1,10-phenanthroline, EDTA and EGTA. This metallopeptidase was active in a broad spectrum of pH, showing maximum activity at pH 6·0 at 37 °C. Casein was also cleaved by this secretory proteolytic enzyme, while bovine serum albumin and haemoglobin were not degraded under these conditions. Fluorescence microscopy and flow cytometry using anti-gp63 antibody against leishmanolysin ofL. amazonensisdemonstrated the presence of similar molecules on the cell-surface ofH. samuelpessoai. Moreover, immunoblot analysis showed the presence of a reactive polypeptide in the cellular extract and in the supernatant fluid ofH. samuelpessoai, which suggests immunological similarities between these two distinct trypanosomatids. The zinc-metallopeptidase inhibitor 1,10-phenanthroline was able to inhibit the secretion of the 66 kDa metallopeptidase in a dose-dependent manner, while the phospholipase C inhibitor (p-CMPS) did not alter the secretion pattern. Additionally, anti-cross-reacting determinant (CRD) antibody failed to recognize any secreted polypeptide fromH. samuelpessoai. Collectively, these results suggest that the gp63-like molecule was released from theH. samuelpessoaisurface by proteolysis instead of phospholipolysis, in a similar mechanism to that observed inLeishmania.

Construction of a New Genetic Engineering Bacterium for Preparation of Superoxide Dismutase with High Productivity

2011 ◽  
Vol 314-316 ◽  
pp. 1973-1976
Author(s):  
Yi Zhao ◽  
Yang Li ◽  
Ren Qiang Li
Keyword(s):  
Genetic Engineering ◽  
Maximum Activity ◽  
Bacterial Protein ◽  
Expression Plasmid ◽  
Gene Encoding ◽  
Good Thermal Stability ◽  
High Productivity ◽  
E Coli ◽  
Bacillus Subtilis Atcc ◽  
Bacillus Spp

A new Mn-SOD gene encoding 202 amino acids was cloned from genomic DNA of Bacillus subtilis ATCC 9372 for construction of a genetic engineering bacterium to produce SOD. Its phylogenetic relationships with other Bacillus spp. revealed that this predicted protein is most closely related to B. atrophaeus NRS-213 (AY197616) and B. subtilis 168. This gene was inserted into expression plasmid pET28a and first successfully expressed in E. coli BL21. The SOD was expressed accounted for approximately 45.6% of total bacterial protein. The activity of the SOD was 2553.211 U/mg, the enzyme showed maximum activity at about pH 8.0 and relatively stable from pH 6.0 to 11.0. This SOD had a good thermal stability with >75% retaining of the relative enzymatic activity after incubation at 50 °C for 90 min. This study demonstrated that a new genetic engineering bacterium to produce SOD with high productivity has been successfully constructed.

scholarly journals Isolation of a Treponema pallidum gene encoding immunodominant outer envelope protein P6, which reacts with sera from patients at different stages of syphilis.

1986 ◽  
Vol 164 (4) ◽  
pp. 1160-1170 ◽  
Author(s):  
K M Peterson ◽  
J B Baseman ◽  
J F Alderete
Keyword(s):  
Envelope Protein ◽  
Treponema Pallidum ◽  
Surface Protein ◽  
Plasmid Vector ◽  
Transformed Cells ◽  
Outer Envelope ◽  
Gene Encoding ◽  
Recombinant Phage ◽  
E Coli ◽  
Outer Membranes

A phage directing the synthesis of an abundant 45-kD Treponema pallidum surface protein was isolated from an EMBL-4 bacteriophage lambda library of T. pallidum DNA. The recombinant phage was identified using an mAb that was directed toward an immunodominant, outer envelope T. pallidum protein designated P6. The recombinant P6 protein possessed the same mol mass as the native treponemal antigen detected from total T. pallidum protein preparations, confirming the cloning of the structural gene for this molecule. Furthermore, E. coli was transformed by a 4.5-kb Eco RI lambda insert fragment subcloned into the plasmid vector pUC19. These transformed cells expressed and translocated the 45-kD protein to their outer membranes. Finally, all sera from patients with different stages of syphilis (primary, secondary, and latent) contained antibody reactive to this protein.

scholarly journals Requirement of Purine and Pyrimidine Synthesis for Colonization of the Mouse Intestine by Escherichia coli

2010 ◽  
Vol 76 (15) ◽  
pp. 5181-5187 ◽  
Author(s):  
Jacqueline Vogel-Scheel ◽  
Carl Alpert ◽  
Wolfram Engst ◽  
Gunnar Loh ◽  
Michael Blaut
Keyword(s):  
Escherichia Coli ◽  
Differentially Expressed ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Pyrimidine Synthesis ◽  
Key Enzyme ◽  
Mouse Intestine ◽  
Gnotobiotic Mice

ABSTRACT To study the adaptation of an intestinal bacterium to its natural environment, germfree mice were associated with commensal Escherichia coli MG1655. Two-dimensional gel electrophoresis was used to identify proteins differentially expressed in E. coli MG1655 collected from either cecal contents or anaerobic in vitro cultures. Fourteen differentially expressed proteins (>3-fold; P < 0.05) were identified, nine of which were upregulated in cecal versus in vitro-grown E. coli. Four of these proteins were investigated further for their role in gut colonization. After deletion of the corresponding genes, the resulting E. coli mutants were tested for their ability to colonize the intestines of gnotobiotic mice in competition with the wild-type strain. A mutant devoid of ydjG, which encodes a putative NADH-dependent methylglyoxal reductase, reached a 1.2-log-lower cecal concentration than the wild type. Deletion of the nanA gene encoding N-acetylneuraminate lyase affected the colonization and persistence of E. coli in the intestines of the gnotobiotic mice only slightly. A mutant devoid of 5′-phosphoribosyl 4-(N-succinocarboxamide)-5-aminoimidazole synthase, a key enzyme of purine synthesis, displayed intestinal cell counts >4 logs lower than those of the wild type. Deletion of the gene encoding aspartate carbamoyltransferase, a key enzyme of pyrimidine synthesis, even resulted in the washout of the corresponding mutant from the mouse intestinal tract. These findings indicate that E. coli needs to synthesize purines and pyrimidines to successfully colonize the mouse intestine.

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