Survey, purification, and properties of sugar phosphate phosphohydrolase among microorganisms

1983 ◽  
Vol 61 (12) ◽  
pp. 1292-1303 ◽  
Author(s):  
Francis Y. M. Choy ◽  
Ya-Pin Lee
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Physiological Role ◽  
Lactobacillus Bulgaricus ◽  
Sugar Phosphate ◽  
Phosphotransferase System ◽  
Enzyme Preparations ◽  
E Coli ◽  
Apparent Homogeneity

Sugar phosphate phosphohydrolase was purified approximately 500- to 600-fold to apparent homogeneity from Escherichia coli B, Escherichia coli C, Escherichia coli var. communior, Escherichia acidilactici, Enterobacter aerogenes, Neisseria meningitidis, and Saccharomyces cereviseae. The molecular weights of the enzyme as estimated by gel filtration ranged from 97 × 103 to 101 × 103. The enzyme was composed of two subunits with the same molecular weight which ranged from 50 × 103 to 52 × 103, as determined by sodium dodecyl sulfate gel electrophoresis. Homogeneous enzyme preparations hydrolyse all the tested α-D-aldohexose 1-phosphate, D-(keto or aldo)hexose 6-phosphate, and pentose phosphate substrates significantly. When the microorganisms were transferred from growth medium with 1% glucose to that without glucose, there were dramatic increases in both the specific and total enzyme activities. At least three isozymes appeared to be present in S. cereviseae, and two appeared to be present in E. coli B, E. coli var. communior, and N. meningitidis. Rabbit antiserum immunized against sugar phosphate phosphohydrolase purified from E. coli B cross-reacted with both the crude extracts and purified preparations of the enzyme from the other microorganisms. The presence of neither sugar phosphate phosphohydrolase activity nor immunocross-reacting material was detected in the following microorganisms: Aspergillus niger, Azotobacter chroococcum, Bacillus subtilis, Bacillus pumilis, Citrobacter freundii, Clostridium butyricum, Corynebacterium xerosis, Flavobacterium aquatile, Flavobacterium synxanthum, Lactobacillus bulgaricus, Micrococcus coralinus, Neisseria perflava, Neurospora crassa, Penicilium expansum, Penicilium notatum, Proteus mirabilis, Proteus vulgaris, Pseudomonas fluorescens, Saccharomyces fermenti, Sarcina lutea, and Streptomyces antibioticus. At present, no conclusive relationship can be established between the phosphoenolpyruvate phosphotransferase system and the enzyme sugar phosphate phosphohydrolase among microorganisms. The physiological role of sugar phosphate phosphohydrolase as a transferase and regulatory enzyme is discussed.

scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

scholarly journals Identification of a Phosphotransferase System of Escherichia coli Required for Growth on N-Acetylmuramic Acid

2004 ◽  
Vol 186 (8) ◽  
pp. 2385-2392 ◽  
Author(s):  
Ulrike Dahl ◽  
Tina Jaeger ◽  
Bao Trâm Nguyen ◽  
Julia M. Sattler ◽  
Christoph Mayer
Keyword(s):  
Escherichia Coli ◽  
Energy Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Glucose Transport System ◽  
Single Polypeptide

ABSTRACT We report here that wild-type Escherichia coli grows on N-acetylmuramic acid (MurNAc) as the sole source of carbon and energy. Analysis of mutants defective in N-acetylglucosamine (GlcNAc) catabolism revealed that the catabolic pathway for MurNAc merges into the GlcNAc pathway on the level of GlcNAc 6-phosphate. Furthermore, analysis of mutants defective in components of the phosphotransferase system (PTS) revealed that a PTS is essential for growth on MurNAc. However, neither the glucose-, mannose/glucosamine-, nor GlcNAc-specific PTS (PtsG, ManXYZ, and NagE, respectively) was found to be necessary. Instead, we identified a gene at 55 min on the E. coli chromosome that is responsible for MurNAc uptake and growth. It encodes a single polypeptide consisting of the EIIB and C domains of a so-far-uncharacterized PTS that was named murP. MurP lacks an EIIA domain and was found to require the activity of the crr-encoded enzyme IIA-glucose (EIIAGlc), a component of the major glucose transport system for growth on MurNAc. murP deletion mutants were unable to grow on MurNAc as the sole source of carbon; however, growth was rescued by providing murP in trans expressed from an isopropylthiogalactopyranoside-inducible plasmid. A functional His6 fusion of MurP was constructed, isolated from membranes, and identified as a polypeptide with an apparent molecular mass of 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Close homologs of MurP were identified in the genome of several bacteria, and we believe that these organisms might also be able to utilize MurNAc.

scholarly journals Characterization of Soluble Enzyme II Complexes of the Escherichia coli Phosphotransferase System

2004 ◽  
Vol 186 (24) ◽  
pp. 8453-8462 ◽  
Author(s):  
Mohammad Aboulwafa ◽  
Milton H. Saier
Keyword(s):  
Escherichia Coli ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Kinetic Properties ◽  
Phosphotransferase System ◽  
E Coli ◽  
Before And After ◽  
Oligomeric Form ◽  
Triton X

ABSTRACT Plasmid-encoded His-tagged glucose permease of Escherichia coli, the enzyme IIBCGlc (IIGlc), exists in two physical forms, a membrane-integrated oligomeric form and a soluble monomeric form, which separate from each other on a gel filtration column (peaks 1 and 2, respectively). Western blot analyses using anti-His tag monoclonal antibodies revealed that although IIGlc from the two fractions migrated similarly in sodium dodecyl sulfate gels, the two fractions migrated differently on native gels both before and after Triton X-100 treatment. Peak 1 IIGlc migrated much more slowly than peak 2 IIGlc. Both preparations exhibited both phosphoenolpyruvate-dependent sugar phosphorylation activity and sugar phosphate-dependent sugar transphosphorylation activity. The kinetics of the transphosphorylation reaction catalyzed by the two IIGlc fractions were different: peak 1 activity was subject to substrate inhibition, while peak 2 activity was not. Moreover, the pH optima for the phosphoenolpyruvate-dependent activities differed for the two fractions. The results provide direct evidence that the two forms of IIGlc differ with respect to their physical states and their catalytic activities. These general conclusions appear to be applicable to the His-tagged mannose permease of E. coli. Thus, both phosphoenolpyruvate-dependent phosphotransferase system enzymes exist in soluble and membrane-integrated forms that exhibit dissimilar physical and kinetic properties.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

scholarly journals Antimicrobial Interventions to Reduce Shiga Toxin-Producing Escherichia coli (STEC) Surrogate Populations on Beef Striploins Intended for Blade Tenderization

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
C. L. Thomas ◽  
H. Thippareddi ◽  
M. Rigdon ◽  
S. Kumar ◽  
R. W. McKee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Antimicrobial Treatment ◽  
Sodium Chlorite ◽  
Beef Industry ◽  
Anterior Portion ◽  
E Coli ◽  
Group Data ◽  
Blade Tenderization ◽  
Antimicrobial Interventions

ObjectivesBlade tenderization (BT) is used in the beef industry to improve tenderness of steaks prepared from subprimals but can translocate surface pathogens to the interior of meat. Application of antimicrobial solutions on the surface of subprimals prior to blade tenderization can reduce the risk of translocation of surface microorganisms. The objectives of this research were: 1) evaluate the efficacy of antimicrobial interventions applied to inoculated (surrogate Escherichia coli) beef striploins prior to blade tenderization; and 2) examine the transfer of E. coli from inoculated striploins to subsequent non-inoculated subprimals.Materials and MethodsThe anterior portion of whole muscle beef striploins (30.48 cm) were inoculated (lean side) across a 10 cm band with an approximately 8.00 log CFU/mL cocktail containing non-pathogenic, rifampicin-resistant surrogate STEC strains (BAA-1427, BAA-1428, BAA-1429, BAA-1430, and BAA-1431). The inoculated striploins were sprayed with (1) levulinic acid (5.0%) + sodium dodecyl sulfate (0.50%) (LVA+SDS), (2) peroxyacetic acid (2000 ppm; PAA; FCN 1666), (3) acidified sodium chlorite (1200 ppm; ASC), or (4) lactic acid (4.5%; LA) by passing through a spray cabinet and blade tenderized, along with an inoculated, non-sprayed control (CON). To evaluate the potential for cross-contamination of subsequent subprimals, an inoculated striploin (for each treatment) was blade tenderized followed by a non-inoculated beef striploin. For each striploin, surface and subsurface samples (2.54 cm wide) were collected from three different locations including the anterior, middle, and posterior end of each striploin. A total of 30 striploins across three replications were randomly assigned to treatment stratification. Sponge samples were also collected from the blade tenderizer (plate of the blade unit and blades) after each treatment group. Data were analyzed using Proc Mixed (SAS Inst., v.9.4; Cary, NC) as a completely randomized split-plot design. Microbial counts for all samples were log transformed and then analyzed for the main effects of antimicrobial treatment, location (anterior to posterior and surface or interior), and their interaction. Differences were considered significant at α ≤ 0.05.ResultsPAA was more effective in reducing E. coli populations (1.80 log CFU/g; P ≤ 0.05) and had lowest recovery of the microorganism from the striploin subsurface compared to other treatments, followed by LVA+SDS (1.00 log CFU/g). E. coli populations gradually decreased (P ≤ 0.05) on the surface and subsurface as sampling moved anterior to posterior. However, E. coli populations were similar (P > 0.05) on the posterior end of inoculated striploins and the anterior end of the subsequent, non-inoculated striploins, indicating transfer of microorganisms from one striploin to the following striploin. E. coli populations of 3.03 log CFU/cm2 and 2.47 log CFU/cm2 were recovered from the plate of the blade unit and the blades of the blade tenderizer. E. coli populations recovered from the plastic plate (3.46 log CFU/cm2) and blades (2.87 log CFU/cm2) of the blade tenderizer were the similar (P > 0.05) for all treatment groups except for PAA (1.41 log CFU/cm2 and 0.97 log CFU/cm2, respectively).ConclusionThese results showed that PAA and LVA+SDS can be used to improve the safety of blade tenderized beef.

scholarly journals The Effect Ultrasound and Surfactants on Nanobubbles Efficacy against Listeria innocua and Escherichia coli O157:H7, in Cell Suspension and on Fresh Produce Surfaces

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2154
Author(s):  
Shamil Rafeeq ◽  
Reza Ovissipour
Keyword(s):  
Escherichia Coli ◽  
Cell Suspension ◽  
Sodium Dodecyl ◽  
Listeria Innocua ◽  
Wash Water ◽  
Tween 20 ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Bacterial Removal ◽  
The Impact

Removing foodborne pathogens from food surfaces and inactivating them in wash water are critical steps for reducing the number of foodborne illnesses. In this study we evaluated the impact of surfactants on enhancing nanobubbles’ efficacy on Escherichia coli O157:H7, and Listeria innocua removal from spinach leaves. We evaluated the synergistic impact of nanobubbles and ultrasound on these two pathogens inactivation in the cell suspension. The results indicated that nanobubbles or ultrasound alone could not significantly reduce bacteria in cell suspension after 15 min. However, a combination of nanobubbles and ultrasonication caused more than 6 log cfu/mL reduction after 15 min, and 7 log cfu/mL reduction after 10 min of L. innocua and E. coli, respectively. Nanobubbles also enhanced bacterial removal from spinach surface in combination with ultrasonication. Nanobubbles with ultrasound removed more than 2 and 4 log cfu/cm2 of L. innocua and E. coli, respectively, while ultrasound alone caused 0.5 and 1 log cfu/cm2 of L. innocua and E. coli reduction, respectively. No reduction was observed in the solutions with PBS and nanobubbles. Adding food-grade surfactants (0.1% Sodium dodecyl sulfate-SDS, and 0.1% Tween 20), did not significantly enhance nanobubbles efficacy on bacterial removal from spinach surface.

scholarly journals Altered Utilization of N-Acetyl-d-Galactosamine by Escherichia coli O157:H7 from the 2006 Spinach Outbreak

2007 ◽  
Vol 190 (5) ◽  
pp. 1710-1717 ◽  
Author(s):  
Amit Mukherjee ◽  
Mark K. Mammel ◽  
J. Eugene LeClerc ◽  
Thomas A. Cebula
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Culture Collection ◽  
Base Substitution ◽  
Phosphotransferase System ◽  
Single Nucleotide ◽  
E Coli ◽  
Single Nucleotide Change ◽  
Phenotype Comparison ◽  
In Silico Analyses

ABSTRACT In silico analyses of previously sequenced strains of Escherichia coli O157:H7, EDL933 and Sakai, localized the gene cluster for the utilization of N-acetyl-d-galactosamine (Aga) and d-galactosamine (Gam). This gene cluster encodes the Aga phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of Aga. As the complete coding sequences for enzyme IIA (EIIA)Aga/Gam, EIIBAga, EIICAga, and EIIDAga of the Aga PTS are present, E. coli O157:H7 strains normally are able to utilize Aga as a sole carbon source. The Gam PTS complex, in contrast, lacks EIICGam, and consequently, E. coli O157:H7 strains cannot utilize Gam. Phenotypic analyses of 120 independent isolates of E. coli O157:H7 from our culture collection revealed that the overwhelming majority (118/120) displayed the expected Aga+ Gam− phenotype. Yet, when 194 individual isolates, derived from a 2006 spinach-associated E. coli O157:H7 outbreak, were analyzed, all (194/194) displayed an Aga− Gam− phenotype. Comparison of aga/gam sequences from two spinach isolates with those of EDL933 and Sakai revealed a single nucleotide change (G:C→A:T) in the agaF gene in the spinach-associated isolates. The base substitution in agaF, which encodes EIIAAga/Gam of the PTS, changes a conserved glycine residue to serine (Gly91Ser). Pyrosequencing of this region showed that all spinach-associated E. coli O157:H7 isolates harbored this same G:C→A:T substitution. Notably, when agaF + was cloned into an expression vector and transformed into six spinach isolates, all (6/6) were able to grow on Aga, thus demonstrating that the Gly91Ser substitution underlies the Aga− phenotype in these isolates.

scholarly journals Escherichia coli NsrR Regulates a Pathway for the Oxidation of 3-Nitrotyramine to 4-Hydroxy-3-Nitrophenylacetate

2008 ◽  
Vol 190 (18) ◽  
pp. 6170-6177 ◽  
Author(s):  
Linda D. Rankin ◽  
Diane M. Bodenmiller ◽  
Jonathan D. Partridge ◽  
Shirley F. Nishino ◽  
Jim C. Spain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Physiological Role ◽  
Growth Conditions ◽  
Growth Defect ◽  
E Coli ◽  
Mutant Phenotypes ◽  
Culture Supernatants ◽  
Chip Chip

ABSTRACT Chromatin immunoprecipitation and microarray (ChIP-chip) analysis showed that the nitric oxide (NO)-sensitive repressor NsrR from Escherichia coli binds in vivo to the promoters of the tynA and feaB genes. These genes encode the first two enzymes of a pathway that is required for the catabolism of phenylethylamine (PEA) and its hydroxylated derivatives tyramine and dopamine. Deletion of nsrR caused small increases in the activities of the tynA and feaB promoters in cultures grown on PEA. Overexpression of nsrR severely retarded growth on PEA and caused a marked repression of the tynA and feaB promoters. Both the growth defect and the promoter repression were reversed in the presence of a source of NO. These results are consistent with NsrR mediating repression of the tynA and feaB genes by binding (in an NO-sensitive fashion) to the sites identified by ChIP-chip. E. coli was shown to use 3-nitrotyramine as a nitrogen source for growth, conditions which partially induce the tynA and feaB promoters. Mutation of tynA (but not feaB) prevented growth on 3-nitrotyramine. Growth yields, mutant phenotypes, and analyses of culture supernatants suggested that 3-nitrotyramine is oxidized to 4-hydroxy-3-nitrophenylacetate, with growth occurring at the expense of the amino group of 3-nitrotyramine. Accordingly, enzyme assays showed that 3-nitrotyramine and its oxidation product (4-hydroxy-3-nitrophenylacetaldehyde) could be oxidized by the enzymes encoded by tynA and feaB, respectively. The results suggest that an additional physiological role of the PEA catabolic pathway is to metabolize nitroaromatic compounds that may accumulate in cells exposed to NO.

scholarly journals Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1202-1206 ◽  
Author(s):  
MG Bolyard ◽  
ST Lord
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Dependent Manner ◽  
Beta Chain ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
E Coli ◽  
Sds Page

Abstract The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

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