scholarly journals Characterization of Methylglyoxal Synthase fromClostridium acetobutylicum ATCC 824 and Its Use in the Formation of 1,2-Propanediol

1999 ◽  
Vol 65 (7) ◽  
pp. 3244-3247 ◽  
Author(s):  
Ke-xue Huang ◽  
Frederick B. Rudolph ◽  
George N. Bennett
Keyword(s):  
Escherichia Coli ◽  
Clostridium Acetobutylicum ◽  
Glycerol Dehydrogenase ◽  
Dihydroxyacetone Phosphate ◽  
Gene Encoding ◽  
E Coli ◽  
Native Molecular Mass ◽  
Optimum Ph ◽  
Methylglyoxal Synthase

ABSTRACT A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The Km andV max values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min−1μg−1, respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.

scholarly journals Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

2011 ◽  
Vol 77 (20) ◽  
pp. 7104-7112 ◽  
Author(s):  
Maria Karczmarczyk ◽  
Yvonne Abbott ◽  
Ciara Walsh ◽  
Nola Leonard ◽  
Séamus Fanning
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Gene Array ◽  
Multidrug Resistant ◽  
Genetic Determinants ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

scholarly journals Purification and characterization of the class-II D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes' strain)

1978 ◽  
Vol 169 (3) ◽  
pp. 633-641 ◽  
Author(s):  
S A Baldwin ◽  
R N Perham ◽  
D Stribling
Keyword(s):  
Escherichia Coli ◽  
Common Ancestor ◽  
Class Ii ◽  
Purification And Characterization ◽  
Thiol Compounds ◽  
E Coli ◽  
Optimum Ph ◽  
Class Ii Aldolase ◽  
Fructose 1,6 Bisphosphate

A new form of the class-II D-fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) of Escherichia coli (Crookes' strain) was isolated from an extract of glycerol-grown bacteria. It has a higher molecular weight (approx. 80000)than previous preparations of the enzyme and closely resembles the typical class-II aldolase from yeast in size and amino acid composition. On the other hand, its kinetic behaviour is not typical of a class-II aldolase. The enzyme has no requirement for thiol compounds either for stability or activity, added K+ ions have no effect, and the optimum pH for the cleavage activity is unusually high. The class-II enzymes from the prokaryote E. coli and the eukaryote yeast show no immunological identity. However, the similarity of their structures suggests that they have evolved from a common ancestor.

scholarly journals Identification of the rrmA Gene Encoding the 23S rRNA m1G745 Methyltransferase in Escherichia coli and Characterization of an m1G745-Deficient Mutant

1998 ◽  
Vol 180 (2) ◽  
pp. 359-365 ◽  
Author(s):  
Claes Gustafsson ◽  
Britt C. Persson
Keyword(s):  
Escherichia Coli ◽  
Polypeptide Chain ◽  
Chain Elongation ◽  
23S Rrna ◽  
Drastic Reduction ◽  
Gene Encoding ◽  
E Coli ◽  
Loosely Coupled ◽  
Rich Media

ABSTRACT An Escherichia coli mutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83–100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. TherrmA gene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on the E. colichromosome and identified to be identical to the previously sequenced gene yebH.

scholarly journals Identification and Characterization of Integron-Mediated Antibiotic Resistance among Shiga Toxin-Producing Escherichia coli Isolates

2001 ◽  
Vol 67 (4) ◽  
pp. 1558-1564 ◽  
Author(s):  
Shaohua Zhao ◽  
David G. White ◽  
Beilei Ge ◽  
Sherry Ayers ◽  
Sharon Friedman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Shiga Toxin ◽  
Antibiotic Resistance Gene ◽  
Gene Encoding ◽  
Gene Cassettes ◽  
E Coli ◽  
Class 1 Integrons ◽  
Class 1

ABSTRACT A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one E. coli O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similaraadA gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the E. coli O111:H8 isolate contained three genes, dfrXII,aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found inCitrobacter freundii and Klebsiella pneumoniae. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E. coli O157:H7 and to several strains of Hafnia alvei. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E. coliO157:H7.

scholarly journals DETEKSI GEN PENYANDI ADHESIN PADA VEROCYTOTOXIGENIC Escherichia coli (VTEC) ISOLAT SAPI (Detection of Gene Encoding Adhesin of Verocytotoxigenic Escherichia coli (VTEC) Isolated from Cattles)

2016 ◽  
Vol 10 (2) ◽  
pp. 186-191
Author(s):  
Wahyu Prihtiyantoro ◽  
Hartatik Hartatik ◽  
Mitra Slipranata ◽  
Novra A. Sandi
Keyword(s):  
Escherichia Coli ◽  
Gene Encoding ◽  
E Coli ◽  
Cattle Faeces ◽  
Vtec Strain

This study was aimed to perform phenotypic and genothypic characterization of Escherichia coli (E. coli), particularly VTEC strain isolated from cattle faeces. In this study, 25 E.coli isolated from faeces specimens and faeces base fertilizer of dairy and beef cattles were used. Examination were carried out using phenotypic and genothypic characterization which is specified for E coli VTEC strain. The result showed that 20 % samples of fresh faeces specimens were detected as VTEC strains and none of isolate was detected from faeces base fertilizer samples. From VTEC strains, could detect 16 % VT1 gene, 12 % VT2 genes and 8% of both. Detection on gene pyelonephritis-associated pilli (pap), S fimbrial adhesion (sfa), and afimbrial adhesion (afa) were found about 60%, 80% and 80%, respectively.

scholarly journals Opening a Novel Biosynthetic Pathway to Dihydroxyacetone and Glycerol in Escherichia coli Mutants through Expression of a Gene Variant (fsaAA129S) for Fructose 6-Phosphate Aldolase

2020 ◽  
Vol 21 (24) ◽  
pp. 9625
Author(s):  
Emma Guitart Font ◽  
Georg A. Sprenger
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Catalytic Efficiency ◽  
Glycerol Dehydrogenase ◽  
Gene Encoding ◽  
E Coli ◽  
Glycerol Formation ◽  
Type Gene ◽  
K 12 ◽  
Glucose 6 Phosphate Dehydrogenase

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h−1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h−1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.

Metabolic Engineering of a 1,2-Propanediol Pathway in Escherichia coli

1999 ◽  
Vol 65 (3) ◽  
pp. 1180-1185 ◽  
Author(s):  
Nedim E. Altaras ◽  
Douglas C. Cameron
Keyword(s):  
Escherichia Coli ◽  
Renewable Resources ◽  
Glycerol Dehydrogenase ◽  
Nonrenewable Resource ◽  
Bioprocess Engineering ◽  
Enantiomerically Pure ◽  
E Coli ◽  
Anaerobic Production ◽  
Methylglyoxal Synthase

ABSTRACT 1,2-Propanediol (1,2-PD) is a major commodity chemical that is currently derived from propylene, a nonrenewable resource. A goal of our research is to develop fermentation routes to 1,2-PD from renewable resources. Here we report the production of enantiomerically pure R-1,2-PD from glucose inEscherichia coli expressing NADH-linked glycerol dehydrogenase genes (E. coli gldA or Klebsiella pneumoniae dhaD). We also show that E. colioverexpressing the E. coli methylglyoxal synthase gene (mgs) produced 1,2-PD. The expression of either glycerol dehydrogenase or methylglyoxal synthase resulted in the anaerobic production of approximately 0.25 g of 1,2-PD per liter. R-1,2-PD production was further improved to 0.7 g of 1,2-PD per liter when methylglyoxal synthase and glycerol dehydrogenase (gldA) were coexpressed. In vitro studies indicated that the route to R-1,2-PD involved the reduction of methylglyoxal to R-lactaldehyde by the recombinant glycerol dehydrogenase and the reduction ofR-lactaldehyde to R-1,2-PD by a nativeE. coli activity. We expect that R-1,2-PD production can be significantly improved through further metabolic and bioprocess engineering.

scholarly journals Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli

1991 ◽  
Vol 276 (2) ◽  
pp. 541-546 ◽  
Author(s):  
K Aisaka ◽  
A Igarashi ◽  
K Yamaguchi ◽  
T Uwajima
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Genetically Engineered ◽  
E Coli ◽  
Sds Page ◽  
Optimum Ph ◽  
Molecular Masses ◽  
Related Compounds ◽  
Optimum Ph And Temperature

N-Acetylneuraminate lyase produced by Escherichia coli was purified and crystallized from a genetically engineered strain (E. coli SF8/pNAL1). The enzyme showed apparent molecular masses of 105,000 Da on gel filtration and 35,000 Da on SDS/PAGE, suggesting that the enzyme is a trimer. The apparent optimum pH and temperature were found to be 6.5-7.0 and 80 degrees C respectively. The Km values for N-acetylneuraminate and N-glycollylneuraminate were 3.3 and 3.3 mM respectively. The enzyme was inhibited by reduction with NaBH4 in the presence of the substrate, indicating that the enzyme belongs to the Schiff-base-forming Class I aldolases. The enzyme was strongly inhibited by Cu2+ ions, p-chloromercuribenzoate and N-bromosuccinimide, and also inhibited competitively by the reaction product, pyruvate, and its structurally related compounds, dihydroxyacetone and DL-glyceraldehyde.

scholarly journals The dhnA gene of Escherichia coli encodes a Class I fructose bisphosphate aldolase

1998 ◽  
Vol 331 (2) ◽  
pp. 437-445 ◽  
Author(s):  
Graeme J. THOMSON ◽  
Geoffrey J. HOWLETT ◽  
Alison E. ASHCROFT ◽  
Alan BERRY
Keyword(s):  
Escherichia Coli ◽  
Lysine Residue ◽  
Site Directed Mutagenesis ◽  
Class I ◽  
Fructose Bisphosphate ◽  
Gene Encoding ◽  
E Coli ◽  
Native Molecular Mass ◽  
Fructose Bisphosphate Aldolase ◽  
Fructose 1,6 Bisphosphate

The gene encoding the Escherichia coli Class I fructose-1,6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBank™ database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBank™]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8–10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure–function studies.

Sign in / Sign up

Export Citation Format

Share Document