A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum.

1. On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction. The rate became approximately constant after declining to 4-5% of the initial value. Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate. The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH. 2. Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution. At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min. 3. Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy. 4. 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective. 5. The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation. 6. Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8. 7. The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates. 8. The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues.     

Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli.

An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK.     

Detection of Escherichia coli in sewage and sludge by polymerase chain reaction.

A method in which the polymerase chain reaction (PCR) was used was developed to amplify either a uidA gene fragment or a 16S rRNA gene fragment from Escherichia coli in sewage and sludge. Because of interference caused by humic acidlike substances, crude DNA extracts were purified with a Sephadex G-200 spun column before the PCR was begun. A Southern analysis in which a nonradioactive chemiluminescent method was used was performed to confirm the presence of PCR products. The sensitivity of detection for PCR products when the chemiluminescent method was used was determined to be 30 ag of E. coli genomic DNA template. In seeded sludge, the PCR amplified the target DNA from 80 E. coli cells per g of sludge and 50 Shigella dysenteriae cells per g of sludge. Because only 0.05 aliquot of a sludge extract was used for the PCR, we deduced that the PCR detected target DNA equivalent to the DNA of 2.5 to 4 cells in the extract. The PCR amplified the uidA fragment from diluted sewage influents and effluents containing E. coli cells. Therefore, the PCR performed with a chemiluminescent gene probe can be used to detect the presence of potentially pathogenic microorganisms in sewage and sludge. This technique can be expanded to permit direct detection of pathogenic microorganisms in water samples, thus leading to enhanced public health protection.     

Cloning and expression in Escherichia coli of the glutamate dehydrogenase gene,gdh, from Corynebacterium melassecola

A hybrid plasmid containing a fragment of the Corynebacterium melassecola chromosome cloned into pBR325 restored growth of glutamate auxotrophs of Escherichia coli strains that have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 3.1-kilobase pair region was shown by complementation analysis and enzyme measurements to carry the glutamate dehydrogenase gene, gdh. Glutamate dehydrogenase encoded by gdh carried on recombinant plasmids was elevated over 100-fold in E. coli cells. The gdh promoter was located by in vitro fusion to a promoter-deficient galK gene.     

Complete nucleotide sequence of the glutamate dehydrogenase gene from Escherichia coli K-12

A 2.3-kb PstI-ClaI chromosomal DNA segment, carrying the complete coding region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, has been sequenced. The complete amino acid sequence (447 residues) of the GDH monomer has been deduced, and comparisons are made with reported amino acid sequences of GDH from other organisms.     

The nucleotide sequence of the pepN gene and its over-expression in Escherichia coli

The complete nucleotide sequence has been determined for the pepN gene of Escherichia coli K-12. The product of this gene, peptidase N, is apparently 870 amino acids in length. The coding sequence is followed by a tandem pair of stop codons and then a sequence capable of forming a stem-and-loop structure in the pepN mRNA. In the process of subcloning the pepN gene we constructed a plasmid which causes peptidase N to be produced at a level of 50% of total protein. The peptidase is fully active and completely soluble and these overproducing cells appear otherwise normal.     

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase

Abstract Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated M _r of 143 808. The deduced amino acid sequence of the E. colli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.     

Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli.

A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids, pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grow anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pP58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent Mr of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH.     

Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization

The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap II phage library of C. thermocellum genomic DNA. In one positive clone, an open reading frame of 948 base pairs corresponded to C. thermocellum ldh gene encoding for the predicted 315-residue protein. The ldh gene was successfully expressed in E. coli FMJ39 (ldh mutant) under the lac promoter. The recombinant enzyme was partially purified from E. coli cell extracts and its kinetic properties were determined. Clostridium thermocellum LDH was shown to catalyze a highly reversible reaction and to be an allosteric enzyme that is activated by fructose-1,6-diphosphate (FDP). For pyruvate, partially purified LDH had Km and Vmax values of 7.3 mmol/L and 87 micromol/min, respectively, and in the presence of FDP, a 24-fold decrease in Km and a 5.7-fold increase in Vmax were recorded. The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas Km and Vmax values were 59.5 mmol/L and 52 micromol/min, respectively, in its presence. The enzyme did not lose activity when incubated at 65 degrees C for 5 min.     

Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli

A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids, pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grow anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pP58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent Mr of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH.     

Enhancing long-term thermal stability in mesophilic glutamate dehydrogenase from Clostridium symbiosum by eliminating cysteine residues

Glutamate dehydrogenase from Clostridium symbiosum has two cysteine residues, C144 and C320. The single mutant C320S and a double mutant with both cysteines replaced by serine have been compared with one another in terms of long-term stability and other properties. Specific activities and kinetic parameters were relatively little affected, but stability was improved—e.g. at 25 °C sterile, sealed samples of wild-type enzyme, C320S and the double mutant at 0.1 mg/ml in 0.1 M phosphate buffer, pH 7 lost 50%, 42% and 32% of activity over 60 days. For the first two proteins this loss was partly reversible with dithiothreitol. When wild-type enzyme was deliberately contaminated with 1 μM Cu²⁺ it became less stable and formed aggregates, whereas the double mutant was not affected. The double mutation thus removes a source of instability through –SH oxidation that would be accentuated by any heavy metal contamination of solutions.     

Detection of urovirulence factors in Escherichia coli by multiplex polymerase chain reaction

Abstract Primers to amplify the genes encoding the virulence factors of uropathogenic Escherichia coli , such as pilus associated with pyelonephritis ( pap ), haemolysin ( hly ), aerobactin ( aer ) and cytotoxic necrotizing factor 1 ( cnf 1) genes, were designed. The above primers along with previously reported primers for S fimbriae ( sfa ) and afimbrial adhesin I ( afaI ) genes were combined to develop a multiplex polymerase chain reaction (PCR) for detection of the respective virulence factors and for the identification of uropathogenic E. coli . The multiplex PCR to detect pap, sfa, afa I, hly, aer and cnf 1 genes was highly specific and the sensitivity was found to be about 5 × 10³ colony forming units of E. coli per ml. A total of 194 E. coli strains isolated from patients with simple acute cystitis were examined by the multiplex PCR and the results were in complete agreement with that obtained by DNA colony hybridization test. The multiplex PCR developed was, therefore, concluded to be a useful, sensitive and rapid assay system to identify uropathogenic E. coli .     

Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.     

Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli

A 6-kb fragment of DNA, which complemented defects in the alcohol dehydrogenase (ADH)-encoding gene (adhE) of Escherichia coli, was cloned into a multicopy vector. Both ADH and coenzyme-A-linked acetaldehyde dehydrogenase (ACDH) activities were encoded by the plasmid, pHIL8. The adhE gene was identified as an open reading frame of 891 codons encoding an Mr 96,008 protein (minus the initiating methionine). Codon usage analysis indicates that adhE should be highly expressed. This gene shows no significant homology to any previously sequenced ADH-encoding gene.     

Microbial source tracking by DNA sequence analysis of the Escherichia coli malate dehydrogenase gene

Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse>dog>deer>human and gull. For in-library isolates, blind analysis of fecal isolates (n=12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.     

Detection of Escherichia coli serogroup O103 by real-time polymerase chain reaction

AIMS: The aims of the study were to identify the specific genes of O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O103 and to provide the basis for a specific real-time PCR test for rapid detection of E. coli O103. METHODS AND RESULTS: The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E. coli and related species, were used to amplify the 12-kbp O103 O-antigen biosynthesis locus of STEC O103. A DNA library representative of this cluster allowed two O103-specific probes to be identified in the flippase (wzx) and UDP-galactose-4-epimerase (galE) genes. Two specific O103 serotyping real-time PCR tests based on these two genes were successfully developed. CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen real-time PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of real-time PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.