Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.     

Isolation and expression of the Escherichia coli gene encoding malate dehydrogenase.

An oligodeoxynucleotide specific for a pentapeptide sequence corresponding to amino acid residues 32 through 36 of Escherichia coli malate dehydrogenase was chemically synthesized and used to identify the mdh gene on plasmid pLC32-38 from the Clarke-Carbon recombinant library. Cells transformed with this plasmid exhibited a 10-fold increase in malate dehydrogenase activity. A 1.2-kilobase PvuII fragment which hybridized with the oligodeoxynucleotide probe was subcloned, and the identity of the mdh structural gene was confirmed by partial nucleotide sequence analysis. The expression of the mdh gene, as measured by both Northern blotting and enzyme assays, was found to vary over a 20-fold range with different culture conditions.     

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed.     

Expression and function of heterologous forms of malate dehydrogenase in yeast.

The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.     

Nucleotide sequence of the malate dehydrogenase gene of Thermus flavus and its mutation directing an increase in enzyme activity

The nucleotide sequence of the malate dehydrogenase (mdh) gene from a thermophilic bacterium, Thermus flavus, was determined. The amino acid sequence of the Thermus malate dehydrogenase resembled that of the porcine heart cytoplasmic enzyme to a certain extent, and Asp-159 and His-187 were identified as possible essential residues for the catalytic function. The mutated mdh gene was also cloned from a spontaneous mutant of T. flavus containing a higher activity of the enzyme. Its mutation point was determined to be a single nucleotide exchange from C to T which caused Thr-190 to be substituted by isoleucine. The mutated enzyme showed resistance to substrate inhibition, an increase in both kcat and Km, and a shift toward a more acid optimum pH for the enzyme reaction.     

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.     

Malate dehydrogenase from the mesophile Chlorobium vibrioforme and from the mild thermophile Chlorobium tepidum: molecular cloning, construction of a hybrid, and expression in Escherichia coli.

The genes (mdh) encoding malate dehydrogenase (MDH) from the mesophile Chlorobium vibrioforme and the moderate thermophile C. tepidum were cloned and sequenced, and the complete amino acid sequences were deduced. When the region upstream of mdh was analyzed, a sequence with high homology to an operon encoding ribosomal proteins from Escherichia coli was found. Each mdh gene consists of a 930-bp open reading frame and encodes 310 amino acid residues, corresponding to a subunit weight of 33,200 Da for the dimeric enzyme. The amino acid sequence identity of the two MDHs is 86%. Homology searches using the primary structures of the two MDHs revealed significant sequence similarity to lactate dehydrogenases. A hybrid mdh was constructed from the 3' part of mdh from C. tepidum and the 5' part of mdh from C. vibrioforme. The thermostabilities of the hybrid enzyme and of MDH from C. vibrioforme and C. tepidum were compared.     

A partial defect in carbon catabolite repression in mutants of Saccharomyces cerevisiae with reduced hexose phosphyorylation

Summary Mutants of Saccharomyces cerevisiae with reduced glucose phosphorylation were investigated. They were all recessive and belonged to one gene HEX1, mutant designation hex1. Carbon catabolite repression of alpha-glucosidases, invertase and part of the total malate dehydrogenase was reduced. Repression of the glyoxylate cycle enzymes, isocitrate lyase and malate synthetase, as well as that of gluconeogenetic fructose-1, 6-bisphosphatase was normal. A slight effect on repression of succinate: cytochrome c oxidoreductase and respiration was to be detected. The effect on repression by fructose was much less pronounced but still clear. However, there was a paradoxical effect of hexose concentration with higher concentrations repressing less. Maltose was also less repressing in the mutant. Growth on all sugars degraded via the hexose phosphorylation reaction was reduced and more strongly so at higher concentrations. Intracellular concentrations of glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-bisphosphate were largely the same in mutant and wild type. The only striking difference between mutant and wild type was a fourfold higher intracellular glucose concentration in maltose grown mutants cells. The data obtained do not support the contention that carbon catabolite repression of the enzymes studied is triggered by intracellular hexoses or their metabolites alone. They rather suggest that it is some component of the hexose phosphorylating system that contributes to carbon catabolite repression.     

cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression.

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.     

Population genetics of Escherichia coli in a natural population of native Australian rats

Escherichia coli , a normal inhabitant of the intestinal tract of mammals and birds, is a diverse species. Most studies on E. coli populations involve organisms from humans or human-associated animals. In this study, we undertook a survey of E. coli from native Australian mammals, predominantly Rattus tunneyi , living in a relatively pristine environment in the Bundjalung National Park. The genetic diversity was assessed and compared by multilocus enzyme electrophoresis (MLEE), sequence analysis of the mdh (malate dehydrogenase) gene and biotyping using seven sugars. Ninety-nine electrophoretic types were identified from the 242 isolates analysed by MLEE and 15 sequences from the mdh genes sequenced from 21 representative strains. The Bundjalung isolates extend the diversity represented by the E. coli reference (ECOR) set , with new MLEE alleles found in six out of 10 loci. Many of the Bundjalung isolates fell into a discrete group in MLEE. Other Bundjalung strains fell into the recognized E. coli ECOR set groups, but tended to be at the base of both the MLEE and mdh gene trees, implying that these strains are derived independently from ancestral forms of the ECOR groups and that ECOR strains represent only a subset of E. coli adapted to humans and human-associated animals. Linkage disequilibrium analysis showed that the Bundjalung population has an 'epidemic' population structure. The Bundjalung isolates were able to utilize more sugars than the ECOR strains, suggesting that diet plays a prominent role in adaptation of E. coli .     

Mutants of Salmonella typhimurium That Are Insensitive to Catabolite Repression of Proline Degradation

In Salmonella typhimurium the two enzymes of proline catabolism, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid dehydrogenase, are subject to catabolite repression when the cells are grown in the presence of glucose. Mutants partially relieved of catabolite repression (PutR) for the proline catabolic enzymes have been isolated by selection on agar plates containing glucose and proline. The specificity of the catabolite repression-insensitive character for the enzymes of proline utilization has been confirmed by an analysis of other unrelated catabolic enzymes. Histidase and amylomaltase of the mutant strains are equally as sensitive to glucose repression as are the enzymes from the wild type. All four PutR mutants exhibit higher induced and higher basal levels of proline oxidase as compared with the corresponding wild-type levels. The mutations of three strains tested are cotransducible with constitutive, pleiotrophic-negative and structural gene mutations of the put region. Three-factor crosses indicate that two putR mutations are located at one end of the cluster of put mutations.     

Molecular cloning, DNA structure and expression of the Escherichia coli D-xylose isomerase.

The D-xylose isomerase (EC 5.3.1.5) gene from Escherichia coli was cloned and isolated by complementation of an isomerase-deficient E. coli strain. The insert containing the gene was restriction mapped and further subcloning located the gene in a 1.6-kb Bg/II fragment. This fragment was sequenced by the chain termination method, and showed the gene to be 1002 bp in size. The Bg/II fragment was cloned into a yeast expression vector utilising the CYCl yeast promoter. This construct allowed expression in E. coli grown on xylose but not glucose suggesting that the yeast promoter is responding to the E. coli catabolite repression system. No expression was detected in yeast from this construct and this is discussed in terms of the upstream region in the E. coli insert with suggestions of how improved constructs may permit achievement of the goal of a xylose-fermenting yeast.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

Malate dehydrogenase: a useful phylogenetic marker for the genus Aeromonas

The reconstruction of correct genealogies among biological entities, the estimation of the divergence time between organisms or the study of the different events that occur along evolutionary lineages are not always based on suitable genes. For reliable results, it is necessary to look at full-length sequences of genes under stabilizing selection (neutral or purifying) and behaving as good molecular clocks. In bacteria it has been proved that the malate dehydrogenase gene (mdh) can be used to determine the inter- and intraspecies divergence, and hence this gene constitutes a potential marker for phylogeny and bacterial population genetics. We have sequenced the full-length mdh gene in 36 type and reference strains of Aeromonas. The species grouping obtained in the phylogenetic tree derived from mdh sequences was in agreement with that currently accepted for the genus Aeromonas. The maximum likelihood models applied to our sequences indicated that the mdh gene is highly conserved among the Aeromonas species and the main evolutionary force acting on it is purifying selection. Only two sites under potential diversifying selection were identified (T 108 and S 193). In order to determine if these two residues could have an influence on the MDH structure, we mapped them in a three-dimensional model constructed from the sequence of A. hydrophila using the human mitochondrial MDH as a template. The presence of purifying selection together with the linear relationship between substitutions and gene divergence makes the mdh an excellent candidate gene for a phylogeny of Aeromonas and probably for other bacterial groups.     

The use of phenylmethylsulfonyl fluoride in the study of catabolite inactivation and repression in intact cells of Saccharomyces cerevisiae

Catabolite inactivation of fructose-1,6-bisphosphatase, isocitrate lyase, phosphoenolpruvate carboxykinase and malate dehydrogenase in intact cells could be prevented by phenylmethylsulfonyl fluoride added 40 min prior to the addition of glucose. Protein synthesis, fermentative and respiratory activity and catabolite repression were not affected. Elimination of catabolite inactivation by the addition of PMSF revealed that catabolite repression started at different times for different enzyme.Abbreviation PMSF phenylmethylsulfonyl fluoride     

New host-vector system for Thermus spp. based on the malate dehydrogenase gene

A Thermus thermophilus HB27 strain was constructed in which the malate dehydrogenase (mdh) gene was deleted. The Deltamdh colonies are recognized by a small-colony phenotype. Wild-type phenotype is restored by transformation with Thermus plasmids or integration vector containing an intact mdh gene. The wild-type phenotype provides a positive selection tool for the introduction of plasmid DNA into Thermus spp., and because mdh levels can be readily quantified, this host-vector system is a convenient tool for monitoring gene expression.     

Cloning chromosomal lac genes of Klebsiella pneumoniae

The chromosomal gene for beta-galactosidase from Klebsiella pneumoniae strain T17R1 and associated regulatory genes have been cloned as a 5-kb HindIII fragment in the pBR322 plasmid vector. The beta-galactoside permease gene is not present in a functional form in the 5-kb fragment. The K. pneumoniae genes are expressed in an Escherichia coli host. The synthesis of beta-galactosidase is inducible by isopropyl-beta-D-galactosidase (IPTG) and is sensitive to catabolite repression. There appears to be greater homology between the K. pneumoniae and E. coli structural genes for beta-galactosidase than there is between the respective repressor genes.