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D H Calhoun

Publications and source records attributed to D H Calhoun.

At least 37 records · Page 2Linked to original sources
PubMed1983

Role of cryptic genes in microbial evolution.

Cryptic genes are phenotypically silent DNA sequences, not normally expressed during the life cycle of an individual. They may, however, be activated in a few individuals of a large population by mutation, recombination, insertion elements, or other genetic mechanisms. A consideration of the microbial literature concerning biochemical evolution, physiology, and taxonomy provides the basis for a hypothesis of microbial adaptation and evolution by mutational activation of cryptic genes. Evidence is presented, and a mathematical model is derived, indicating that powerful and biologically important mechanisms exist to prevent the loss of cryptic genes. We propose that cryptic genes persist as a vital element of the genetic repertoire, ready for recall by mutational activation in future generations. Cryptic genes provide a versatile endogenous genetic reservoir that enhances the adaptive potential of a species by a mechanism that is independent of genetic exchange.

Biological Evolution↗
PubMed1982

DNA sequence fine-structure analysis of ilvG (IlvG+) mutations of Escherichia coli K-12.

Six ilvG (IlvG+) mutations of Escherichia coli K-12 were transferred to recombinant plasmids, and the DNA sequence of each mutation was determined. This analysis confirmed that expression of the ilvG gene product (acetohydroxy acid synthase II) requires the deletion of a single base pair or the addition of two base pairs within ilvG to displace a frameshift site present in wild-type E. coli K-12. This system should be useful in the analysis of potential frameshift mutagens.

Acetolactate Synthase↗
PubMed1982

Physical and genetic localization of ilv regulatory sites in lambda ilv bacteriophages.

A set of nine lambda dilv phages were used to transduce bacterial recipients containing point mutations or deletions in the ilv genes located at 84 min on the Escherichia coli K-12 chromosome. This genetic analysis indicated that two phages carry the entire ilvGEDAC cluster; others carry the complete ilvC gene and, in addition, bacterial DNA that extends to a termination point between ilvA and ilvC, within ilvD, within ilvE, or within ilvG. DNA extracted from the lambda dilv phages was digested with EcoRI, HindIII, KpnI, PstI, SalI, and SmaI. The restriction maps revealed that these phages were generated after insertion at four distinct insertion sites downstream (clockwise) of ilvC. The physical relationships between the various phages were further examined by electron microscopic heteroduplex analysis. The physical maps of the phages thus generated were straightforward and in complete accord with the genetic data. No evidence for genetic rearrangements of ilv DNA in the phage was obtained, thus validating conclusions based on the use of these phages in previous and ongoing research projects. Bacterial cells with deletions of the ilv genes were made lysogenic with lambda dilv phage to examine the regulation of ilv genes present in the phage. The results confirm previous studies showing that one site for control by repression and derepression is upstream (counterclockwise) of ilvG. It was shown, in addition, that the activities of dihydroxy acid dehydrase and threonine deaminase were increased when the prototrophic lysogens were grown with 20 mM leucine. Since this increase was exhibited even when the ilvG-linked control region was not carried by the lambda dilv phage, additional control sites must be located within the ilvEDA region of the ilvGEDA transcription unit.

Amino Acids, Branched-Chain↗
PubMed1982

Absence of significant membrane localization of the proteins coded by the ilvGEDAC genes of Escherichia coli K-12.

We previously characterized a set of lambda dilv phages by genetic, restriction enzyme, and heteroduplex analyses and tentatively correlated isoleucine-valine gene products with specific ilv DNA segments by using cloned ilv segments in maxicells and lambda dilv phage infection of UV-irradiated cells. In this work, the identity of the ilvC gene product, alpha-acetohydroxy acid isomeroreductase, was confirmed by demonstrating its induction by the physiological inducers alpha-acetolactate and alpha-acetohydroxybutyrate. The identity of the ilvE gene product, transaminase, B, was confirmed by antibody precipitation of the purified enzyme. Phage derivatives with ilv regulatory mutations were found to have the predicted effect upon the ilvGEDA and ilvC protein products. The distribution of the ilvGEDA and ilvC gene products in the soluble, periplasmic, inner membrane, and outer membrane fractions was examined, and no significant membrane association was observed. The expression of the ilv genes in the lambda dilv phage from ilv and phage lambda promoters was compared in order to determine the fractional contribution of each to ilv gene expression. An additional protein of 54,000 daltons that was not detected in the previous analysis was observed to be coded by a bacterial gene but was produced only by readthrough from phage promoters.

Bacterial Proteins↗
PubMed1982

Identification of a protein of 15,000 daltons related to isoleucine-valine biosynthesis in Escherichia coli K-12.

The effect of the ilvG671, ilvG468, and ilvG603 mutations (phenotype, IlvG+ Valr; formerly ilvO) upon proteins synthesized was determined by infection of irradiated Escherichia coli K-12 cells, using specifically constructed derivatives of lambda dilv phage. These ilvG alleles are similar to the previously studied ilvG2096(Valr) allele in that they activate the latent ilvG gene which is present in the wild-type strain, leading to the synthesis of a 62,000-dalton protein. In addition, all of these ilvG (Valr) alleles increase the synthesis of a 15,000-dalton protein. To localize the gene coding for the 15,000-dalton protein, the proteins produced in maxicells containing plasmids with specific deletions of ilv and rrnX DNA segments were analyzed. The gene coding for the 15,000-dalton protein was located within a region about 1,000 base pairs long between ilv and trpT. The function of the 15,000-dalton protein is not known.

Bacterial Proteins↗
PubMed1982

Cloning of the ilvA538 gene coding for feedback-hypersensitive threonine deaminase from Escherichia coli K-12.

A variety of experimental results implicate the ilvA gene product, threonine deaminase, as an autoregulatory protein that affects the expression of its own gene and those coding for some related proteins. Some of the most direct evidence comes from the analysis of mutations in the ilvA gene with pleiotropic genetic regulatory effects. The most extensively documented mutation, ilvA538, lowers the expression of and abolishes repression control of the ilvGEDA transcription unit. A pleiotropic effect of the ilvA538 mutation, which may be either incidental or mechanistically related to the loss of repression control, renders threonine deaminase feedback hypersensitive to the inhibition of catalytic activity by the pathway end product, isoleucine. We transferred this mutation to lambda dilv phage and pBR322 derivatives. Direct enzyme assay of the plasmid- and phage-coded ilvA538 gene product in delta ilv hosts confirmed the feedback hypersensitivity of the enzyme product. In conjunction with the ilvG671 (phenotype, ILvG+ Valr; previously designated ilvO671) allele located in cis, high levels of the plasmid and lambda dilv phage-coded mutant enzyme suitable for protein purification were observed. Deletion mapping experiments with lambda dilv phage confirmed that the ilvA538 mutation, and not mutations promoter proximal to ilvD (transcription is from ilvG to ilvA), confer a loss of repression control. These genetic mapping studies indicate, however, that an additional mutation(s) may be present that contributes, at least in part, to the reduced enzyme levels in strains with the ilvA538 mutation.

Cloning, Molecular↗
PubMed1981

Identification of the protein products of the rrnC, ilv, rho region of the Escherichia coli K-12 chromosome.

Two methods have been used to identify the protein products of the Escherichia coli K-12 ilv region at 84 min and the flanking rrnC (counterclockwise) and rho (clockwise) loci. First, a set of lambda dilv specialized transducing phages, including some phages that carry rho and others that carry part of rrnC, was used to infect UV irradiated cells. The proteins produced by the infecting lambda dilv phage were selectively labelled with radioactivity amino acids and identified by SDS gel electrophoresis and autoradiography. Second, restriction enzyme fragments were cloned from the lambda dilv phage into pBR322 and the plasmid specific gene products produced in maxicells were similarly identified by SDS gel electrophoresis and autoradiography. The proteins produced were correlated with specific genes and restriction enzyme fragments present in the lambda dilv phage and the pBR322 derivatives. Several ilv gene products that have previously been refractory to protein purification attempts have been identified for the first time by this technique. The presence of mutations at the ilvO site is shown to activate the cryptic ilvG gene and to result in the production of a 62,000 dalton protein. A 15,000 dalton protein of unknown function is synthesized from a DNA segment between ilv and rrnC. The rho gene was cloned from lambda dilv phage into pBR322 and shown to be dominant to a rho mutation on the host chromosome. The rho gene product and four additional proteins coded by genes near or between rho and ilv have been detected.

Bacterial Proteins↗
PubMed1981

Molecular basis of valine resistance in Escherichia coli K-12.

The relationship of valine resistance to the expression of the ilvGEDA operon of Escherichia coli K-12 has been determined. DNA sequence and in vivo protein analyses indicate that in wild-type E. coli K-12 there is a frameshift site within the gene (ilvG) for valine resistance. The ilvG+2096 (formerly designated ilv02096) mutation displaces this frameshift site, resulting in the expression of ilvG and the relief of transcriptional polarity on the distal genes of this operon. Thus, the "ilv0" mutation, which concomitantly confers valine resistance and increased expression of the ilvEDA genes, is, in fact, the "reversion" of a polar site within the first structural gene of the ilvGEDA operon.

Base Sequence↗
PubMed1979

Deletion mapping of the ilvGOEDAC genes of Escherichia coli K-12.

A set of lambdadilv phage has been examined that carry overlapping segments of isoleucine-valine structural and regulatory genes derived from the ilv cluster at 83 min on the Escherichia coli K-12 chromosome. The ilv genes present in these phage, and their order, have been determined by transduction of auxotrophs, escape synthesis, and deletion mapping. The order of ilv genes in the phage, and hence the order in the host chromosome, was found to be ilvG-ilvO-ilvEDA-ilvC. Lysogens containing lambdadilv phage were constructed for dominance analysis of regulatory mutations in the ilvO and ilvA genes. The ilvO671 allele is cis-dominant to ilvO+, while the ilvA538 allele is trans-recessive to ilvA+. Thus, the ilvO gene, that is identified by cis-dominant regulatory mutations that result in increased ilvG and ilvEDA expression, is situated between and may be contiguous with ilvG and ilvEDA.

Chromosome Mapping↗
PubMed1979

Mapping of ilvO loci of Escherichia coli K-12 with bacteriophage lambda dilv.

A set of lambda dilv phage have been used in a deletion mapping procedure to determine the location of two previously characterized ilvO alleles. In contrast to earlier conclusions derived from three-factor crosses and episome-shortening techniques with phage P1, the order found is ilvG-ilvO-ilvEDA. A three-factor cross with phage P1 is described that is not consistent with this location for an ilvO allele. Further analysis of this particular three-factor cross revealed than an artifact attributable to a mutual syntrophism had skewed the apparent frequency of inheritance of the ilvO locus. The role of mutual syntrophism is discussed as a source of mapping errors for the ilvO locus. The value of this set of lambda dilv phage and this mapping procedure for obtaining comparatively unambiguous data on the locations of the ilv structural and regulatory genes is demonstrated.

Acetolactate Synthase↗
PubMed1978

Increased antimetabolite sensitivity with variation of carbon source during growth.

In Serratia marcescens, analogs of leucine (norleucine), methionine (alpha-methylmethionine), histidine (3-amino-1,2,4-triazolealanine), tyrosine (p-aminophenylalanine), and tryptophan (7-methylindole) are conditional inhibitors of growth; inhibition occurs during the metabolism of some carbon sources but not with others. A further increase in sensitivity to growth inhibition by these analogs can be accomplished through the use of particular combinations of carbon sources present in the inoculum and in the subsequent analog-containing culture medium. Variable sensitivity to analog-mediated inhibition of growth observed during growth on glucose, glycerol, fructose, or citrate correlated inversely with the intracellular pool sizes of the amino acids cognate to the analogs used. The above-cited results, in conjunction with previous results obtained with Pseudomonas aeruginosa and Bacillus subtilis, involve diverse biochemical pathways and suggest that nutritional manipulation to alter the pattern of carbon flow in microorganisms is a generally useful means to accomplish increased sensitivity to growth inhibition by metabolite analogs.

Amino Acids↗
PubMed1978

Suppressors of a genetic regulatory mutation affecting isoleucine-valine biosynthesis in Escherichia coli K-12.

Escherichia coli K-12 mutant PS187 carries a mutation, ilvA538, in the structural gene for the biosynthetic L-threonine deaminase that leads to a leucine-sensitive growth phenotype, an isoleucine- and leucine-hypersensitive L-threonine deaminase, and pleiotropic effects resulting in abnormally low and invariant expression of some of the isoleucine-valine biosynthetic enzymes. Fifty-eight derivatives of strain PS187 were isolated as resistant to growth inhibition by leucine, by valine, or by valine plus glycly-valine and were biochemically, genetically, and physiologically characterized. All of these derivatives produced the feedback-hypersensitive L-threonine deaminase, and thus presumably possess the ilvA538 allele of the parent strain. Elevated synthesis of L-threonine deaminase was observed in 41 of the 58 isolates. Among 18 strains analyzed genetically, only those with mutations linked to the ilv gene clusters at 83 min produced elevated levels of L-threonine deaminase. One of the strains, MSR91, isolated as resistant to valine plus glycyl-valine, was chosen for more detailed study. The locus in strain MSR91 conferring resistance was located in four factor crosses between ilvE and rbs, and is in or near the ilvO gene postulated to be a site controlling the expression of the ilvEDA genes. Synthesis of the ilvEDA gene products in strain MSR91 is constitutive and derepressed approximately 200-fold relative to the parent strain, indicating that the genetic regulatory effects of the ilvA538 allele have been suppressed. Strain MSR91 should be suitable for use in purification of the ilvA538 gene product, since enzyme synthesis is fully derepressed and the suppressor mutation is clearly not located within the ilvA gene.

Chromosome Mapping↗
PubMed1976

Threonine deaminase from Escherichia coli: feedback-hypersensitive enzyme from a genetic regulatory mutant.

A mutation, ilvA538, in the gene coding for the biosynthetic L-threonine deaminase of Escherichia coli K-12 has previously been demonstrated to have pleiotropic regulatory effects leading to low and invariant expression of some of the isoleucine-valine biosynthetic enzyme, and altered expression of the branched-chain aminoacyl-tRNA synthetases. Strain PS187, which carries the ilvA538 allele, has a partial growth requirement for L-isoleucine and is characterized by a sensitivity to growth inhibition by L-leucine. The experiments reported here demonstrate that the L-threonine deaminase produced by strain PS187 is hypersensitive to inhibition by the pathway end product L-isoleucine. In addition, L-leucine, which acts at relatively high concentrations in vitro as an inhibitor of L-threonine deaminase from the wild type, is a more potent inhibitor of the activity of the mutant enzyme. Forty-six derivatives of strain PS187 were isolated as spontaneous mutants resistant to the growth-inhibitory effects of L-leucine. Two of these, strains MSR14 and MSR16, produce an L-threonine deaminase that is more resistant than the wild type to L-isoleucine inhibition, and intermediate between the wild type and strain PS187 with respect to L-leucine inhibition. Strains MSR14 and MSR16 produce L-threonine deaminase and dihydroxyacid dehydrase, the ilvD gene product, at the low levels characteristic of the parent strain. Other L-leucine-resistant derivatives of strain PS187 produce higher levels of the feedback-hypersensitive L-threonine deaminase. Thus, the sensitivity to growth inhibition by L-leucine observed with strain PS187 appears to be related both to the hypersensitivity of L-threonine deaminase to inhibition of catalytic activity and to the low level of ilv gene expression. The results reported here indicated that L-threonine deaminase is structurally altered in strain PS187, and thus provide further support for the proposal that L-threonine deaminase participates as a genetic regulatory element for the expression of the branched-chain amino acid biosynthetic enzymes.

Cell-Free System↗
PubMed1975

Threonine deaminase from Escherichia coli. II. Maturation and physical properties of the enzyme from a mutant altered in its regulation of gene expression.

The biosynthetic L-threonine deaminase (L-threonine hydrolase deaminating, EC 4.2.1.16) has been purified from Escherichia coli K12 regulatory mutant CU18. This mutant has properties that follow the predictions of the autogregulatory model previously proposed for the control of synthesis of the isoleucine-valine biosynthetic enzymes. The autoregulatory model specifies that L-threonine deaminase participates in the control of the expression of the ilv ADE gene cluster as well as the ilv B gene and ilv C gene, which constitute three separate units of regulation. The single mutation in strain CU18 results in altered regulation of ilv gene expression and in the production of an altered L-threonine deaminase. The immature form of the enzyme purified from mutant CU18 exhibits an altered response to L-valine, a maturation-inducing ligand. The native form of the mutant is altered in its apparent Km for L-threonine and in its response to the effects of L-valine and L-isoleucine upon catalytic activity. The mutant and wild type L-threonine deaminases differ in the apoenzyme formed as a consequence of alkaline dialysis. Dialysis of the mutant enzyme yields an apoenzyme mixture, apparently of dimers and monomers, while the wild type enzyme yields only dimers. The CU18 L-threonine deaminase, is however, indistinguishable from the wild type enzyme in molecular weight and subunit composition.

Chromatography, Ion Exchange↗