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.     

Isolation and characterization of dominant mutations resistant to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae.

Seven dominant mutations showing greatly enhanced resistance to the glucose repression of galactokinase synthesis have been isolated from GAL81 mutants, which have the constitutive phenotype but are still strongly repressible by glucose for the synthesis of the Leloir enzymes. These glucose-resistant mutants were due to semidominant mutations at either of two loci, GAL82 and GAL83. Both loci are unlinked to the GAL81- gal4, gal80, or gal7 X gal10 X gal1 locus or to each other. The GAL83 locus was mapped on chromosome V at a site between arg9 and cho1. The GAL82 and GAL83 mutations produced partial resistance of galactokinase to glucose repression only when one or both of these mutations were combined with a GAL81 or a gal80 mutation. The GAL82 and GAL83 mutations are probably specific for expression of the Leloir pathway and related enzymes, because they do not affect the synthesis of alpha-D-glucosidase, invertase, or isocitrate lyase.     

Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae

A total of 37 recessive mutations showing enhanced resistance to the glucose repression of galactokinase synthesis have been isolated by a selection procedure with a GAL81 gal7 double mutant. These mutations were grouped into three different complementation classes. One class, reg1, contains mutants arising from mutations at a site close to, but complementing, the gal3 locus. The reg1 mutant also showed resistance to the glucose repression of invertase synthesis but not to that of alpha-D-glucosidase. The two other classes were identified as arising from recessive mutations at the GAL82 locus and the GAL83 locus, respectively, at which various dominant mutations were isolated previously. When in a constitutive background due to the GAL81 or gal80 mutation, the GAL82 and GAL83 mutations did not show a mutually additive effect on the resistance to glucose repression of galactokinase synthesis, while the reg1 and GAL82 (or GAL83) mutations did. Based upon the specific behavior of cells with various genotypes for the above genes in response to the concentration of galactose and glucose in the medium, we propose a model involving three independent circuits for glucose signals in the regulation of the structural genes for the galactose pathway enzymes.     

Glucose repression of maltase and methanol-oxidizing enzymes in the methylotrophic yeastHansenula polymorpha: Isolation and study of regulatory mutants

Regulation of the synthesis of maltase and methanol-oxidizing enzymes by the carbon source has been analyzed in the methylotrophic yeastHansenula polymorpha. Maltase was shown to be responsible for the growth ofH. polymorpha not only on maltose, but also on sucrose. The affinity of maltase towards maltase substrates decreased in the order: 4-nitrophenyl glucoside (pNPG) <sucrose <maltose. Mutants with glucose repression-insensitive synthesis of alcohol oxidase and maltase were obtained fromH. polymorpha by mutagenesis and subsequent selection on methanol medium in the presence of 2-deoxy-d-glucose. One of the isolated mutants, L63, was studied in more detail. Mutant L63 was recessive and monogenic and it was not deficient in hexokinase. Its analysis revealed thatH. polymorpha most probably has a repressor protein that in the presence of glucose can down-regulate expression of both maltase and enzymes of methanol oxidation.     

Regulatory mutations affecting the synthesis of cellulase in Bacillus pumilus

A wild strain of Bacillus pumilus was investigated for cellulase production, and putative mutants of this strain were screened for catabolite repression insensitivity after chemical mutagenesis using ethyl methanesulphonate (EMS) as a mutagenic agent. Out of four classes of mutants studied and classified according to their cellulase induction rate and level of cellulase production in the presence of high concentrations of glucose (2.6%[w/v]), classes III and IV exhibited cellulase production up to 6.2 mg cellulase and 11.4 mg cellulase per gram of dry cell mass respectively. These mutants were referred to as catabolite repression-insensitive when compared to the wild strain which exhibited a total repression of cellulase synthesis under the same conditions. How EMS triggered the catabolite repression insensitivity in these mutants was not established. However this mutation brought out new strains of cellulase hyperproducers (mutants 6 and 11) in the presence of glucose when compared to other cellulase producers such as Aspergillus terreus, A. nidulans and Trichoderma reesei, which exhibited catabolite repression of cellulase synthesis. These mutants were selected as the most promising candidates for cellulase synthesis even at high glucose concentration.     

Pleiotropic mutations regulating resistance to glucose repression in Saccharomyces carlsbergensis are allelic to the structural gene for hexokinase B.

Previously, we described a mutation glr1-1 in Saccharomyces carlsbergensis which pleiotropically relieves the synthesis of the following enzymes from glucose repression: maltase, galactokinase, alpha-galactosidase, NADH:cytochrome c reductase, and cytochrome c oxidase (C. A. Michels and A. Romanowski, J. Bacteriol, 143:674-679, 1980.) In this report, we demonstrate that glr1-1 and two other alleles, glr1-3 and glr1-16, are also insensitive to the glucose repression of invertase synthesis. Determinations of the levels of hexokinase activity and the rate of glucose transport in these mutants show that both are reduced as compared with the parent strain. Complementation tests and genetic analysis indicate that the glr1 mutations are allelic to HXK2, the structural gene for hexokinase B. The significance of this result is discussed with regard to the mechanism of glucose repression in S. carlsbergensis.     

Fatty Acid Degradation in Escherichia coli: Requirement of Cyclic Adenosine Monophosphate and Cyclic Adenosine Monophosphate Receptor Protein for Enzyme Synthesis

The strong repression of inducible synthesis of the enzymes of fatty acid degradation by glucose can be partially relieved by the addition of cyclic adenosine 3',5' monophosphate (cyclic AMP) to the growth medium. This reversal of the glucose effect by cyclic AMP is not observed in a mutant (K29) that is unable to grow on fatty acids as sole carbon source and that was found to synthesize low levels of several enzymes specified by the fad regulon. In a revertant selected for the ability to grow on oleate these effects are concomitantly relieved. By both genetic (co-transduction of the mutation with the strA locus) and biochemical experiments (an extract of the mutant strain does not show the cyclic AMP-dependent stimulation of the deoxyribonucleic acid-directed in vitro synthesis of the enzymes of the gal operon), it is demonstrated that the mutant lacks functional cyclic AMP receptor protein (CR protein). It is concluded that, like many other inducible enzyme systems, expression of the enzymes of the fad system requires cyclic AMP and the CR protein.     

Demonstration of several independent loci involved in the synthesis of iso-2-cytochrome c in yeast

Summary This study concerns the chromosomal genes controlling the synthesis of cytochrome c in yeast. In the wild type there are two molecular species of cytochrome c : iso-1 (major from) and iso-2 (minor form) which differ in many positions of their amino-acid sequence. A mutation, CY1cy1-1, in the structural gene for iso-1, leads to iso-1 deficiency, while retaining a normal albeit small amount of iso-2-cytochrome c.The cyI-1 mutant does not grow on DL-lactate as sole carbon source, while the wild type does. This property was used for selecting cytochrome c rich revertants (CYT) from cytochrome c deficient strains cy1-1; ca 200 revertants were isolated after extensive nitrous acid mutagenesis from a haploid cy1-1 strain or from a diploid cy1-1/cy1-1 strain and ca 30 of them were analyzed genetically and biochemically. The cytochrome c of seven (CYT) revertants was extracted and characterized; none of them contained iso-1-cytochrome c, but all contained large amount of iso-2-cytochrome csufficient to compensate for the deficiency. It was concluded that none of the revertants resulted from back mutation of cy1-1 and that the cy1-1 mutation is a deletion or some other irreversible aberration. These conclusions were corroborated by genetic analysis. It was shown that every reversion is due to a chromosomal mutation segregating as a single gene. Five unlinked gene loci, CY2A, CY2B, CY2C, CY2D, CY2E, were uncovered in this way. None of them were linked to the CY1 locus. Revertants selected in the diploid strain were dominant or semi-dominant while those selected in the haploid strain were recessive. To the first class belong alleles at loci CY2A, CY2B, CY2C, while to the latter belong alleles at loci CY2D and CY2E.Five unlinked loci are implicated in iso-2-cytochrome c synthesis. Mutations selected at these loci act as suppressors of cytochrome c deficiency caused by a deletion of the CY1 locus. In fact the muations do not restore the synthesis of the deficient protein (iso-1-cytochrome c), but increase the synthesis of an another protein, structurally alike (iso-2-cytochrome c), and having very similar if not identical physiological activity. We propose the term of compensator genes to define this type of mutations. We discuss some possible mechanisms to explain the rarity of compensator mutations and the hypothesis that the locus CY2A could correspond not only to the regulatory gene for iso-2-cytochrome c but also to the structural one.     

A nuclear-encoded function essential for translation of the chloroplast psaB mRNA in chlamydomonas.

We report the analysis of a photosystem I-deficient mutant of Chlamydomonas reinhardtii, F15, that contains a mutation at the TAB1 (for translation of psaB mRNA) nuclear locus. Pulse labeling of chloroplast proteins revealed that the synthesis of the two photosystem I reaction center polypeptides PSAA and PSAB was undetectable in this mutant. The mRNA levels of these proteins were only moderately reduced, suggesting that the primary defect occurs at a step during or after translation. We constructed chimeric genes consisting of the psaA and psaB 5' untranslated region (5' UTR) fused to the aminoglycoside adenyltransferase (aadA) coding sequence, which confers spectinomycin resistance. Insertion of these genes into the chloroplast genome through biolistic transformation and analysis of their expression in the TAB1 mutant nuclear background revealed that the psaB (but not the psaA) 5' UTR is the target of the wild-type TAB1 function. This suggests that TAB1 is required for the initiation of psaB mRNA translation. The dependence of PSAA synthesis or accumulation on PSAB synthesis is strongly suggested by the identification of a suppressor mutation within the psaB 5' UTR. The suppressor specifically restores the synthesis of both proteins in the presence of the tab1-F15 mutation. The location of the suppressor mutation within a putative base-paired region near the psaB initiation codon suggests a role for TAB1 in the activation of translation of the psaB mRNA.     

Effects of transition mutations in the regulatory locus spoIIA on the incidence of sporulation in Bacillus subtilis

We have determined the changes in DNA sequence corresponding to three mutations in the promoter-proximal open reading frame of spoIIA, a locus that regulates sporulation in Bacillus subtilis. All three mutations prevent the synthesis of two sporulation-associated enzymes, but they differ in their effects on spore incidence. We now find that mutation spo-42, which allows spores to be produced at a low incidence, is a transition that changes Gly95 to Asp in the protein encoded by the open reading frame. Mutation spo-69, which blocks sporulation entirely, consists of two transitions: these change Gly62 to Asp and Ala1 16 to Thr. Mutation sas-1, which partially suppresses spo-69, is also a transition: this changes residue 62 (which had become Asp as a result of the spo-69 mutation) to Asn.     

Identification of New Genes Involved in the Regulation of Yeast Alcohol Dehydrogenase II

Recessive mutations in two negative control elements, CRE1 and CRE2, have been obtained that allow the glucose-repressible alcohol dehydrogenase (ADHII) of yeast to escape repression by glucose. Both the cre1 and cre2 alleles affected ADHII synthesis irrespective of the allele of the positive effector, ADR1. However, for complete derepression of ADHII synthesis, a wild-type ADR1 gene was required. Neither the cre1 nor cre2 alleles affected the expression of several other glucose-repressible enzymes. A third locus, CCR4, was identified by recessive mutations that suppressed the cre1 and cre2 phenotypes. The ccr4 allele blocked the derepression of ADHII and several other glucose-repressible enzymes, indicating that the CCR4 gene is a positive control element. The ccr4 allele had no effect on the repression of ADHII when it was combined with the ADR1-5c allele, whereas the phenotypically similar ccr1 allele, which partially suppresses ADR1-5c, did not suppress the cre1 or cre2 phenotype. Complementation studies also indicated that ccr1 and snf1 are allelic. A model of ADHII regulation is proposed in which both ADR1 and CCR4 are required for ADHII expression. CRE1 and CRE2 negatively control CCR4, whereas CCR1 is required for ADR1 function.     

A rice glutamate receptor-like gene is critical for the division and survival of individual cells in the root apical meristem

Glu receptors are known to function as Glu-activated ion channels that mediate mostly excitatory neurotransmission in animals. Glu receptor-like genes have also been reported in higher plants, although their function is largely unknown. We have identified a rice (Oryza sativa) Glu receptor-like gene, designated GLR3.1, in which mutation by T-DNA insertion caused a short-root mutant phenotype. Histology and DNA synthesis analyses revealed that the mutant root meristematic activity is distorted and is accompanied by enhanced programmed cell death. Our results supply genetic evidence that a plant Glu receptor-like gene, rice GLR3.1, is essential for the maintenance of cell division and individual cell survival in the root apical meristem at the early seedling stage.     

Involvement of the CDC25 gene product in the signal transmission pathway of the glucose-induced RAS-mediated cAMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-protein-mediated cAMP signal, which induces a protein phosphorylation cascade. Yeast strains without a functional CDC25 gene were deficient in basal cAMP synthesis and in the glucose-induced cAMP signal. Addition of dinitrophenol, which in wild-type strains strongly stimulates in vivo cAMP synthesis by lowering intracellular pH, did not enhance the cAMP level. cdc25 disruption mutants, in which the basal cAMP level was restored by the RAS2val19 oncogene or by disruption of the gene (PDE2) coding for the high-affinity phosphodiesterase, were still deficient in the glucose- and acidification-induced cAMP responses. These results indicate that the CDC25 gene product is required not only for basal cAMP synthesis in yeast but also for specific activation of cAMP synthesis by the signal transmission pathway leading from glucose to adenyl cyclase. They also show that intracellular acidification stimulates the pathway at or upstream of the CDC25 protein. When shifted to the restrictive temperature, cells with the temperature sensitive cdc25-5 mutation lost their cAMP content within a few minutes. After prolonged incubation at the restrictive temperature, cells with this mutation, and also those with the temperature sensitive cdc25-1 mutation, arrested at the 'start' point (in G1) of the cell cycle, and subsequently accumulated in the resting state G0. In contrast with cdc25-5 cells, however, the cAMP level did not decrease and normal glucose- and acidification-induced cAMP responses were observed when cdc25-1 cells were shifted to the restrictive temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of decreased levels of Niemann-Pick C1 intracellular cholesterol transport on obesity is reversed in the C57BL/6J, metabolic syndrome mouse strain: a metabolic or an inflammatory effect?

We have previously shown that decreased dosage of Niemann-Pick C1 (Npc1) protein, caused by heterozygosity at the null mutation, Npc1 (nih), locus, causes altered lipid metabolism in mice. When studied on the "lean" BALB/cJ genetic background, the decreased protein was associated with no weight changes in either males or females when on a regular diet but increased weights and adiposity when on a high fat diet Jelinek et al. (Obesity 18: 1457-1459, 2010, Gene 491:128-134, 2012). When the heterozygotes were studied on a mixed C57BL/6J, BALB/cJ background, increased weight and adiposity were also found on a regular diet (sexes pooled Jelinek et al. [Hum Molec Genet 20:312-321, 2011]). We find somewhat different results when the hypomorphic Npc1 mutation, Npc1 (nmf164), is studied on a pure C57BL/6J, "metabolic syndrome" genetic background with male, but not female, heterozygotes having lower weights on the regular diet. The result does not seem to be due to the difference in the two mutations as heterozygous Npc1 (nmf164) mice on the BALB/cJ background acted like the null mutant heterozygotes. Studies of glucose tolerance, liver enzymes, liver triglycerides and fat deposition, and adipose tissue caveolin 1 levels did not disclose reasons for these differing results.     

Altered expression of biodegradative threonine dehydratase in Escherichia coli mutants.

A number of strains of Escherichia coli K-12 failed to synthesize significant amounts of biodegradative threonine dehydratase (EC 4.2.1.16) when grown anaerobically in tryptone-yeast extract medium, a condition which is optimal for the induction of this enzyme. However, the addition of 10 mM potassium nitrate to the culture medium enabled a few of these strains, notably MB201, to induce the enzyme. An examination of the kinetic parameters, modifier sensitivity, and immunological cross-reactivity revealed that the enzyme produced by MB201 in nitrate-supplemented medium appeared indistinguishable from the dehydratase of a wild-type strain. The reduced expression of threonine dehydratase in MB201 appeared highly specific; the synthesis of two other inducible enzymes, D-serine deaminase and tryptophanase, and two "anaerobic" proteins, namely, fumarate reductase and cytochrome c551, remained unaffected. The mutation (tdcI) responsible for the altered expression of the dehydratase in MB201 was located at min 91 on the E. coli chromosome and appeared to tightly linked to if not identical with pgi, the gene encoding phosphoglucose isomerase, as judged by growth experiments on glucose and fructose, direct assay of phosphoglucose isomerase activity, spontaneous and simultaneous reversion of MB201 (tdcI) to TdcI+ and Pgi+ phenotype, and cosegregation of the two loci during transduction with P1 phage. Because not all strains lacking the dehydratase showed nitrate-dependent enzyme synthesis or had lesions at the pgi locus, it appears that mutations at multiple loci on the E. coli chromosome may influence the expression of the enzyme in vivo.     

The role of glutathione reductase in the interplay between oxidative stress response and turnover of cytosolic NADPH in Kluyveromyces lactis

The phosphoglucose isomerase mutant of the respiratory yeast Kluyveromyces lactis (rag2) is forced to metabolize glucose through the oxidative pentose phosphate pathway and shows an increased respiratory chain activity and reactive oxygen species production. We have proved that the K. lactis rag2 mutant is more resistant to oxidative stress (OS) than the wild type, and higher activities of glutathione reductase (GLR) and catalase contribute to this phenotype. Resistance to OS of the rag2 mutant is reduced when the gene encoding GLR is deleted. The reduction is higher when, in addition, catalase activity is inhibited. In K. lactis, catalase activity is induced by peroxide-mediated OS but GLR is not. We have found that the increase of GLR activity is correlated with that of glucose-6-phosphate dehydrogenase (G6PDH) activity that produces NADPH. G6PDH is positively regulated by an active respiratory chain and GLR plays a role in the reoxidation of the NADPH from the pentose phosphate pathway in these conditions. Cytosolic NADPH is also used by mitochondrial external alternative dehydrogenases. Neither GLR overexpression nor induction of the OS response restores growth on glucose of the rag2 mutant when the mitochondrial reoxidation of cytosolic NADPH is blocked.     

Regulation of Repressible Acid Phosphatase by Cyclic Amp in SACCHAROMYCES CEREVISIAE

One of the cyr 1 mutants (cyr 1-2) in yeast produced low levels of adenylate cyclase and cyclic AMP at 25 degrees and was unable to derepress acid phosphatase. Addition of cyclic AMP to the cyr1-2 cultures elevated the level of repressible acid phosphatase activity. The bcy1 mutation, which suppresses the cyr1-2 mutation by allowing activity of a cyclic AMP-independent protein kinase, also allows acid phosphatase synthesis without restoring adenylate cyclase activity. The CYR3 mutant had structurally altered cyclic AMP-dependent protein kinase and was unable to derepress acid phosphatase. The cyr1 locus was different from pho2, pho4 and pho81, which were known to regulate acid phosphatase synthesis. Mutants carrying cyr1-2 and pho80, PHO81c, PHO82 or pho85 mutations, which confer constitutive synthesis of repressible acid phosphatase, produced acid phosphatase. The cyr1-2 mutant produced significantly low levels of invertase and alpha-D-glucosidase. These results indicated that cyclic AMP-dependent protein kinase exerts its function in the synthesis of repressible acid phosphatase and other enzymes.     

Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase

The SUC2 gene of yeast (Saccharomyces) encodes two forms of invertase: a secreted, glycosylated form, the synthesis of which is regulated by glucose repression, and an intracellular, nonglycosylated enzyme that is produced constitutively. The SUC2 gene has been cloned and shown to encode two RNAs (1.8 and 1.9 kb) that differ at their 5′ ends. The stable level of the larger RNA is regulated by glucose; the level of the smaller RNA is not. A correspondence between the presence of the 1.9 kb RNA and the secreted invertase, and between the 1.8 kb RNA and the intracellular invertase, was observed in glucose-repressed and -derepressed wild-type cells. In addition, cells carrying a mutation at the SNF1 locus fail to derepress synthesis of the secreted invertase and also fail to produce stable 1.9 kb RNA during growth in low glucose. Glucose regulation of invertase synthesis thus is exerted, at least in part, at the RNA level. A naturally silent allele (suc2°) of the SUC2 locus that does not direct the synthesis of active invertase was found to produce both the 1.8 and 1.9 kb RNAs under normal regulation by glucose. A model is proposed to account for the synthesis and regulation of the two forms of invertase: the larger, regulated mRNA contains the initiation codon for the signal sequence required for synthesis of the secreted, glycosylated form of invertase; the smaller, constitutively transcribed mRNA begins within the coding region of the signal sequence, resulting in synthesis of the intracellular enzyme.