New mutants resistant to glucose repression affected in the regulation of the NADH reoxidation

Spontaneous mutants resistant to vanadate, arsenate or thiophosphate were isolated from a haploid strain of Saccharomyces cerevisiae. These three anions have an inhibitory effect on some mitochondrial functions and at the level of glyceraldehyde 3-phosphate dehydrogenase, a glycolysis enzyme. All the selected mutants had the same phenotype: they were deficient in alcohol dehydrogenase I, the terminal enzyme of the glycolysis, and possessed a high content of cytochrome c oxidase, the terminal enzyme of the respiratory chain. Moreover, cytochrome c oxidase biosynthesis had become insensitive to the catabolite repression, while the biosynthesis of the other enzymes sensitive to this phenomenon were always inhibited by glucose. Metabolic effects of this pleiotropic mutation manifested themselves in the following ways. 1. Growth rate and final cell mass were enhanced, compared to the wild type, when cells were grown on glucose or on glycerol, but not on lactate or ethanol. 2. Growth under anaerobiosis was nil and mutants did not ferment. 3. Mitochondrial respiration of the mutant strains was identical to the wild type with succinate or 2-oxo-glutarate as substrate, and weak with ethanol. But with added NADH, respiration rate of the mutants was higher than that of the wild type and partially insensitive to antimycin, even when cells were grown in repression conditions. It is postulated that in mutants strains, NADH produced at the level of glyceraldehyde 3-phosphate dehydrogenase, failing to be reoxidized via alcohol dehydrogenase, could be reoxidized with a high turnover owing to the enhancement of the amount of cytochrome c oxidase. Since NADH reoxidation is partially insensitive to antimycin, a secondary pathway going from external NADH dehydrogenase to cytochrome c oxidase is suggested.     

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.     

Chromosomal Location of Pleiotropic Negative Sporulation Mutations in BACILLUS SUBTILIS

Genetic analysis by PBS-1 transduction and transformation of a large group of pleiotropic negative sporulation mutants has shown that mutations of this phenotype may be located in five genetically distinct regions. The first group of mutant sites, spoA mutations, is located in the terminal region of the chromosome and linked to the lys-1 marker by PBS-1 transduction. The second group, spoB mutations, is located between phe-1 and the attachment site for the lysogenic bacteriophage ϕ 105. Fine structure analysis of the mutant sites within the spoB locus has been accomplished. A third location for mutants of this phenotype, spoE mutants, was found between the metC3 and ura-1 markers. Two mutants were found at this site and both were capable of sporulation, in contrast to the rest of the pleiotropic sporulation mutants. A fourth chromosomal site, spoH mutations, was found near the ribosomal and RNA polymerase loci. A large group of mutant sites, spoF mutations, was found to be linked to each other by recombination index analysis in transformation but unlinked to any of the known auxotrophic mutations comprising the chromosomal map. All mutants analyzed showing a pleiotropic negative phenotype were found to map within one of these five regions. Interspecific transformation with Bacillus amyloliquefaciens as donor has shown that all of the pleiotropic negative sporulation mutations are conserved relative to a selected group of auxotrophic markers. The degree of conservation in decreasing order is: spoH > spoF = spoB > spoA.     

Regulation and genetic enhancement of beta-amylase production in Clostridium thermosulfurogenes.

We studied the general mechanism for regulation of beta-amylase synthesis in Clostridium thermosulfurogenes. beta-Amylase was expressed at high levels only when the organism was grown on maltose or other carbohydrates containing maltose units. Three kinds of mutants altered in beta-amylase production were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained starch-glucose agar plates. beta-Amylase was produced only when maltose was added to cells growing on sucrose in wild-type and catabolite repression-resistant mutant strains, but the differential rate of enzyme synthesis in constitutive mutants was constant regardless of the presence of maltose. In carbon-limited chemostats of wild-type and catabolite repression-resistant mutant stains, beta-amylase was expressed on maltose but not on glucose or sucrose. beta-Amylase synthesis was immediately repressed by the addition of glucose. Therefore, we concluded that beta-amylase synthesis in C. thermosulfurogenes was inducible and subject to catabolite repression. The addition of cAMP did not eliminate the repressive effect of glucose. The mutants were generally characterized in terms of beta-amylase production, growth properties, fermentation product formation, and alterations in glucose isomerase and glucoamylase activities. A hyperproductive mutant produced eightfold more beta-amylase on starch medium than the wild type and more rapidly fermented starch to ethanol.     

Relaxation of catabolite repression in streptomycin-dependent Escherichia coli

Acetohydroxy acid synthetase, which is sensitive to catabolite repression in wild-type Escherichia coli B, was relatively resistant to this control in a streptomycin-dependent mutant. The streptomycin-dependent mutant was found to be inducible for beta-galactosidase in the presence of glucose, although repression of beta-galactosidase by glucose occurred under experimental conditions where growth of the streptomycin-dependent mutant was limited. Additional glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and isocitrate dehydrogenase) were found to be insensitive to the carbon source in streptomycin-dependent mutants: these enzymes were formed by streptomycin-dependent E. coli B in equivalent quantities when either glucose or glycerol was the carbon source. Two enzymes, glucokinase and glucose 6-phosphate dehydrogenase, that are glucose-insensitive in wild-type E. coli B were formed in equivalent quantity on glucose or glycerol in both streptomycin-sensitive and streptomycin-dependent E. coli B. The results indicate a general decrease or relaxation of catabolite repression in the streptomycin-dependent mutant. The yield of streptomycin-dependent cells from glucose was one-third less than that of the streptomycin-sensitive strain. We conclude that the decreased efficiency of glucose utilization in streptomycin-dependent E. coli B is responsible for the relaxation of catabolite repression in this mutant.     

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.     

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.     

Effect of exogenous cycllc amp on catabolite repression of cellulase formation in aspergillus nidulans

The addition of 1 mM cyclic AMP to induced and repressed cultures of Aspergillus nidulans and its mutant strain (CRR 141) resistant to catabolite repression was fully capable of releasing the wild type from catabolite repression while it caused hyperproduction of cellulases in glycerol repressed cultures. The relief of the catabolite repression was also accompanied by a dramatic drop in enhanced protease levels, thereby indicating that the synthesis of proteases (during the catabolite repression) is under the control of cyclic AMP.     

Glycolytic enzymes and intermediates in carbon catabolite repression mutants of Saccharomyces cerevisiae

Summary Glycolytic parameters were determined in recessive yeast mutants with partial defects in carbon catabolite repression. Specific activities of pyruvate kinase and pyruvate decarboxylase in glucose grown cells of all mutant and wild type stains were 4–5 times higher than in ethanol grown cells. Mutants of gene HEX1 had a reduced hexose phosphorylating activity on allmedia wheras those of gene HEX2 had elevated levels but only in glucose grown cells. Mutants of gene CAT80 were normal in this respect. All other glycolytic enzymes were normal in all mutants. This was also true for glycolytic intermediates. Only hexlmutants showed a reduced fermentation of repressing sugars. The three genes appear to be involved in catabolite repression of several but not of all repressible enzymes. Even though all three types of mutants show a limited overlap in their effects on certain enzymes, they still are distinctly different in their action spectra. Carbon catabolite repression apparently does not depend on the sole accumulation of glycolytic intermediales. The activity of the products of the three genes HEX1, HEX2 and CAT80 are required directly or indirectly for triggering carbon catabolite repression. Even a small segment of carbon catabolite repression is controlled by several genes with regulatory functions indicating that the entire regulatory circuit is highly complex.     

Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae

The escape of several enzymes from “ammonia catabolite repression” in gdhA^? (NADP-linked glutamate-dehydrogenase-less) mutants, as well as in gdhCR mutants of Saccharomyces cerevisiae, does not involve glutamine synthetase, either as a positive or as a negative control element. A glutamine-synthetase-less mutant (gln^?) was used in this demonstration.In addition to its derepressing effect on the NAD-linked glutamate dehydrogenase, the gdhCR mutation releases “nitrogen catabolite repression” on arginase and allatoinase, as well as glutamine repression on glutamine synthetase. A gdhCS mutation was used to demonstrate that these effects are not mediated through the NAD-linked glutamate dehydrogenase.     

Isolation and characterization of catabolite-resistant mutants in the D-serine deaminase system of Escherichia coli K-12.

Two classes of D-serine deaminase (Dsdase)-specific secondary mutants of Escherichia coli K-12 were isolated from a Dsdase low constitutive nonhyperinducible mutant as types which could grow in the presence of both D-serine and glucose. These strains contain cis dominant, nonsuppressible mutations in the dsdO (operator-initiator) region. In the first class of mutants (e.g., FB4010), Dsdase synthesis is completely insensitive to catabolite repression, and synthesis occurs at a high constitutive rate in the absence of cyclic adenosine 5'-monophosphate. In the second class (e.g., FB4005), Dsdase synthesis is partially insensitive to catabolite repression, and catabolite repression is reversed by the addition of cyclic adenosine 5'-monophosphate. Dsdase synthesis in strain FB4005 is partially independent of the cyclic adenosine 5'-monophosphate binding protein, as constitutive synthesis is reduced only 65% (relative to the cap+ strain) in strains unable to synthesize the cyclic adenosine 5'-monophosphate binding protein. Surprisingly, the constitutive rate of Dsdase synthesis is fourfold higher in all mutants of both classes than in the parent, indicating a close interrelationship between the sites of response to induction and catabolite repression.     

Catabolite repression-resistant mutants of Bacillus subtilis.

Mutants of Bacillus subtilis that are able to sporulate under the condition of catabolite repression were isolated by a simple selection technique. The mutants used in the present study were able to grow normally on minimal medium with ammonium sulphate as the nitrogen source and glucose as the carbon source. Studies carried out with these mutants show that there is no close relation between catabolite repression of an inducible enzyme, acetoin dehydrogenase, and that of sporulation. Certain mutants are able to sporulate in the presence of all the carbon sources tested but some mutants are resistant only to the carbon source used in isolation. It is suggested that several metabolic steps may be affected in catabolite repression of sporulation.     

Histidine utilization by Alcaligenes eutrophus: Regulation of histidase formation under heterotrophic conditions of growth

Histidine supported good growth of Alcaligenes eutrophus strain H 16 as a nitrogen source, but only poor growth as a carbon and energy source. The facultative chemolithoautotrophic bacterium was also able to utilize urocanic acid, the first intermediate of histidine catabolism. The products of histidine degradation were ammonium, formate and glutamate. Three enzymes of the pathway, histidase, urocanase and formiminoglutamate hydrolase, were present in histidine-grown cells. Two types of spontaneous mutants, derived from the wild type, were characterized by an increased growth rate on histidine. One of these types was found to produce histidase constitutively and at a higher activity compared with the parental strain. The second type of mutant had apparently gained an improved histidine uptake system, which is supposed to be growth rate-limiting in the wild type. From the physiological studies the conclusion was drawn that the control of histidine-degrading enzymes is based on induction by urocanate and catabolite repression by carbon sources supporting fast growth, such as succinate or pyruvate. Ammonium was found not to affect catabolite repression, however, we obtained evidence that histidine uptake is subject to a nitrogen control.Abbreviation CTAB hexadecyltrimethylammonium bromide     

Beta-fructofuranosidase production by 2-deoxyglucose resistant mutants of aspergillus niger in submerged and solid-state fermentation

Aspergillus niger produces extracellular beta-fructofuranosidase under submerged (SmF) and solid state fermentation (SSF) conditions. After UV mutagenesis of conidiospores of A. niger, 2-deoxyglucose (10 g/l) resistant mutants were isolated on Czapek's minimal medium containing glycerol as a carbon source and the mutants were examined for improved production of beta-fructofuranosidase in SmF and SSF conditions. One of such mutant DGRA-1 overproduced beta-fructofuranosidase in both SmF and SSF conditions. In SmF, the mutant DGRA-1 showed higher beta-fructofuranosidase productivity (110.8 U/l/hr) than the wild type (48.3 U/l/hr). While in SSF the same strain produced 322 U/l/hr of beta-fructofuranosidase, 2 times higher than that of wild type (154.2 U/l/hr). In SmF, both wild type and mutants produced relatively low level of beta-fructofuranosidase in medium containing sucrose with glucose than from the sucrose medium. However in SSF, the DGRA-1 mutant grown in sucrose and sucrose+ glucose did not show any difference with respect to beta-fructofuranosidase production. These results indicate that the catabolite repression of beta-fructofuranosidase synthesis is observed in SmF whereas in SSF such regulation was not prominent.     

Catabolite repression of Pseudomonas aeruginosa amidase: isolation of promotor mutants.

Among mutants of Pseudomonas aeruginosa isolated from fluoroacetamide medium were some which synthesized amidase at about 5% of the rate of the parent constitutive strain, PAC101. Seven fluoroacetamide-resistant mutants with low amidase activity gave rise to secondary mutant strains on succinate+butyramide plates. One appeared to be an 'up-promotor' mutant and synthesized amidase at a high rate. This mutant, PAC433, was not stimulated by cyclic-AMP and was much less sensitive to catabolite repression by succinate. The mutation conferring resistance to catabolite repression was cotransduced at a frequency of 96% (26/27) with the amidase genes amiR, amiE. Five other revertants had catabolite repression-resistance mutations which were linked to the amidase genes and these also were probably promotor mutants. One strain had a mutation conferring resistance to catabolite repression which was unlinked to the amidase genes.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.     

Isolation and phenotypic characterization of Pseudomonas aeruginosa pseudorevertants containing suppressors of the catabolite repression control-defective crc-10 allele

The amiE gene encodes an aliphatic amidase capable of converting fluoroacetamide to the toxic compound fluoroacetate and is one of many genes whose expression is subject to catabolite repression control in Pseudomonas aeruginosa. The protein product of the crc gene, Crc, is required for repression of amiE and most other genes subject to catabolite repression control in this bacterium. When grown in a carbon source such as succinate, wild-type P. aeruginosa is insensitive to fluoroacetamide (due to repression of amiE expression). In contrast, mutants harboring the crc-10 null allele cannot grow in the presence of fluoroacetamide (due to lack of repression of amiE). Selection for succinate-dependent, fluoroacetamide-resistant derivatives of the crc-10 mutant yielded three independent pseudorevertants containing suppressors that restored a degree of catabolite repression control. Synthesis of Crc protein was not reestablished in these pseudorevertants. All three suppressors of crc-10 were extragenic, and all three also suppressed a Delta crc::tetA allele. In each of the three pseudorevertants, catabolite repression control of amidase expression was restored. Catabolite repression control of mannitol dehydrogenase production was also restored in two of the three isolates. None of the suppressors restored repression of glucose-6-phosphate dehydrogenase or pyocyanin production.     

Effect of carbon source on enzymes involved in glycerol metabolism inNeurospora crassa

Specific activities of eight enzymes involved in glycerol metabolism were determined in crude extracts of three strains ofNeurospora crassa after growth on six different carbon sources. One of the strains was wild type, which grew poorly on glycerol as sole carbon source; the other two were mutant strains which were efficient glycerol utilizers. A possible basis for this greater effeciency of glycerol utilization was catabolite repression of glyceraldehyde kinase by glycerol in wild type, and two-fold higher glycerate kinase activity in the mutant strains after growth on glycerol, thus apparently allowing two routes for glyceraldehyde to enter the glycolytic pathway in the mutant strains but only one in wild type. The preferential entry of glyceraldehyde to the glycolytic pathway through glycerate was suggested by the lack of glyceraldehyde kinase in all three strains after growth on one or more of the carbon sources and the generally higher levels of aldehyde dehydrogenase and of glycerate kinase than of glyceraldehyde kinase.