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.     

Biogenesis of mitochondria. The effects of catabolite repression on the in vitro phospholipid synthetic capacities of subcellular fractions of respiratory-competent and respiratory-deficient strains of Saccharomyces cerevisiae.

A respiratory-competent wild-type strain and a nuclear isogenic, mitochondrial DNA-less, petite mutant strain of Saccharomyces cerevisiae were grown under conditions of catabolite repression in batch cultures and under conditions of catabolite derepression in chemostat cultures. Subcellular fractions were isolated and the capacity of these fractions to incorporate sn-[2-³H]glycerol 3-phosphate into phospholipids was studied. Neither catabolite repression nor loss of mitochondrial DNA appreciably altered the total in vitro lipid synthesized by mitochondrial fractions during the incubation. Mitochondria isolated from catabolite-derepressed wild-type and petite cells had approximately the same specific activity in vitro for the synthesis of phosphatidylinositol. phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, and neutral lipids. Mitochondria isolated from the petite cells retained the capacity to synthesize phosphatidylglycerol and diphosphatidylglycerol, although the synthesis of these phospholipids was far less extensive than that by the mitochondria isolated from the wild-type cells. In both cases, mitochondria prepared from catabolite-repressed cells synthesized a greater proportion of phosphatidylserine than did mitochondria from catabolite-derepressed cells. The proportions of phospholipid species synthesized in vitro by the microsomal fractions studied were not grossly affected by catabolite repression or loss of mitochondrial DNA.     

Chlorophyll b to chlorophyll a conversion precedes chlorophyll degradation in Hordeum vulgare L.

This study reveals by in vivo deuterium labeling that in higher plants chlorophyll (Chl) b is converted to Chl a before degradation. For this purpose, de-greening of excised green primary leaves of barley (Hordeum vulgare) was induced by permanent darkness in the presence of heavy water (80 atom % (2)H). The resulting Chl a catabolite in the plant extract was subjected to chemical degradation by chromic acid. 3-(2-Hydroxyethyl)-4-methyl-maleimide, the key fragment that originates from the Chl catabolite, was isolated. High resolution (1)H-, (2)H-NMR and mass spectroscopy unequivocally demonstrates that a fraction of this maleimide fragment consists of a mono-deuterated methyl group. These results suggest that Chl b is converted into Chl a before degradation. Quantification proves that the initial ratio of Chl a:Chl b in the green plant is preserved to about 60-70% in the catabolite composition isolated from yellowing leaves. The incorporation of only one deuterium atom indicates the involvement of two distinguishable redox enzymes during the conversion.     

Catabolite repression of Pseudomonas aeruginosa amidase: the effect of carbon source on amidase synthesis.

Synthesis of the Pseudomonas aeruginosa aliphatic amidase was repressed severely by succinate and malate and less severely by glucose, acetate or lactate. Amidase synthesis in inducible and constitutive strains was stimulated by cyclic AMP, which also gave partial relief to catabolite repression produced by the addition of lactate to cultures growing in pyruvate medium. Mutants which were resistant to catabolite repression were isolated from succinate+lactamide medium.     

Cloning, expression, and carbon catabolite repression of the bamM gene encoding beta-amylase of Bacillus megaterium DSM319

The bamM gene from Bacillus megaterium DSM319 encoding an extracellular beta-amylase was isolated and completely sequenced. Chromosomal inactivation by deletion mutagenesis resulted in total loss of amylolytic activity, indicative of a single starch-degrading enzyme. Functional characterization of the expressed protein revealed a maltogenic enzyme exhibiting optimal activities at pH 7.5 and 50 degrees C. Amylase expression is subject to catabolite repression by glucose. A putative cis-acting catabolite-responsive element (CRE) was identified; it is located within the bamM coding region, matching the position of the predicted signal peptide processing site. Base substitutions introduced by site-directed mutagenesis within the bamM-CRE--retaining unchanged the amino acid sequence--provoked a remarkable relief from carbon catabolite repression (CCR), thereby proving functionality of the CRE for CCR.     

Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast

Summary Mutants with reduced hexokinase activity previously isolated as resistant to carbon catabolite repression of invertase and maltase (Zimmermann and Scheel, 1977) were allele tested with mutant strains of Lobo and Maitra (1977) which had defects in one or several of the genes coding for glucokinase and the two unspecific hexokinases. It could be demonstrated, that the mutation abolishing carbon catabolite repression had occurred in a gene allelic to the structural gene of hexokinase PII. Moreover, the defective mutant allele for hexokinase PII isolated by Lobo and Maitra (1977) was also defective in carbon catabolite repression. Neither glucokinase nor hexokinase PI showed any effect on this regulatory system. Biochemical analysis in crude extracts also showed altered kinetic properties of hexokinases in the hex1 mutants. The results directly support the hypothesis previously put forward, that one of the hexokinases is not only active as a catalytic, but also as a regulatory protein.     

The effect of alpha-ketoglutaric acid on amino acid utilization by nonstarter Lactobacillus spp. isolated from Cheddar cheese

AIMS: To examine the effect of alpha-ketoglutaric acid (alpha-KG) on the utilization and catabolism of amino acids by strains of nonstarter lactobacilli isolated from Cheddar cheese. METHODS AND RESULTS: The effect of alpha-KG in the growth medium of nonstarter lactobacilli on amino acid metabolism, catabolite levels, peptide hydrolase and aminotransferase activities was examined. The pattern of amino acid utilization, catabolite formation and aminotransferase activity was affected by keto acid. CONCLUSIONS: Amino acid conversion into cheese aroma and flavour compounds by nonstarter lactobacilli is enhanced in the presence of alpha-ketoglutarate. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing the availability of alpha-ketoglutarate in cheese offers a possible method of reducing the maturation period by accelerating the rate of character compound formation from amino acids by the nonstarter lactobacilli.     

Evidence for non-vacuolar proteolytic catabolite inactivation of yeast fructose-1,6-bisphosphatase

Immunoblotting was used to study whether proteolytic degradation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in yeast cells during catabolite inactivation occurs intra- or extravacuolarly. The 40-kDa subunits of both the phosphorylated and the non-phosphorylated fructose-1,6-bisphosphatase are rapidly degraded by an extract from isolated vacuoles to a 32-kDa intermediate which accumulates and is then slowly further degraded. However, in intact cells, neither the 32-kDa nor any other intermediate reacting with the fructose-1,6-bisphosphatase antibodies is observed following glucose-induced degradation of the enzyme. These observations are discussed as evidence against intravacuolar degradation of fructose-1,6-bisphosphatase during proteolytic catabolite inactivation.     

Differentially regulated malate synthase genes participate in carbon and nitrogen metabolism of S. cerevisiae.

We have isolated a second gene (MLS1), which in addition to DAL7, encodes malate synthase from S. cerevisiae. Expression of the two genes is specific for their physiological roles in carbon and nitrogen metabolism. Expression of MLS1, which participates in the utilization of non-fermentable carbon sources, is sensitive to carbon catabolite repression, but nearly insensitive to nitrogen catabolite repression. DAL7, which participates in catabolism of the nitrogenous compound allantoin, is insensitive to carbon catabolite repression, but highly sensitive to nitrogen catabolite repression. Results obtained with null mutations in these genes suggest that S. cerevisiae contains at least one and perhaps two additional malate synthase genes.     

Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.     

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.     

Comparative proteome analysis of alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. N16-5 grown on different carbon sources

To determine the impact of carbohydrates on the metabolic pathway in alkaliphiles, proteomes were obtained from cultures containing different carbohydrates and were resolved on two-dimensional gel electrophoresis (2-DE). The proteomes were compared to determine differentially expressed proteins. A novel alkaliphilic bacterium (alkaliphilic Bacillus sp. N16-5 isolated from Wudunur Soda Lake, China) was isolated in media with five different carbon sources (glucose, mannose, galactose, arabinose, and xylose). Comparative proteome analysis identified 61 differentially expressed proteins, which were mainly involved in carbohydrate metabolism, amino acid transport, and metabolism, as well as energy production and conversion. The comparison was based on the draft genome sequence of strain N16-5. The abundance of enzymes involved in central metabolism was significantly changed when exposed to various carbohydrates. Notably, catabolite control protein A (CcpA) was up-regulated under all carbon sources compared with glucose. In addition, pentose exhibited a stronger effect than hexose in CcpA-mediated carbon catabolite repression. These results provided a fundamental understanding of carbohydrate metabolism in alkaliphiles.     

Characterization of a mutant of the yeast Candida maltosa defective in catabolite inactivation of gluconeogenetic enzymes

A spontaneous mutant of the yeast Candida maltosa SBUG 700 was isolated showing pseudohyphal marphology under all growth conditions tested. The C. maltosa PHM mutant takes up glucose with the kinetics of C. maltosa SBUG 700 and starved cells contain the same cyclic AMP concentration. Addition of glucose to the PHM mutant does not result in an increase of the intracellular cyclic AMP level and in catabolite inactivation of fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. However, addition of 2,4-dinitrophenol is followed by a rapid, transient increase of the cyclic AMP level in the mutant cells, but not by catabolite inactivation. These results show that a common mechanism might be responsible for catabolite inactivation and glucose-induced cAMP signaling or that glucose-induced cAMP signaling is required for catabolite inactivation in C. maltosa.     

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.     

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.     

Glucose kinase-dependent catabolite repression in Staphylococcus xylosus.

By transposon Tn917 mutagenesis, 16 mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild-type strain. The transposons were found to reside in three adjacent locations in the genome of S. xylosus. The nucleotide sequence of the chromosomal fragment affected by the Tn917 insertions yielded an open reading frame encoding a protein with a size of 328 amino acids with a high level of similarity to glucose kinase from Streptomyces coelicolor. Weaker similarity was also found to bacterial fructokinases and xylose repressors of gram-positive bacteria. The gene was designated glkA. Immediately downstream of glkA, two open reading frames were present whose deduced gene products showed no obvious similarity to known proteins. Measurements of catabolic enzyme activities in the mutant strains grown in the presence or absence of sugars established the pleiotropic nature of the mutations. Besides beta-galactosidase activity, which had been used to detect the mutants, six other tested enzymes were partially relieved from repression by glucose. Reduction of fructose-mediated catabolite repression was observed for some of the enzyme activities. Glucose transport and ATP-dependent phosphorylation of HPr, the phosphocarrier of the phosphoenolpyruvate:carbohydrate phosphotransferase system involved in catabolite repression in gram-positive bacteria, were not affected. The cloned glkA gene fully restored catabolite repression in the mutant strains in trans. Loss of GlkA function is thus responsible for the partial relief from catabolite repression. Glucose kinase activity in the mutants reached about 75% of the wild-type level, indicating the presence of another enzyme in S. xylosus. However, the cloned gene complemented an Escherichia coli strain in glucose kinase. Therefore, the glkA gene encodes a glucose kinase that participates in catabolite repression in S. xylosus.     

Glucose effect in tgl mutant of Escherichia col K12 defective in methyl-alpha-D-glucoside transport.

1. The dependence of the rate of accumulation of methyl-alpha-D-glucoside on its extracellular concentration was studied in the tgl mutant of Escherichia coli K12, isolated earlier. It has been shown that the kinetics of methyl-alpha-D-glucoside transport differ sharply from those in wild-type bacteria. 2. The beta-galactosidase synthesis in tgl strain is much less sensitive both to permanent and transient glucose catabolite repression. The level of cyclic AMP in mutant cells under the conditions of glucose catabolite repression is several times higher than in the parent strain. 3. The tgl mutation does not affect the manifestation of catabolite inhibition and inducer exclusion with glucose. 4. The data obtained are discussed in the light of a hypothesis concerning the existence of two sites, binding and pecific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system. The tgl mutation alters the first site, and the second one is damaged by the pgt mutation. 5. It is suggested that the products of the tgl and gpt genes are necessary for the manifestation of the phenomena of glucose permanent and transient repression. The effects of catabolite inhibition and inducer exclusion are realized irrespective of the existence or absence of the tgl product.     

Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.

The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria.