Absence of glucose-induced cAMP signaling in the Saccharomyces cerevisiae mutants cat1 and cat3 which are deficient in derepression of glucose-repressible proteins

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae induces a transient, specific cAMP signal. Intracellular acidification in these cells, as caused by addition of protonophores like 2,4-dinitrophenol (DNP) causes a large, lasting increase in the cAMP level. The effect of glucose and DNP was investigated in glucose-repressed wild type cells and in cells of two mutants which are deficient in derepression of glucose-repressible proteins, cat1 and cat3. Addition of glucose to cells of the cat3 mutant caused a transient increase in the cAMP level whereas cells of the cat1 mutant and in most cases also repressed wild type cells did not respond to glucose addition with a cAMP increase. The glucose-induced cAMP increase in cat3 cells and the cAMP increase occasionally present in repressed wild type cells however could be prevented completely by addition of a very low level of glucose in advance. In derepressed wild type cells this does not prevent the specific glucose-induced cAMP signal at all. These results indicate that repressed cells do not show a true glucose-induced cAMP signal. When DNP was added to glucose-repressed wild type cells or to cells of the cat1 and cat3 mutants no cAMP increase was observed. Addition of a very low level of glucose before the DNP restored the cAMP increase which points to lack of ATP as the cause for the absence of the DNP effect. These data show that intracellular acidification is able to enhance the cAMP level in repressed cells. The glucose-induced artefactual increase occasionally observed in repressed cells is probably caused by the fact that their low intracellular pH is only restored after the ATP level has increased to such an extent that it is no longer limiting for cAMP synthesis. It is unclear why the artefactual increases are not always observed. Measurement of glucose- and DNP-induced activation of trehalase confirmed the physiological validity of the changes observed in the cAMP level. Our results are consistent with the idea that the glucose-induced signaling pathway contains a glucose-repressible protein and that the protein is located before the point where intracellular acidification triggers activation of the pathway.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - Mes 4-morpholineethanesulfonic acid     

Growth of Escherichia coli in iron-enriched medium increases HPI catalase activity

Escherichia coli has two catalases, HPI and HPII. HPI is induced during logarithmic growth in response to low concentrations of hydrogen peroxide. This induction is OxyR-dependent. On the other hand, HPII is not peroxide-inducible but is induced in entry to the stationary phase. We demonstrate here that E. coli displayed higher HPI catalase activity when compared to the cultures that were grown in a normal medium, if grown in a medium supplemented with iron-citrate. Iron supplementation had no effect on HPII catalase. This increase of HPI activity was OxyR-independent and not observed in a Deltafur mutant. The physiological significance of the increase of HPI activity is unclear, but it appears that the katG gene that codes for HPI catalase is among the genes that are regulated by Fur.     

Catalases HPI and HPII in Escherichia coli are induced independently

Three strains of Escherichia coli differing only in the catalase locus mutated by transposon Tn10 were constructed. These strains produced only catalase HPI (katE::Tn10 and katF::Tn10 strains) or catalase HPII (katG::Tn10). HPI levels increased gradually about twofold during logarithmic growth but did not increase during growth into stationary phase in rich medium. HPII levels, which were initially threefold lower than HPI levels, did not change during logarithmic growth but did increase tenfold during growth into stationary phase. HPI levels increased in response to ascorbate or H2O2 being added to the medium but HPII levels did not. In minimal medium, any carbon source derived from the tricarboxylic acid cycle caused five- to tenfold higher HPII levels during logarithmic growth but had very little effect on HPI levels. Active electron transport did not affect either HPI or HPII levels.     

The inducible microsomal fatty alcohol oxidase of Candida (Torulopsis) apicola

In the transition phase of Candida apicola IMET 43747 from logarithmic to stationary growth a pyridine-nucleotide-independent alcohol oxidase was induced coinciding with the beginning of sophorose lipid production. This enzyme was not repressed by glucose and was measurable in stationary cells grown on glucose or on a mixture of n-hexadecane and glucose. An NAD⁺-dependent aldehyde dehydrogenase behaved in the same way. Both enzymes were localized in the microsomal fraction. The alcohol oxidase accepted long-chain (fatty) aliphatic alcohols (C₈ to at least C₁₆) and diols starting from decanediol. Trace activities were found with -hydroxy fatty acids. Aromatic, secondary and tertiary alcohols were not oxidized. In the stationary growth phase the substrate specificity of the alcohol oxidase tends to be changed to more hydrophobic substrates. The physiological role of both enzymes, the alcohol oxidase and aldehyde dehydrogenase, is discussed including their possible involvement in the synthesis of sophorose lipid. Correspondence to: R. K. Hommel     

Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.     

The RpoS-Mediated Regulation of Isocitrate Dehydrogenase Gene Expression in Escherichia coli

The Escherichia coli NADP⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42), encoded by an icd gene, is a tricarboxylic acid (TCA) cycle enzyme responsible for the oxidative decarboxylation of isocitrate to α-ketoglutarate. In order to examine how the icd gene expression is regulated, an icd-lacZ reporter fusion was constructed. While the icd gene was induced in exponential growth phase, it was repressed in stationary growth phase. Genetic inactivation of an rpoS gene, whose product is an alternative sigma factor, induced the icd gene expression approximately 4.8 times more in the stationary phase and the IDH enzyme activity in the rpoS mutant was 3.2 times higher than that in the wild type, indicating that the RpoS factor acts as a negative regulator of the icd gene expression in the stationary phase.     

Glucose regulation of specific gene expression is altered in a glucokinase-deficient mutant of Tetrahymena

Summary Expression of the galactokinase gene in Tetrahymena thermophila can be repressed by glucose, glucose analogs, and epinephrine, each apparently acting through increased intracellular levels of adenosine 3′:5′-cyc lic monophosphate (cAMP) (1). To characterize further the initial steps in the control of galactokinase gene-expression by glucose, we have analyzed mutants which are defective in the metabolism of this sugar; these mutants were selected for their resistance to the glucose analog, 2-deoxyglucose (2). In one such mutant that is deficient in glucokinase, the synthesis of galactokinase is totally resistant to repression by glucose or its analogs, while repression by exogenous catecholamines or dibutyryl cAMP is unaffected. Radiochromatographic analyses of extracts of wild-type cells incubated with [¹⁴C]-deoxyglucose reveal intracellular conversio to several deoxyglucose metabolites, principally deoxyglucose-6-P and smaller amounts of deoxyglunose 1-P and 2-deoxygluconate; extracts of glucokinase-deficient cells prepared in a similar manner contain only trace amounts of deoxyglucose-6-P. The glucose analog 3-O-methylglucose, which is transported but not phosphorylated in wild-type cells, also cannot maintain repression of galactokinase. These results establish that the transport and subsequent phosphorylation of glucose are required for glucose-initiated repression of galactokinase gene expression, possibly acting by modulation of catecholamine or cyclic AMP levels. Additionally, we show unequivocally that: (a) cells containing derepressed levels of galactokinase are repressed upon the addition of glucose by inhibition of the synthesis of new enzyme and dilution of preformed enzyme concomitant with cell division, rather than through selective inactivation or degradation of galactokinase; and (b) glycerol kinase, glucokinase and fructokinase activities also are repressed by glucose in wild-type Tetrahymena, indicating that the glucose repression phenomenon is pleiotropic. Because the glucose repression of the synthesis of each of these enzymes is abolished in cells deficient in glucokinase, the regulatory mechanisms elucidated for repression of galactokinase synthesis are likely to be of wide significance.     

Pitfalls in the measurement of membrane potential in yeast cells using tetraphenylphosphonium

The uptake of the lipophilic cation tetraphenylphosphonium (Ph₄P⁺) by Saccharomyces cerevisiae was measured using yeast grown on glucose and harvested either at the logarithmic or at the stationary phase of growth. When yeast was collected at the stationary phase, Ph₄P⁺ uptake proceeded steadily during several hours until an equilibrium was reached. When yeast was collected in the logarithmic phase of growth, a biphasic uptake was observed. The second phase of uptake began when the glucose of the incubation medium had been exhausted. From experiments in the presence of cycloheximide or chloramphenicol it is concluded that the second phase of Ph₄P⁺ uptake is dependent on the synthesis of some protein(s) repressed by glucose but unrelated with the existence of functional mitochondria. The addition of compounds which collapse the membrane potential provokes an efflux from the yeast cells of the Ph₄P⁺ accumulated both during the first phase and the second phase of uptake. It is concluded that accumulation of Ph₄P⁺ in yeast cells is a complex process and that Ph₄P⁺ cannot be used to give a quantitative measure of the yeast plasma membrane potential.     

Cytochrome P-450 in the sophorose-lipid-producing yeast Candida (Torulopsis) apicola

The appearance of cytochrome P-450 and of cytochrome oxidase aa₃ were determined in the sophorose lipid producing yeast Candida (Torulopsis) apicola IMET 43 747 grown on a mixture of glucose and n-hexadecane. Cytochrome P-450, detectable in both the logarithmic and the stationary growth phase was not repressed by glucose. At the end of the logarithmic growth phase the content of cytochrome P-450 was three- to fivefold increased, which was connected with initiation of sophorose lipid biosynthesis. After that it dropped to the basal level, which remained constant during sophorose lipid biosynthesis. Cytochrome P-450 from logarithmic cells was cross-reactive with an antibody derived against cytochrome P-450alk from C. tropicalis. With microsomal proteins of stationary cells no cross-reactivity was obtained. The microsomal hydroxylase system of stationary cells seem to be regulated by the carbohydrate used as carbon source. Correspondence to: R. K. Hommel     

DYNAMICS OF CELLULAR AND EXTRACELLULAR cAMP IN ANABAENA FLOS-AQUAE (CYANOPHYTA): INTRINSIC CULTURE VARIABILITY AND CORRELATION WITH METABOLIC VARIABLES1

The production and extracellular release of cyclic adenosine 3′: 5′-monophosphate (cAMP) by the blue-green alga Anabaena flos-aquae (Lyngb.) Breb. varied greatly within and between active growth phase and stationary phase and under differing nutrient regimes. Enhanced cellular cAMP production was found in actively growing Anabaena inoculated into media deficient in nitrate or phosphate, or into fresh media containing non-limiting nutrient concentrations. In stationary phase Anabaena, but not actively growing cells, the concentrations of intra-cellular cAMP present in cells grown under a variety of nutrient regimes could be significantly correlated to [¹⁴C]-bicarbonate uptake by an exponential relationship.     

Alkaline phosphatase production during sporulation ofBacillus cereus

Cell-bound alkaline phosphatase ofBacillus cereus was produced during vegetative growth and sporulation in a complex medium. Addition of glucose repressed the sporulation process and the amount of enzyme synthesized increased. The time course of alkaline phosphatase production is very similar in both sporulating and non-sporulating cells. Irrespective of sporulation, alkaline phosphatase level shows a peak of activity in the exponential phase, and another in the stationary phase of growth. This preliminary data indicates differences betweenB. cereus, andB. subtilis in alkaline phosphatase characteristics.     

Caloric restriction controls stationary phase survival through Protein Kinase A (PKA) and cytosolic pH

Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pH_c) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pH_c and the central nutrient signalling cAMP‐Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pH_c correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pH_c showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.     

Synthesis of the arginine dihydrolase pathway enzymes inLactobacillus buchneri

The formation of the arginine dihydrolase pathway enzymes inLactobacillus buchneri NCDO₁₁₀, a heterofermentative organism, was investigated. The specific activities of arginine deiminase, ornithine transcarbamylase, and carbamate kinase were higher in galactose-grown cells than in glucose- or sucrose-grown cells in the early stationary phase of growth. The addition of arginine to growing cells increased the specific activity of these three enzymes with all growth sugars. The specific activities of the enzymes decreased during the stationary phase of growth when the sugar-grown cells was galactose. When glucose was virtually exhausted from the medium, the activities of the three enzymes were not altered. This enzymic system was not repressed by glucose, and these results are different from those obtained withL. leichmanni, homofermentative organism.Dedicated to Dr. Luis F. Leloir on the occasion of his 80th birthday, 6 September 1986.Member of the Scientific Researcher's Career of theConsejo Nacional de Investigaciones Cientificas Ténicas (CONICET) Argentina.     

A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon

Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP‐amy‐pgm genes. Expression of the glg operon and glycogen accumulation were carbon source‐ and growth phase‐dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log‐phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose‐grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen‐branching enzyme) mutants are glycogen‐deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.     

Succinate-mediated catabolite repression of enzymes of glucose metabolism in root-nodule bacteria

Enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways were detected in strains ofRhizobium andBradyrhizobium cultured on glucose. The enzymes, except glyceraldehyde-3-phosphate dehydrogenase, were present only in trace amounts in succinategrown cells. The enzymes of the pentose phosphate pathway, being absent inBradyrhizobium, were detected only in glucose-grown cells ofRhizobium. The presence of the glucose-catabolic enzymes in cells only during growth on glucose suggests that they are inducible in nature. Succinate repressed the glucose catabolic enzymes, and the repression appeared to be similar to catabolite repression. Exogenous addition of cAMP caused no change in the activity of these enzymes, demonstrating that the repression was unlikely to be mediated via cAMP.