Altered end-product patterns and catabolite repression in Escherichia coli

Dobrogosz, Walter J. (North Carolina State University, Raleigh). Altered end-product patterns and catabolite repression in Escherichia coli. J. Bacteriol. 91:2263-2269. 1966.-End products formed during growth of Escherichia coli ML30 on glucose were examined under various conditions known to promote or prevent catabolite repression of the inducible beta-galactosidase system in this organism. Cultures were grown under these conditions in the presence of C(14)-glucose or C(14)-pyruvate. The products formed were assayed isotopically after separation on columns of silicic acid. Under conditions known to promote catabolite repression, glucose was degraded primarily to acetate and CO(2). When repression was turned off by anaerobic shock, glucose metabolism was characterized by the accumulation of ethyl alcohol in addition to acetate and CO(2). The results presented in this report indicate that oxidative decarboxylation of pyruvate may markedly affect the amount of energy that can be derived from glucose catabolism. In turn, the amount of energy derived from catabolic processes may play a key role in the mechanism of catabolite repression.     

Effect of Amino Sugars on Catabolite Repression in Escherichia coli

N-acetylglucosamine was found to be a good repressor source for catabolite repression of the beta-galactosidase system in Escherichia coli. It was found capable of increasing the severity of repression by glucose or gluconate when included in the medium with either of these substrates. N-acetylglucosamine was shown to be assimilated under these conditions, but had no effect on culture growth rates. Its influence on catabolite repression was not altered by growth in the presence of inhibiting levels of penicillin. These findings indicated that catabolite repression may be associated with certain reactions of amino sugar metabolism. A working model has been formulated along these lines and will be used to explore this possible relationship further.     

Catabolite repression of tryptophanase in Escherichia coli

Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of beta-glactosidase. Induction of tryptophanase and beta-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for beta-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3',5'-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of beta-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, beta-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either beta-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of beta-galactosidase in both strains. Addition of 2.5 x 10(-3)m cAMP appeared partially to reverse the inhibitory effect of the EDTA treatment on enzyme induction but did not restore normal growth.     

Derepression of an NAD-linked dehydrogenase that serves an Escherichia coli mutant for growth on glycerol

An Escherichia coli mutant using an NAD-linked dehydrogenase instead of an ATP-dependent kinase as the first enzyme for glycerol dissimilation excreted dihydroxyacetone during the initial phase of growth. The intermediate was salvaged as growth of the culture advanced. The transient loss of the intermediate into the medium appeared to be partly determined by variation of the level of glycerol dehydrogenase with growth conditions. With up to 2% casein hydrolysate as the carbon and energy source, the cellular level of the dehydrogenase increased 1 order of magnitude at the end of growth. This increase was probably caused by the depletion of certain metabolites and was prevented by the addition of pyruvate or glucose to the growth medium. The repressive effect of these compounds was not lifted by the addition of cyclic AMP. Diminution of oxygen tension in the culture medium with increased cell density was not directly responsible for the increase of the enzyme level. Thus, neither catabolite repression nor respiratory repression was implicated as an important control mechanism in the synthesis of this enzyme. Since increases in the specific activity of the enzyme in cell extracts reflected increases in the concentration of the enzyme protein, post-translational control was also not involved. A novel kind of regulation of gene expression is indicated.     

cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression.

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.     

A quantitative approach to catabolite repression in Escherichia coli

A dynamic mathematical model was developed to describe the uptake of various carbohydrates (glucose, lactose, glycerol, sucrose, and galactose) in Escherichia coli. For validation a number of isogenic strains with defined mutations were used. By considering metabolic reactions as well as signal transduction processes influencing the relevant pathways, we were able to describe quantitatively the phenomenon of catabolite repression in E. coli. We verified model predictions by measuring time courses of several extra- and intracellular components such as glycolytic intermediates, EII-ACrr phosphorylation level, both LacZ and PtsG concentrations, and total cAMP concentrations under various growth conditions. The entire data base consists of 18 experiments performed with nine different strains. The model describes the expression of 17 key enzymes, 38 enzymatic reactions, and the dynamic behavior of more than 50 metabolites. The different phenomena affecting the phosphorylation level of EIIACrr, the key regulation molecule for inducer exclusion and catabolite repression in enteric bacteria, can now be explained quantitatively.     

Effects of aerobic and anaerobic shock on catabolite repression in cyclic AMP suppressor mutants of Escherichia coli.

Cultures of Escherichia coli K-12 grown on glucose or gluconate under aerobic conditions exhibited catabolite repression of beta-galactosidase synthesis. Depression occurred when these cultures were subjected to anaerobic shock. These states of repression and depression were found to be associated with low and high differential rates of cyclic AMP synthesis, respectively. This observation is consistent with the view that cyclic AMP plays a central role in the catabolite repression phenomenon. We report here, however, that identical stages of repression and derepression occur in mutant strains possessing cya crp(Csm) genotypes and therefore unable to synthesize cyclic AMP. These results suggest that cyclic AMP is not the sole regulator involved in catabolite repression.     

Carbon catabolite repression in bacteria.

Carbon catabolite repression (CCR) is a regulatory mechanism by which the expression of genes required for the utilization of secondary sources of carbon is prevented by the presence of a preferred substrate. This enables bacteria to increase their fitness by optimizing growth rates in natural environments providing complex mixtures of nutrients. In most bacteria, the enzymes involved in sugar transport and phosphorylation play an essential role in signal generation leading through different transduction mechanisms to catabolite repression. The actual mechanisms of regulation are substantially different in various bacteria. The mechanism of lactose-glucose diauxie in Escherichia coli has been reinvestigated and was found to be caused mainly by inducer exclusion. In addition, the gene encoding HPr kinase, a key component of CCR in many bacteria, was discovered recently.     

Catabolite repression in Enterococcus faecalis

Metabolism of citrate, pyruvate and sugars by Enterococcus faecalis E-239 and JH2-2 and an isogenic, catabolite derepressed mutant of JH2-2, strain CL4, was investigated. The growth rates of E. faecalis E-239 on citrate and pyruvate were 0.58 and 0.63 h(-1), respectively, indicating that both acids were used as energy sources. Fructose and glucose prevented the metabolism of citrate until all the glucose or fructose had been metabolised. Diauxie growth was not observed but growth on glucose and fructose was much faster than on citrate. In contrast, citrate was co-metabolized with galactose or sucrose and pyruvate with glucose. When glucose was added to cells growing on citrate, glucose metabolism began immediately but inhibition of citrate utilisation did not begin for approximately 1.5 h. Growth rates of E. faecalis JH2-2 and its isogenic, catabolite derepressed mutant, strain CL4, on citrate, were 0.41 and 0.36 h(-1), respectively. The catabolite derepressed mutant was able to co-metabolise citrate and glucose at all concentrations of glucose tested (3-25 mM), while its parent, could only metabolise citrate once all the glucose had been consumed. In strains JH2-2 and E-239, the growth rate on citrate decreased as the glucose concentration increased and, in 25 mM glucose, consumption of citrate was inhibited for several hours after glucose had been consumed. These results indicate that catabolite repression by glucose and fructose occurs in enterococci.     

The role of mitochondria in carbon catabolite repression in yeast

Summary The role of mitochondria in carbon catabolite repression in Saccharomyces cerevisiae was investigated by comparing normal, respiratory competent (RHO) strains with their mitochondrially inherited, respiratory deficient mutant derivatives (rho). Formation of maltase and invertase was used as an indicator system for the effect of carbon catabolite repression on carbon catabolic reactions. Fermentation rates for glucose, maltose and sucrose were the same in RHO and rho strains. Specific activities of maltase and invertase were usually higher in the rho-mutants. A very pronounced difference in invertase levels was observed when cells were grown on maltose; rho-mutants had around 30 times more invertase than their RHO parent strains.The fact that rho-mutants were much less sensitive to carbon catabolite repression of invertase synthesis than their RHO parents was used to search for the mitochondrial factor(s) or function(s) involved in carbon catabolite repression. A possible metabolic influence of mitochondria on this system of regulation was tested after growth of RHO strains under anaerobic conditions (no respiration nor oxidative phosphorylation), in the presence of KCN (respiration inhibited), dinitrophenol (uncoupling of oxidative phosphorylation) and of both inhibitors anaerobic conditions and dinitrophenol had no effect on the extent of invertase repression. KCN reduced the degree of repression but not to the level found in rho-mutants. A combination of both inhibitors gave the same results as with KCN alone. Erythromycin and chloramphenicol were used as specific inhibitors of mitochondrial protein synthesis. Erythromycin prevented the formation of mitochondrial respiratory systems but did not induce rho-mutants under the conditions used. However, repression of invertase was as strong as in the absence of the inhibitor. Chloramphenicol led only to a slight reduction of the respiratory systems and did not affect invertase levels. A combination of both antibiotics had about the same effect as growth in the presence of KCN.The results showed that mitochondria are involved in carbon catabolite repression and they cause an increase in the degree of repression. These effects cannot be due to mere metabolic activities nor to factors made on the mitochondrial protein synthesizing machinery. This regulatory role of mitochondria is observed as long as an intact mitochondrial genome is maintained.     

Regulation of lac Operon Expression: Reappraisal of the Theory of Catabolite Repression

The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of beta-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3',5'-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of beta-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism.     

Synthesis of oxaloacetate in Bacillus subtilis mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex.

Bacillus subtilis mutants deficient in the 2-ketoglutarate dehydrogenase enzymatic complex required aspartate for growth at wild-type rates on carbon sources for which synthesis of the degradative enzymes is sensitive to catabolite repression (e.g., poor carbon sources), but did not require aspartate for growth on carbon sources which exert catabolite repression (e.g., good carbon sources). Measurement of metabolite pools in a mutant lacking the 2-ketoglutarate dehydrogenase active complex showed that the aspartate requirement for growth on poor carbon sources resulted from a deficiency in intracellular oxaloacetate pools even through pyruvate carboxylase was present at levels corresponding to those in wild-type cells. The oxaloacetate deficiency most likely resulted from the inability of the mutant to regenerate oxaloacetate from citrate due to the enzymatic block in the tricarboxylic acid cycle. Mutants in the enzymes of the dicarboxylic acid half of the citric acid cycle similarly required aspartate for wild-type growth in minimal medium. These results suggested that the complete turning of the tricarboxylic acid cycle is involved in the maintainance of oxaloacetate levels in B. subtilis. The ability of the mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex to grow at wild-type rates on media containing good carbon sources in the absence of exogenous aspartate is not understood.     

D-Fucose as a gratuitous inducer of the L-arabinose operon in strains of Escherichia coli B-r mutant in gene araC

d-Fucose, a nonmetabolizable analogue of l-arabinose, prevents growth of Escherichia coli B/r on a mineral salts medium plus l-arabinose by inhibiting induction of the l-arabinose operon. Mutations giving rise to d-fucose resistance map in gene araC and result in constitutive expression of the l-arabinose operon. Most of these mutations also permit d-fucose to serve as a gratuitous inducer. It is concluded that d-fucose-resistant mutants produce an araC gene product with an altered inducer specificity. Addition of l-arabinose to cells induced with the gratuitous inducer, d-fucose, resulted in severe transient repression of operon expression followed by permanent catabolite repression. Transient repression but no permanent catabolite repression was obtained when cells unable to metabolize l-arabinose were employed. It is concluded that transport of l-arabinose alone is sufficient to achieve transient repression of its own operon, but that metabolism of l-arabinose must occur to achieve permanent catabolite repression of the l-arabinose operon. This general effect has been termed "self-catabolite repression."     

Glucose-lactose diauxie in Escherichia coli

Growth of Escherichia coli in medium containing glucose, at a concentration insufficient to support full growth, and containing lactose, is diauxic. A mutation in the gene, CR, which determines catabolite repression specific to the lac operon, was found to relieve glucose-lactose but not glucose-maltose diauxie. Furthermore, a high concentration of lactose was shown to overcome diauxie in a CR(+) strain. Studies on the induction of beta-galactosidase by lactose suggested that glucose inhibits induction by 10(-2)m lactose. Preinduction of the lac operon was found to overcome this effect. The ability of glucose to prevent expression of the lac operon by reducing the internal concentration of inducer as well as by catabolite repression is discussed.     

Catabolite repression and nitrogen control of allantoin-degrading enzymes in Pseudomonas aeruginosa

The formation of the allantoin-degrading enzymes allantoinase, allantoicase and ureidoglycolase in Pseudomonas aeruginosa was found to be regulated by induction, catabolite repression and nitrogen control. Induction was observed when urate, allantoin or allantoate were included in the growth medium, but not with ureidoglycolate. Tricarboxylic acid cycle intermediates exerted catabolite repression of the synthesis of the three enzymes, while pyruvate and glucose caused less repression. The operation of a nitrogen control mechanism in the regulation of the allantoin-degrading enzymes could be demonstrated with glutamine synthetase-negative mutants, which showed elevated synthesis and escape from catabolite repression when growth was limited for glutamine.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Repression of maleate cis-trans isomerase(maleate isomerase) by carbon sources and its reversal were investigated by using Alcaligenes faecalis IB-14.

The formation of maleate isomerase was induced by malonate favorably in a poor medium, whereas it was repressed in a rich medium by carbon sources such as intermediates of TCA cycle. The repression provoked by dl-malate was accompanied with remarkable promotion of the cell growth and with accumulation of a large amount of pyruvate. The enzyme levels of TCA cycle were elevated several times in the dl-malate repressed cells. It was probable to assume that the formation of maleate isomerase was subject to catabolite repression when a rapid and surplus metabolism of dl-malate via TCA cycle was conducted.

So, as an approach to reveal the chemical nature of the catabolite moiety, reversal of the catabolite repression was studied. It was demonstrated that the repression provoked by dl-malate was reversed by various cultural conditions as follows; addition of higher concentrations of malonate, divided supply of dl-malate, “anaerobic” incubation and addition of higher concentrations of ammonium ion. From physiological significances of these events, it was revealed that catabolite repression of maleate isomerase was reversed by minimizing the functioning of TCA cycle.     

Transcriptional analysis of catabolite repression in Clostridium acetobutylicum growing on mixtures of D-glucose and D-xylose

Clostridium acetobutylicum is a strict anaerobic organism that is used for biotechnological butanol fermentation. It ferments various hexoses and pentoses to solvents but prefers glucose presumably using a catabolite repression mechanism. Accordingly during growth on a mixture of D-glucose and D-xylose a typical diauxic growth pattern was observed. We used DNA microarrays and real-time RT-PCR to study gene expression during growth on D-glucose, D-xylose mixtures on a defined minimal medium together with monitoring substrate consumption and product formation. We identified two putative operons involved in D-xylose degradation. The first operon (CAC1344-CAC1349) includes a transporter, a xylulose-kinase, a transaldolase, a transketolase, an aldose-1-epimerase and a putative xylose isomerase that has been annotated as an arabinose isomerase. This operon is induced by D-xylose but was catabolite repressed by D-glucose. A second operon (CAC2610-CAC2612) consists of a xylulose-kinase, a hypothetical protein and a gene that has been annotated as a L-fucose isomerase that might in fact code for a xylose isomerase. This operon was induced by D-xylose but was not subject to catabolite repression. In accordance with these results we identified a CRE site in the catabolite repressed operon but not in the operon that was not subject to catabolite repression.     

Role of the DNA sequence downstream of the Bacillus subtilis hut promoter in regulation of the hut operon

To identify the role of the downstream region of a hut promoter in regulation of the Bacillus subtilis hut operon, three single-base substitutions (+9G-->A, +14C-->T, and +23T-->G) were introduced into the hut operon. Analysis of expression of the hut operon containing each of these three single-base substitutions and the hut-lacZ fusions with the single-base substitutions at position +14 showed that the position at +14 and probably the position at +23 were required for amino acid repression at the hut promoter, while the position at +14 was not required for catabolite repression at the hut promoter. The position at +9 was required for a histidine-dependent increase of activity of the hut promoter. Analysis of expression of the hut-lacZ fusions and the hut operon in the codY mutant indicated that the position at +14 and probably the position at +23 were involved in CodY-mediated amino acid repression at the hut promoter and that CodY was not required for catabolite repression at the hut promoter.