Induction of Nonpigmented Variants of Erwinia herbicola by Incubation at Supraoptimal Temperatures

As with other inducible enzymes, the induced synthesis of l-arabinose isomerase (l-arabinose ketol isomerase, EC 5.3.1.4) in Salmonella typhimurium is subject to catabolite repression. Of the three catabolite repressors tested, glucose produces maximum repression. Analogues of catabolite repressors like 2-deoxy-d-glucose and d-fucose also inhibit the synthesis of the enzyme. The catabolite repression is completely reversed in the presence of 1.5 x 10(-3)m cyclic 3',5'-adenosine monophosphate (AMP). The maximum repression is produced in glucose-grown cells in glucose-containing induction medium. Cyclic 3',5-AMP reverses this repression provided that the cells are treated with ethylenediaminetetraacetic acid (EDTA). In normal cells, cyclic 3',5'-AMP has no effect on the induction but in EDTA-treated cells the cyclic nucleotide enhances synthesis of the enzyme. The inhibition produced by d-fucose cannot be reversed by cyclic 3',5'-AMP. d-Fucose competes with the inducer l-arabinose in some step(s) involved in the process of induction.     

Regulatory properties of araC(c) mutants in the L-arabinose operon of escherichia coliB/r.

Merodiploids containing a high-constitutive and a low-constitutive araC(c) allele were assayed for constitutive expression of the ara operon. Low-constitutive araC(c) alleles either were unable to repress the constitutive rate of ara operon expression exhibited by by high-constitutive araC(c) alleles or achieved a partial repression of the high-constitutive rate of operon expression. Either mutation to a low-constitutive araC(c) mutant resulted in a partial or complete loss of repressor function, or subunit mixing between the two araC(c) mutant proteins resulted in a partial or complete dominance of the high-constitutive araC(c) allele. Five of the six araC(c) alleles tested allowed a partial induction of the ara operon in cya crp background. In general, a higher level of ara operon induction was achieved in the cya crp background by high araC(c) alleles than by low araC(c) alleles. Furthermore, several araC(c) mutants exhibited decreased sensitivity to catabolite repression, particularly in the presence of inducer. The results suggest a model in which certain araC(c) gene products can achieve ara operon induction in the presence of either arabinose (inducer) or catabolite activator protein-cyclic adenosine monophosphate, whereas the wild-type araC gene product requires the presence of both of these factors for operon expression.     

Interaction between mutant alleles of araC of the Escherichia coli B/r L-arabinose operon.

Strains were constructed that contain mutational alterations affecting two distinct functional domains within the araC gene protein. The araCi (catabolite repression insensitivity) and araCh (catabolite repression hypersensitivity) mutations were used to alter the catabolite repression sensitivity domain, and mutation to D-fucose resistance was used to alter the inducer binding domain. araCh, D-fucose-resistant double mutants never exhibited constitutive ara operon expression, whereas all of the araCi, D-fucose-resistant double mutants did exhibit constitutivity. When L-arabinose was used as an inducer, most of the double mutants exhibited the sensitivity to catabolite repression associated with the araCi or araCh mutation. However, when D-fucose was used as an inducer, changes in sensitivity to catabolite repression were observed that were attributed to interactions between the two protein domains. The roles of catabolite activator protein and araC gene protein in the induction of the araBAD operon were discussed.     

Dominance relationships among mutant alleles of regulatory gene araC in the Escherichia coli B/R L-arabinose operon.

The araBAD operon of Escherichia coli B/r is positively and negatively regulated by the araC+ regulatory protein. Mutations in gene araC can result in a variety of different regulatory phenotypes: araC null mutants (those carrying a null allele exhibiting no repressor or activator activity) are unable to achieve operon induction; araC-constitutive (araCc) mutants are partially constitutive, inducible by D-fucose, and resistant to catabolite repression; araCh mutants are hypersensitive to catabolite repression; and araCi mutants are resistant to catabolite repression. Various mutant alleles of gene araC were cloned into a derivative of plasmid pBR322 by in vivo recombination. Various heterozygous araC allelic combinations were constructed by transformation. Analysis of isomerase (araA) specific activity levels under various growth conditions indicated the following dominance relationships with regard to sensitivity to catabolite repression: araCh greater than araC+ greater than (araCc and araCi) greater than araC. It was concluded that the araCh protein may form a repressor complex that is refractory to removal by cyclic AMP receptor protein-cyclic AMP complex. This was interpreted in terms of the known nucleoprotein interactions between ara regulatory proteins and ara regulatory DNA.     

Regulation of the galactose-inducible lac operon and the histidine utilization operons in pts mutants of Klebsiella aerogenes.

Galactose appears to be the physiological inducer of the chromosomal lac operon in Klebsiella aerogenes. Both lactose and galactose are poor inducers in strains having a functional galactose catabolism (gal) operon, but both are excellent inducers in gal mutants. Thus the slow growth of K. aerogenes on lactose reflects the rapid degradation of the inducer. Several pts mutations were characterized and shown to affect both inducer exclusion and permanent catabolite repression. The beta-galactosidase of pts mutants cannot be induced at all by lactose, and pts mutants appear to have a permanent and constitutive inducer exclusion phenotype. In addition, pts mutants show a reduced rate of glucose metabolism, leading to slower growth on glucose and a reduced degree of glucose-mediated permanent catabolite repression. The crr-type pseudorevertants of pts mutations relieve the constitutive inducer exclusion for lac but do not restore the full level of glucose-mediated permanent catabolite repression and only slightly weaken the glucose-mediated inducer exclusion. Except for weakening the glucose-mediated permanent catabolite repression, pts and crr mutations have no effect on expression of the histidine utilization (hut) operons.     

Galactose transport in Saccharomyces cerevisiae. I. Nonmetabolized sugars as substrates and inducers of the galactose transport system

The inducible galactose transport system in bakers' yeast carries out the facilitated diffusion of the nonmetabolized galactose analogues d-fucose and l-arabinose. This capacity depends on the activity of the Ga 2 gene. In some strains, d-fucose and l-arabinose are also gratuitous inducers. Mutants in which the inducibility of the galactose pathway enzymes is altered show a parallel alteration of the inducibility of the galactose transport system.     

Catabolite repression in the D-serine deaminase system of Escherichia coli K-12

The induced synthesis of d-serine deaminase in Escherichia coli is subject to three catabolic effects: inhibition on inducer uptake, transient repression, and catabolite repression. Inhibition on d-serine uptake is not significant at the d-serine concentration normally used for induction. Transient repression and catabolite repression of d-serine deaminase synthesis are abolished by mutations in dsdCy, which appears to be an operator locus. The decline in the rate of constitutive synthesis observed in dsdCx mutants growing with glycerol as carbon source at temperatures above 37 C is due to catabolite repression. The low level of constitutivity at 37 C and the partial cis dominance of dsdCx mutants are not artifacts of catabolite repression. It is suggested that a product of one of the genes of the dsd operon may regulate the expression of the operon.     

Transient Repression of the lac Operon

Severe transient repression of constitutive or induced beta-galactosidase synthesis occurs upon the addition of glucose to cells of Escherichia coli growing on glycerol, succinic acid, or lactic acid. Only mutants particularily well adapted to growth on glucose exhibit this phenomenon when transferred to a glucose-containing medium. No change in ribonucleic acid (RNA) metabolism was observed during transient repression. We could show that transient repression is pleiotropic, affecting all products of the lac operon. It occurs in a mutant insensitive to catabolite repression. It is established much more rapidly than catabolite repression, and is elicited by glucose analogues that are phosphorylated but not further catabolized by the cell. Thus, transient repression is not a consequence of the exclusion of inducer from the cell, does not require catabolism of the added compound, and does not involve a gross change in RNA metabolism. We conclude that transient repression is distinct from catabolite repression.     

Mutations in the L-arabinose operon of Escherichia coli B-r that result in hypersensitivity to catabolite repression

Two independent mutants resistant to l-arabinose inhibition only in the presence of d-glucose were isolated from an l-arabinose-sensitive strain containing the araD139 mutation. Preliminary mapping studies indicate that these mutations are closely linked to the araIOC region. Addition of d-glucose to growing cultures of these mutants results in a 95 to 98% repression of ara operon expression, as compared to a 50% repression of the parental control. Since cultures of both mutant and parental strains undergo a 50% repression of lac operon expression upon addition of glucose, the hypersensitivity to catabolite repression exhibited by these mutants is specific for the ara operon. Addition of cyclic adenosine monophosphate reverses the catabolite repression of the ara operon in both mutant and parent strains to 70 to 80% of the control. It is suggested that in these mutants the affinity of the ara operon initiator region for the cAMP-catabolite-activator protein complex may have been altered.     

Hyperinducibility as a result of mutation in structural genes and self-catabolite repression in the ara operon

Mutations in gene araB producing an l-arabinose-negative phenotype cause either an increase (hyperinducible), decrease (polar), or have no effect at all on the inducible rate of expression of the l-arabinose operon. Fourteen araB gene mutants exhibiting such effects were shown to be the result of: nonsense, frameshift, or missense mutations. All missense mutants were hyperinducible, exhibiting approximately a twofold increase in rate of l-arabinose isomerase production. All frameshift and most nonsense mutants exhibited polar effect. One nonsense mutant was hyperinducible. The cis-dominant polar effect of nonsense and frameshift mutants (as compared to induced wild type) were more pronounced in arabinose-utilizing merodiploids and in araBaraC(c) double mutants where inducible and constitutive enzyme levels are respectively determined. On the other hand, in arabinose-utilizing merodiploids, missense mutations no longer exhibited hyperinducibility but displayed a wild-type level of operon expression. Increases in the wild type-inducible rate of expression of the operon were found when growth rate was dependent on the concentration of l-arabinose. Cyclic 3',5'-adenosine monophosphate also stimulated expression of the operon with the wild type in a mineral l-arabinose medium. These observations are explained on the basis that the steady-state expression of the l-arabinose operon OIBAD is dependent on the concentration of (i) l-arabinose, the effector of this system, which stimulates the expression of the operon, and (ii) catabolite repressors, produced from l-arabinose, which dampen the expression of the operon. We have termed the latter phenomenon "self-catabolite" repression.     

Repression and catabolite gene activation in the araBAD operon.

Catabolite gene activation of the araBAD operon was examined by using catabolite gene activator protein (CAP) site deletion mutants. A high-affinity CAP-binding site between the divergently orientated araBAD and araC operons has been previously identified by DNase I footprinting techniques. Subsequent experiments disagreed as to whether this site is directly involved in stimulating araBAD expression. In this paper, we present data showing that deletions generated by in vitro mutagenesis of the CAP site led to a five- to sixfold reduction in single-copy araBAD promoter activity in vivo. We concluded that catabolite gene activation of araBAD involves this CAP site. The hypothesis that CAP stimulates the araBAD promoter primarily by relieving repression was then tested. The upstream operator araO2 was required for repression, but we observed that the magnitude of CAP stimulation was unaffected by the presence or absence of araO2. We concluded that CAP plays no role in relieving repression. Other experiments showed that when CAP binds it induces a bend in the ara DNA; similar bending has been reported upon CAP binding to lac DNA. This conformational change in the DNA may be essential to the mechanism of CAP activation.     

Oxalurate transport in Saccharomyces cerevisiae.

Oxalurate, the gratuitous inducer of the allantoin degradative enzymes, was taken into the cell by an energy-dependent active transport system with an apparent Km of 1.2 mM. Efflux of previously accumulated oxalurate was rapid, with a half-life of about 2 min. The oxalurate uptake system appears to be both constitutively produced and insensitive to nitrogen catabolite repression. The latter observations suggest that failure of oxalurate to bring about induction of allophanate hydrolase in cultures growing under repressive conditions does not result from inducer exclusion, but rather from repression of dur1,2 gene expression.     

Involvement of the lac regulatory genes in catabolite repression in Escherichia coli

1. Acute transient catabolite repression of beta-galactosidase synthesis, observed when glucose is added to glycerol-grown cells of Escherichia coli (Moses & Prevost, 1966), requires the presence of a functional operator gene (o) in the lactose operon. Total deletion of the operator gene abolished acute transient repression, even in the presence of a functional regulator gene (i). 2. Regulator constitutives (i(-)) also show transient repression provided that the operator gene is functional. Regulator deletion mutants (i(del)), with which to test specifically the role of the i gene, have not so far been available. 3. The above mutants, showing various changes in the lactose operon, show no alteration in the effect of glucose on induced tryptophanase synthesis. Glucose metabolism, as measured in terms of the release of (14)CO(2) from [1-(14)C]glucose and [6-(14)C]glucose, also showed no differences between strains exhibiting or not exhibiting transient repression. This suggests no change in the operation of the pentose phosphate cycle, a metabolic activity known to be of paramount importance for glucose repression of beta-galactosidase synthesis (Prevost & Moses, 1967). 4. Chronic permanent repression by glucose of beta-galactosidase synthesis (less severe in degree than acute transient repression) persists in strains in which transient repression has been genetically abolished. Constitutive alkaline-phosphatase synthesis, which shows no transient repression, also demonstrates chronic permanent repression by glucose. 5. Chloramphenicol repression also persists in mutants with no transient repression, and also affects alkaline phosphatase. It is suggested that chronic permanent repression and chloramphenicol repression are non-specific, and that they do not influence beta-galactosidase synthesis via the regulatory system of the lactose operon.     

Galactose repression of beta-galactosidase induction in Escherichia coli

Beggs, William H. (University of Minnesota, Minneapolis), and Palmer Rogers. Galactose repression of beta-galactosidase induction in Escherichia coli. J. Bacteriol. 91:1869-1874. 1966.-Galactose repression of beta-galactosidase induction in Escherichia coli was investigated to determine whether the galactose molecule itself is the catabolite repressor of this enzyme system. Without exception, beta-galactosidase induction by cells grown in a synthetic salts medium with lactate or glycerol as the carbon source was more strongly repressed by glucose than by galactose. This relationship existed even when the organism was previously grown in the synthetic medium containing galactose as the source of carbon. Two observations suggested that the ability of galactose to repress beta-galactosidase formation by Escherichia coli depends directly upon the cells' capacity to catabolize galactose. First, galactose repression of beta-galactosidase synthesis was markedly enhanced in bacteria tested subsequent to gratuitous induction of the galactose-degrading enzymes with d-fucose. Second, galactose failed to exert a repressive effect on beta-galactosidase in a galactose-negative mutant lacking the first two enzymes involved in galactose catabolism. Glucose completely repressed enzyme formation in this mutant. This same mutant, into which the genes for inducible galactose utilization had been introduced previously by transduction, again exhibited galactose repression. Pyruvate was found to be at least as effective as galactose in repressing beta-galactosidase induction by cells grown in synthetic salts medium plus glycerol. It is concluded that the galactose molecule itself is not the catabolite repressor of beta-galactosidase, but that repression is exerted through some intermediate in galactose catabolism.     

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.     

Regulation of galactose operon expression: glucose effects and role of cyclic adenosine 3',5'-monophosphate.

We studied the following two aspects of the glucose effect on galactose operon expression in Escherichia coli K-12: catabolite repression and inducer exclusion. Using both inducible and constitutive strains and measuring the rate of promoter-proximal enzyme synthesis, we found that the galactose operon did not seem to exhibit catabolite repression. The only glucose effect on galactose operon expression which we observed was inducer exclusion, as shown by the existence of diauxic growth in the presence of glucose and galactose. This diauxie was not relieved by cyclic adenosine 3',5'-monophosphate. Cyclic adenosine 3',5'-monophosphate did not seem to be an antagonist of any glucose effect on galactose operon expression; its only effect was to stimulate promoter-distal gene expression.     

Effect of catabolite repression on the mer operon

The plasmid-determined mer operon, which provides resistance to inorganic mercury compounds, was subject to a 2.5-fold decrease in expression when glucose was administered at the same time as the inducer HgCl2. This glucose-mediated transient repression of the operon was overcome by the addition of cyclic AMP. Permanent catabolite repression of the operon was observed in the 1.6- to 1.9-fold decrease in expression in mutants lacking either adenyl cyclase (cya) or the catabolite activator protein (crp). The effect of the cya mutation on mer expression could be overcome by the addition of cyclic AMP at the time of induction, In addition to these effects on the whole cells of a wild-type strains, we examined the effect of catabolite repression on the expression of the mercuric ion [Hg(II)] reductase enzyme, assayable in cell extracts, and on the Hg(II) uptake system, assayable in a mutant strain which lacked reductase activity. There was a two- to threefold effect of repression on the Hg(II) reductase enzyme assayable in vitro after induction under catabolite repressing conditions (either with glucose or in the crp and cya mutants). We did not find a similar repressing effect on the induction of the Hg(II) uptake system, which is also determined by the mer operon.     

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.     

Production of phenotypically epimeraseless yeast by L-arabinose

The previous report from this laboratory that l-arabinose is a gratuitous inducer of the galactose transport system has been found to be an artefact resulting from the combination of galactose contamination of commercial samples of l-arabinose and inhibition by l-arabinose of galactose metabolism by inactivation of uridine-diphosphate-glucose-4-epimerase. As a result of l-arabinose inhibition of the metabolism of the contaminating galactose, galactose itself serves as a gratuitous inducer, producing phenotypically epimeraseless yeast.     

Constitutive mutations in the controlling site region of the araBAD operon of Escherichia coli B/r that decrease sensitivity to catabolite repression.

Strains of Escherichia coli B/r containing a deletion of the regulatory gene araC are Ara-. Slow-growing revertants of these strains were isolated and designated aralc because they contain a second mutation in a controlling site, aral, that allows for a low level of constitutive expression of the araBAD operon (Englesbert et al., 1969). We mutagenized aralc delta C strains and selected mutants that grow faster in mineral L-arabinose medium. The new mutations, called araXc, map very close to the original aralc mutations and are in the controlling site region between araB and araC. The aralcXc delta C strains have a higher constitutive level of expression of the araBAD operon than the aralc delta C parents. The araXc mutations are cis acting and decrease the araBAD operon's sensitivity to catabolite repression. The araBAD operon is expressed equally well in ara delta C and ara C cya crp backgrounds. The repressor form of ara C protein is able to repress the constitutive synthesis due to the ara Xc allele.