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.     

Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli

Loomis, William F., Jr. (Massachusetts Institute of Technology, Cambridge, Mass.), and Boris Magasanik. Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli. J. Bacteriol. 92:170-177. 1966.-Many carbon sources were found to give rise to catabolite repression of beta-galactosidase in a mutant strain of Escherichia coli lacking hexose phosphate isomerase activity. Compounds containing glucose or galactose cannot be formed from several of these carbon sources in this mutant strain, and, therefore, appear not to be required for catabolite repression of beta-galactosidase. Glucose was observed to elicit catabolite repression of beta-galactosidase in another mutant strain under conditions in which the formation of compounds of the citric acid cycle is inhibited. If catabolite repression of the lac operon is mediated by a single compound, it appears that the compound is related to the pentoses and trioses of intermediary metabolism. The repression of beta-galactosidase by galactose in galactokinase negative strains was shown to be independent of the gene, CR, which determines catabolite sensitivity of the lac operon, and to be dependent on a functional i gene.     

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.     

Corepressor system for catabolite repression of the lac operon in Escherichia coli

Acetylated amino sugars, normally used in the biosynthesis of cell walls and cell membranes, were found to play a role as corepressors for catabolite repression of the lac operon in Escherichia coli. This conclusion was derived from studies conducted on mutants of E. coli that were able to assimilate an exogenous source of N-acetylglucosamine (AcGN) but were unable to dissimilate or grow on this compound. At concentrations less than 10(-4)m, AcGN caused severe catabolite repression of beta-galactosidase synthesis in cultures grown under either nonrepressed or partially repressed conditions. This repression occurred in the absence of any effect of AcGN on either the carbon and energy metabolism or the growth of the organism. In addition, this repression by AcGN occurred in a mutant strain that is constitutive for beta-galactosidase production, demonstrating that the AcGN effect does not involve the uptake of inducer. This model for the corepressor system of catabolite repression is discussed in relation to the existing theories on repression of the lac operon.     

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.     

Catabolite repression of the lac operon. Effect of mutations in the lac promoter

1. Several lac diploid strains of Escherichia coli were constructed and tested to discover whether mutations in the lac promoter alleviate catabolite repression. 2. In each of these diploids the chromosome carries one of the promoter mutations, L8, L29 or L1; so that the rate of synthesis of the enzymes of the lac operon is only 2-6% of the fully induced wild-type. Each diploid harbours the episome F'lacM15 that specifies the synthesis of thiogalactoside transacetylase under the control of intact regulator, promoter and operator regions, but has a deletion in the structural gene for beta-galactosidase. In each diploid more than 90% of the thiogalactoside transacetylase is synthesized from the episome, and 100% of the beta-galactosidase is synthesized from the chromosome, and comparison of the extent of catabolite repression that the two enzymes suffered indicated whether the chromosomal promoter mutation relieves catabolite repression. 3. In the strains in which the promoter carries either of the point mutations L8 or L29 the enzymes were equally repressed, suggesting that neither L8 nor L29 affects catabolite repression. 4. In a diploid strain harbouring the same episome but carrying deletion L1 on the chromosome, synthesis of beta-galactosidase suffered much less repression than that of thiogalactoside transacetylase. 5. In a diploid strain in which the chromosome carries L1 and also a second mutation that increases the rate of expression of lac to that permitted by L8 or L29, the synthesis of beta-galactosidase again suffered much less repression than the synthesis of thiogalactoside transacetylase. 6. The effect of L1 (which deletes the boundary between the i gene and the lac promoter) is ascribed to its bringing the expression of lac under the control of the promoter of the i gene. 7. Even in strains carrying L1, some catabolite repression persists; this is not due to a trans effect from the episome since it occurs equally in a haploid strain with L1.     

Transport regulation of recombinant gene expression in E. coli and B. subtilis

Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances.     

Control of mixed-substrate utilization in continuous cultures of Escherichia coli

The chemostat culture technique was used to study the control mechanisms which operate during utilization of mixtures of glucose and lactose and glucose and l-aspartic acid by populations of Escherichia coli B6. Constitutive mutants were rapidly selected during continuous culture on a mixture of glucose and lactose, and the beta-galactosidase level of the culture increased greatly. After mutant selection, the specific beta-galactosidase level of the culture was a decreasing function of growth rate. In cultures of both the inducible wild type and the constitutive mutant, glucose and lactose were simultaneously utilized at moderate growth rates, whereas only glucose was used in the inducible cultures at high growth rates. Catabolite repression was shown to be the primary mechanism of control of beta-galactosidase level and lactose utilization in continuous culture on mixed substrates. In batch culture, as in the chemostat, catabolite repression acting by itself on the lac enzymes was insufficient to prevent lactose utilization or cause diauxie. Interference with induction of the lac operon, as well as catabolite repression, was necessary to produce diauxic growth. Continuous cultures fed mixtures of glucose and l-aspartic acid utilized both substrates at moderate growth rates, even though the catabolic enzyme aspartase was linearly repressed with increasing growth rate. Although the repression of aspartase paralleled the catabolite repression of beta-galactosidase, l-aspartic acid could be utilized even at very low levels of the catabolic enzyme because of direct anabolic incorporation into protein.     

Paradoxical effect of weak inducers on the lac operon of Escherichia coli

Previously, we reported the existence of a group of compounds whose function in the regulation of the lac operon was "paradoxical" in that they acted as either inducers or repressors depending on the circumstances. We now show that this group of compounds does not repress the lac operon by catabolite repression, transient repression, or by preventing the uptake of inducers. A model is presented which shows that "paradoxical" behavior is to be expected if a weak inducer is present at a concentration that is high relative to its binding affinity for the regulatory macromolecule. This model depends on the assumptions that the regulatory macromolecule is an allosteric protein which undergoes a transition between two conformational states and that the rate of enzyme synthesis depends on the fraction of protein molecules in each state. The previous observations on the responses of lac regulatory mutants to weak inducers have been extended to a series of such mutants. Weak inducers repress beta-galactosidase synthesis in several i(-) mutants. When this happens, enzyme synthesis can be reinduced by using a strong inducer such as isopropyl-beta-d-thiogalactoside. These compounds induce operator constitutives and the i(t) mutant more easily than they induce a wild-type strain.     

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.     

Quality and position of the three lac operators of E. coli define efficiency of repression.

Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation. We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome. We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators. A short distance of 92 bp allows effective repression by two very weak operators (O3, O3). The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration. This increase in concentration depends on flexible DNA which allows loop formation.     

Requirements for Macromolecular Synthesis in the Establishment of β-Galactosidase Repression in Zygotes

Inhibitors of protein synthesis do not consistently prevent formation of the lac operon repressor, according to several published reports, although direct evidence indicates that the repressor is a protein. Inhibition of ribonucleic acid (RNA) synthesis has never been shown to block lactose repression. These results have raised the possibility that repressor is synthesized in some unusual fashion. We have studied the effect of various inhibitors upon the establishment of repression in zygotes, utilizing conditions which minimize catabolite repression. Inhibition of protein synthesis by either chloramphenicol treatment or tryptophan deprivation blocked repressor formation in our experiments. Sodium borate and 6-azauracil are compounds reported to be specific inhibitors of RNA synthesis, and their behavior in control experiments is consistent with this specificity. Both delayed the establishment of repression. Thymine deprivation, either by starvation of a thymine auxotroph or by treatment with 5-fluorodeoxyuridine, did not delay the onset of repression. We conclude that repressor formation requires RNA synthesis and probably utilizes the usual protein-forming mechanisms.     

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.     

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.