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Results for “Lac Operon”

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At least 19 recordsLinked to original sources

PubMed1989

Clonal analysis of the lac operons from Klebsiella M5al and the Lac plasmid (pRE2) from Klebsiella V9A.

The chromosomal lac region of the coliform bacterium Klebsiella M5al was cloned into the multicopy plasmid pBR322 to give pHE7 and pHE8. pHE8 contains 12.6 kb of M5al DNA, including its complete lac operon, and pHE7 contains 2.5 kb of M5al DNA and includes the complete lac Y gene and a small segment of lacZ. The M5al operon has the same gene order as the Escherichia coli lac operon. The lac genes of the Lac plasmid of Klebsiella V9A were cloned into pBR322 to give pHE1 and pHE2, of approximately 39 and 43 kb. Both plasmids were unstable in an E. coli RecA-strain, in contrast to the stability of pHE8. Polyacrylamide gel electrophoresis tests suggested that the M5al beta-galactosidase monomer is about 5% longer, i.e. has about 50 more amino acids, than that of the E. coli Z gene. Tests made on the enzymes coded by the lac operons of M5al, another Klebsiella strain (V9A) and its resident Lac plasmid, and several Lac+ Enterobacteria, led to the conclusion that only Escherichia coli among the Enterobacteria contains an active lacA gene.

Acetyltransferases↗

PubMed1970

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.

Acyltransferases↗

PubMed1977

Physiological regulation of a decontrolled lac operon.

The expression of the lac operon was studied under a variety of growth conditions in induced and in constitutive cells of Escherichia coli that carried different catabolite-insensitive lac promoters. Use of such "decontrolled" lac operons permitted a study of the expression of an operon that was presumably subject only to passive control. Since the use of toluenized cells was demonstrated not to be completely reliable, all enzyme assays were performed on sonic supernatant fluids. The cells contained different catabolite-insensitive promoters, which included the L1 and UV5 lac promoters, as well as others isolated in this study. There were three major observations. First, small but real carbon source effects were seen. Second, there was only a small change in beta-galactosidase specific activity with changes in the growth rate. This result implies a limited transcription and/or translation capacity within the cell. Third, at rapid growth rates, most promoters exhibited a decreased expression. The UV5 promoter, which was the "strongest" promoter, was an exception. A mechanism to explain this promoter-dependent control is discussed.

Acetylesterase↗

PubMed1988

[Study of the expression of the cya gene in Escherichia coli K12 on a model of the hybrid cya-lac operon].

Bacteriophage Mud1 (Ap, lac) was used to construct the cya-lac operon fusion in Escherichia coli chromosome. The maximal cya expression was observed by measuring the levels of beta-galactosidase in the strains lacking the cAMP. The inhibition of cya transcription was proportional either to the amount of cAMP added or to the copy number of cya gene in the cell. Functional cAMP receptor protein (CRP) was necessary for the effect. The cya gene expression was low in cAMP-independent crp mutant even in the absence of the cAMP.

Cloning, Molecular↗

PubMed1990

The three operators of the lac operon cooperate in repression.

We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one 'pseudo-operator' O2 or O3 decreases repression by wild-type tetrameric Lac repressor approximately 2- to 3-fold; absence of both 'pseudo-operators' decreases repression greater than 50-fold. O1 alone represses under these conditions only approximately 20-fold. Dimeric active Lac repressor (iadi) represses the wild-type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.

Base Sequence↗

PubMed1986

opp-lac Operon fusions and transcriptional regulation of the Escherichia coli trp-linked oligopeptide permease.

The transcriptional regulation of the Escherichia coli trp-linked opp operon that encodes the oligopeptide permease was investigated by using lambda plac Mu51-generated lac operon fusions. Synthesis of beta-galactosidase by strains harboring oppA-lac, oppB-lac, and oppD-lac fusions occurred at a basal level when the fusion-containing strains were grown in minimal medium. The addition of L-leucine or L-alanine to exponentially growing, aerobic cultures or shifting the aerobic fusion-containing strains to anaerobic growth medium increased the synthesis of beta-galactosidase from all opp-lac fusions. When transcription of the opp operon was induced by L-leucine, the differential rate of beta-galactosidase synthesis from each opp-lac fusion increased 8- to 10-fold; this increased rate of lacZ expression from the opp-lac fusions resulted in a 5- to 6-fold increase in total beta-galactosidase activity after maximum expression was achieved. Importantly, when F'123 derivatives harboring independently isolated E. coli opp-lac operon fusions were introduced into E. coli and Salmonella typhimurium, the data clearly demonstrated that the E. coli opp operon was expressed identically and responded to the same transcriptional regulatory signals in both E. coli and S. typhimurium. A comparison of beta-galactosidase synthesis by E. coli strains harboring an opp-lac operon fusion and either an oppE+ locus or an oppE mutation demonstrated that the reduction in peptide transport produced by the oppE mutation does not result from a decrease in the level of opp operon transcription.

Alanine↗

PubMed1969

Catabolite repression of the lac operon. Separt epressionof two enzymes.

1. Catabolite repression of beta-galactosidase and of thiogalactoside transacetylase was studied in several strains of Escherichia coli K 12, in an attempt to show whether a single site within the structural genes of the lac operon co-ordinately controls translational repression for the two enzymes. In all experiments the rate of synthesis of the enzymes was compared in glycerol-minimal medium and in glucose-minimal medium. 2. In a wild-type strain, glucose repressed the synthesis of the two enzymes equally. 3. The possibility that repression was co-ordinate was investigated by studies of mutant strains that carry deletions in the genes for beta-galactosidase or galactoside permease or both. In all of the strains with deletions, the repression of thiogalactoside transacetylase persisted, and it is concluded that there is no part of the structural gene for beta-galactosidase that is essential for catabolite repression of thiogalactoside transacetylase. 4. Subculture of one strain through several transfers in rich medium greatly increased its susceptibility to catabolite repression by glucose. It is concluded that unknown features of the genotype can markedly affect sensitivity to catabolite repression. 5. These results make it clear that one cannot draw valid conclusions about the effect of known genotypic differences on catabolite repression from a comparison of two separate strains; to study the effect of a particular genetic change in a lac operon it is necessary to construct a partially diploid strain so that catabolite repression suffered by one lac operon can be compared with that suffered by another. 6. Four such partial diploids were constructed. In all of them catabolite repression of beta-galactosidase synthesized by one operon was equal in extent to catabolite repression of thiogalactoside transacetylase synthesized by the other. 7. Taken together, these results suggest that catabolite repression of beta-galactosidase and thiogalactoside transacetylase is separate but equal.

Acyltransferases↗

PubMed1987

Specific destruction of the second lac operator decreases repression of the lac operon in Escherichia coli fivefold.

The second operator of the lac operon, located within the 5'-coding region of the lacZ gene, was specifically destroyed by means of oligonucleotide-directed mutagenesis. Eight of its bases were exchanged without altering the wild-type amino acid sequence of beta-galactosidase. The mutation was transferred onto an F'lac+I+O+Z+pro+ episome. We observed a fivefold decrease in repression of beta-galactosidase expression compared to that in the wild-type.

Enzyme Repression↗

PubMed1968

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.

Enzyme Induction↗

PubMed1986

[Construction of the hybrid crp-lac operon and study of the role of CRP-cAMP complex in its regulation in Escherichia coli].

Phage Mu d1(Ap,lac) was used to construct the hybrid crp-lac operon in Escherichia coli cells possessing crps mutation which increases sensitivity of bacterial growth to exogenous cAMP. Transcription of the structural lac genes in this hybrid operon was initiated from the promoter region of the crp gene. By measuring levels of beta-galactosidase enzyme, the maximal transcription of the crp gene was obtained in strains with cya or crp mutations causing inactivation of the CRP - cAMP complex. Restoration of cya+crp+ genotype resulted in 5 to 7 times decrease of the lacZ gene activity. At the concentration of exogenous cAMP which induces catabolic derepression in the wild-type strain, the expression of crp-lac operon increased 2-3 times. Using F'ts114lac-mediated chromosome mobilization, the direction of transcription of the crp gene was found to be counterclockwise, relative to the E. coli genetic map.

Escherichia coli↗

PubMed1992

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and beta-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.

Amino Acid Sequence↗

PubMed1969

Catabolite repression of the lac operon. The contribution f trascriptional repression.

1. Experiments were carried out to distinguish the contributions of transcriptional and translational repression to catabolite repression of the lac operon. 2. In strain EZ16-3-G of Escherichia coli the synthesis of thiogalactoside transacetylase is directed by a gene situated on an episome, and the operator, promotor and regulator genes that lay cis to this gene have been deleted, so that the normal mechanism for controlling transcription is abolished. The extent of catabolite repression in this strain was much less than that in wild-type strains. 3. The same episome is responsible for the synthesis of thiogalactoside transacetylase in strain RM32/F'd25, and in this strain a second lac operon directs the synthesis of beta-galactosidase under the control of a wild-type operator-promotor-regulator system. The extent of catabolite repression of thiogalactoside transacetylase in strain RM32/F'd25 was substantially more than in strain EZ16-3-G, but less than that of beta-galactosidase in strain RM32/F'd25. 4. Since the synthesis of thiogalactoside transacetylase in these organisms is presumably subject to translational repression only, it is concluded that in strain RM32/F'd25 the synthesis of beta-galactosidase is subject to both transcriptional and translational repression. It is also concluded that the extent of translational repression varies between strains.

Acyltransferases↗

PubMedDate not supplied

Detection in vivo of protein-DNA interactions within the lac operon of Escherichia coli.

Studies of the sequence-specific binding of proteins to DNA have so far relied on in vitro experiments using cloned restriction fragments containing the relevant DNA sequences. We have applied the genomic sequencing technique of Church and Gilbert to show that the interactions observed in vitro occur in vivo. We use this approach to study the binding of regulatory proteins to the lac operon in vivo and detect changes in the reactivity (inhibition or enhancement) of guanines to methylation by dimethyl sulphate caused by the proximity of proteins to the N-7 atom of these guanines. We can detect the simultaneous binding of the catobolite gene activator protein (CAP) and the Lac repressor to their specific recognition sequences, and following induction of the lac operon we observe effects that are related to RNA polymerase binding or RNA elongation. We have successfully used oligonucleotide probes as short as 17 bases to display genomic sequence.

Base Sequence↗

PubMed1988

ack::Mu d1-8 (Apr lac) operon fusions of Salmonella typhimurium LT2.

Several ack::Mu d1-8 insertions were isolated from Salmonella typhimurium on MacConkey-glucose-trimethylamine oxide medium. They could not produce gas from glucose in the absence of exogenous formate. Two of the mutants expressed beta-galactosidase activity and were shown to be ack::Mu d1-8 fusions by genetic analysis. These insertions were characterized and located on the genetic map at the ack locus by co-transduction with zei::Tn10. The gene order zei::Tn10-ack-pta was established. The direction of transcription of ack was clockwise on the genetic map. Expression of the lac operon in ack::Mu d1-8 was increased twofold by anaerobiosis. Addition of formate, pyruvate, and acetate did not affect the anaerobic expression of the lac operon in ack::Mu d1-8.

Acetate Kinase↗

PubMed1991

Genomic hsd-Mu(lac) operon fusion mutants of Escherichia coli K-12.

Genomic (chromosomal)hsd-Mu(lac) operon fusions have been constructed in two strains of Escherichia coli K-12 for the three hsd genes, hsdRK, hsdMK and hsdSK, using MudX and lambda placMu53. Expression of hsdK mutants ranged from 16 to 74 units (u) (with a mean of 52 u) for fusions to promoter pres and ranged from 26-75 u (also with a mean of 52 u) for fusions to promoter pmod. The expression of the two hsdK promoters was measured in different stages of growth. The pres fusion mutant showed a lag in beta-galactosidase (beta Gal) production, as compared to the pmod fusion mutant. One r-Km-K mutant (JR205) showed more than ten times the beta Gal activity of other insertion mutants. The activity of this mutant decreased by 20-fold upon the transfer of F101-102, which includes the wild-type hsd region. Positive gene-dosage effect was observed using F' plasmids containing the hsd-lacZ region.

Bacteriophage mu↗

PubMed1972

Polycistronic effects of catabolite repression on the lac operon.

The catabolite repression caused by glucose and glucose-6-phosphate has been studied for both beta-galactosidase and thiogalactoside transacetylase, the products of the operator proximal and distal cistrons of the lac operon, respectively. We find that both cistrons are affected coordinately by this form of repression. We also find that a single alteration at the lac promoter region is sufficient to abolish sensitivity to repression of both cistrons. From this, we conclude that there is only one target site for catabolite repression in the lac operon.

Acetyltransferases↗

PubMed1969

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.

Carbon Isotopes↗

PubMed1979

Tight-binding repressors of the lac operon: selection system and in vitro analysis.

The isolation and characterization of altered repressors of the lac operon which have an increased affinity for an operator should give useful clues about the molecular basis for the very tight and specific interaction between repressor and operator. A selection system has been devised which allows the isolation of such repressor mutants. This system selects for mutant repressors which can overcome lac operator-constitutive (Oc) mutations. By using in vivo assays, 24 candidates were obtained which, compared with wild type, have an increased trans effect of their repressor on one or several Oc operators. Three of these candidates have been investigated in vitro; the affinity of their repressor for inducer was unchanged, whereas the affinity for wild-type operator was increased 15-, 86-, and 262-fold, respectively.

Bacterial Proteins↗