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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↗
PubMed2004

Dynamics and bistability in a reduced model of the lac operon.

It is known that the lac operon regulatory pathway is capable of showing bistable behavior. This is an important complex feature, arising from the nonlinearity of the involved mechanisms, which is essential to understand the dynamic behavior of this molecular regulatory system. To find which of the mechanisms involved in the regulation of the lac operon is the origin of bistability, we take a previously published model which accounts for the dynamics of mRNA, lactose, allolactose, permease and beta-galactosidase involvement and simplify it by ignoring permease dynamics (assuming a constant permease concentration). To test the behavior of the reduced model, three existing sets of data on beta-galactosidase levels as a function of time are simulated and we obtain a reasonable agreement between the data and the model predictions. The steady states of the reduced model were numerically and analytically analyzed and it was shown that it may indeed display bistability, depending on the extracellular lactose concentration and growth rate.

Computer Simulation↗
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↗
PubMed2006

In silico evolved lac operons exhibit bistability for artificial inducers, but not for lactose.

Bistability in the lac operon of Escherichia coli has been widely studied, both experimentally and theoretically. Experimentally, bistability has been observed when E. coli is induced by an artificial, nonmetabolizable, inducer. However, if the lac operon is induced with lactose, the natural inducer, bistability has not been demonstrated. We derive an analytical expression that can predict the occurrence of bistability both for artificial inducers and lactose. We find very different conditions for bistability in the two cases. Indeed, for artificial inducers bistability is predicted, but for lactose the condition for bistability is much more difficult to satisfy. Moreover, we demonstrate that in silico evolution of the lac operon generates an operon that avoids bistability with respect to lactose, but does exhibit bistability with respect to artificial inducers. The activity of this evolved operon strikingly resembles the experimentally observed activity of the operon. Thus our computational experiments suggest that the wild-type lac operon, which regulates lactose metabolism, is not a bistable switch. Nevertheless, for engineering purposes, this operon can be used as a bistable switch with artificial inducers.

Computer Simulation↗
PubMed2005

RNA polymerase structure and function at lac operon.

Transcription of E. coli lac operon by RNA polymerase (RNAP) is a classic example of how the basic functions of this enzyme, specifically the ability to recognize/bind promoters, melt the DNA and initiate RNA synthesis, is positively regulated by transcription activators, such as cyclic AMP-receptor protein, CRP, and negatively regulated by lac-repressor, LacI. In this review, we discuss the recent progress in structural and biochemical studies of RNAP and its binary and ternary complexes with CRP and lac promoter. With structural information now available for RNAP and models of binary and ternary elongation complexes, the interaction between these factors and RNAP can be modeled, and possible molecular mechanisms of their action can be inferred.

Cyclic AMP Receptor Protein↗
PubMedDate not supplied

Mathematical model of the lac operon: inducer exclusion, catabolite repression, and diauxic growth on glucose and lactose.

A mathematical model of the lactose (lac) operon was developed to study diauxic growth on glucose and lactose. The model includes catabolite repression, inducer exclusion, lactose hydrolysis to glucose and galactose, and synthesis and degradation of allolactose. Two models for catabolite repression were tested: (i) cyclic AMP (cAMP) synthesis inversely correlated with the external glucose concentration and (ii) synthesis inversely correlated with the glucose transport rate. No significant differences in the two models were observed. In addition to synthesis, degradation and secretion of cAMP were also included in the model. Two models for the phosphorylation of the glucose produced from lactose hydrolysis were also tested: (i) phosphorylation by intracellular hexokinase and (ii) secretion of glucose and subsequent phosphorylation upon transport back into the cell. The latter model resulted in weak catabolite repression when the glucose produced from lactose was transported out of the cell, whereas the former model showed no catabolite repression during growth on lactose. Parameter sensitivity analysis indicates the importance of key parameters to lac operon expression and cell growth: the lactose and allolactose transformation rates by beta-galactosidase and the glucose concentrations that affect catabolite repression and inducer exclusion. Large values of the allolactose hydrolysis rate resulted in low concentrations of allolactose, low-level expression of the lac operon, and slow growth due to limited import and metabolism of lactose; small values resulted in a high concentration of allolactose, high-level expression of the lac operon, and slow growth due to a limiting concentration of glucose 6-phosphate formed from allolactose. Changes in the rates of all beta-galactosidase-catalyzed reactions showed similar behavior, but had more drastic effects on the growth rate. Changes in the glucose concentration that inhibited lactose transport could extend or contract the diauxic growth period during growth in the presence of glucose and lactose. Moreover, changes in the glucose concentration that affected catabolite repression affected the cAMP levels and lac operon expression, but had a lesser effect on the growth rate.

Biological Transport↗
PubMed2001

An experimental and theoretical study of the inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides.

Previously, we have developed a genetically structured mathematical model to describe the inhibition of Escherichia coli lac operon gene expression by antigene oligos. Our model predicted that antigene oligos targeted to the operator region of the lac operon would have a significant inhibitory effect on beta-galactosidase production. In this investigation, the E. coli lac operon gene expression in the presence of antigene oligos was studied experimentally. A 21-mer oligo, which was designed to form a triplex with the operator, was found to be able to specifically inhibit beta-galactosidase production in a dose-dependent manner. In contrast to the 21-mer triplex-forming oligonucleotide (TFO), several control oligos showed no inhibitory effect. The ineffectiveness of the various control oligos, along with the fact that the 21-mer oligo has no homology sequence with lacZYA, and no mRNA is transcribed from the operator, suggests that the 21-mer oligo inhibits target gene expression by an antigene mechanism. To simulate the kinetics of lac operon gene expression in the presence of antigene oligos, a genetically structured kinetic model, which includes transport of oligo into the cell, growth of bacteria cells, and lac operon gene expression, was developed. Predictions of the kinetic model fit the experimental data quite well after adjustment of the value of the oligonucleotide transport rate constant (9.0 x 10(-)(3) min(-)(1)) and oligo binding affinity constant (1.05 x 10(6) M(-)(1)). Our values for these two adjusted parameters are in the range of reported literature values.

Biological Transport↗
PubMed1984

Isolation and analysis of aroFo mutants by using an aroF-lac operon fusion.

The lac structural genes were fused to the regulatory region of the aroF-tyrA operon so that the expression of beta-galactosidase was regulated by the tyrR+ gene product. Transducing phage carrying the aroF-lac fusion were isolated, and a lambda aroF-lac lysogen was used to select for aroFo mutants. A plasmid vector was constructed onto which the aroFo mutations were transferred by recombination in vivo.

Coliphages↗
PubMed2004

Influence of catabolite repression and inducer exclusion on the bistable behavior of the lac operon.

A mathematical model of the lac operon which includes all of the known regulatory mechanisms, including external-glucose-dependent catabolite repression and inducer exclusion, as well as the time delays inherent to transcription and translation, is presented. With this model we investigate the influence of external glucose, by means of catabolite repression and the regulation of lactose uptake, on the bistable behavior of this system.

Catalysis↗
PubMed1997

The lac operon of Lactobacillus casei contains lacT, a gene coding for a protein of the Bg1G family of transcriptional antiterminators.

The 5' region of the lac operon of Lactobacillus casei has been investigated. An open reading frame of 293 codons, designated lacT, was identified upstream of lacE. The gene product encoded by lacT is related to the family of transcriptional antiterminator proteins, which includes BglG from Escherichia coli, ArbG from Erwinia chrysanthemi, SacT, SacY, and LicT from Bacillus subtilis, and BglR from Lactococcus lactis. Amino acid sequence identities range from 35 to 24%, while similarities range from 56 to 47%. The transcriptional start site of the lac operon was identified upstream of lacT. The corresponding mRNA would contain in the 5' region a sequence with high similarity to the consensus RNA binding site of transcriptional antiterminators overlapping a sequence capable of folding into a structure that resembles a rho-independent terminator. LacT was shown to be active as an antiterminator in a B. subtilis test system using the sacB target sequence. lacT directly precedes lacEGF, the genes coding for enzyme IICB, phospho-beta-galactosidase, and enzyme IIA, and these genes are followed by a sequence that appears to encode a second rho-independent transcription terminator-like structure. Northern hybridizations with probes against lacT, lacE, and lacF revealed transcripts of similar sizes for the lac mRNAs of several L. casei strains. Since the length of the lac mRNA is just sufficient to contain lacTEGF, we conclude that the lac operon of L. casei does not contain the genes of the accessory tagatose-6-phosphate pathway as occurs in the lac operons of Lactococcus lactis, Streptococcus mutans, or Staphylococcus aureus.

Amino Acid Sequence↗
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↗
PubMed2004

Lack of evidence for horizontal transfer of the lac operon into Escherichia coli.

The idea that Escherichia coli gained the lac operon via horizontal transfer, allowing it to invade a new niche and form a new species, has become a paradigmatic example of bacterial nonpathogenic adaptation and speciation catalyzed by horizontal transfer. Surprisingly, empirical evidence for this event is essentially nonexistent. To see whether horizontal transfer occurred, I compared a phylogeny of 14 Enterobacteriaceae based on two housekeeping genes to a phylogeny of a part of their lac operon. Although several species in this clade appear to have acquired some or all of the operon via horizontal transfer, there is no evidence of horizontal transfer into E. coli. It is not clear whether the horizontal transfer events for which there is evidence were adaptive because those species which have acquired the operon are not thought to live in high lactose environments. I propose that vertical transmission from the common ancestor of the Enterobacteriaceae, with subsequent loss of these genes in many species can explain much of the patchy distribution of lactose use in this clade. Finally, I argue that we need new, well-supported examples of horizontal transfer spurring niche expansion and speciation, particularly in nonpathogenic cases, before we can accept claims that horizontal transfer is a hallmark of bacterial adaptation.

Adaptation, Biological↗
PubMed2003

Growth rate regulation of lac operon expression in Escherichia coli is cyclic AMP dependent.

In contrast to the ribosomal RNA gene expression increasing with growth rate, transcription of the lac operon is downregulated by cell growth rate. In continuous culture, growth rate regulation of lac promoter was independent of carbon substrate used and its location on the chromosome. Since the lac operon is activated by cyclic adenosine monophosphate (cAMP), which decreases with increasing cell growth rate, expression of plac-lacZ reporter fusion was analyzed in cya mutant under various growth conditions. The results demonstrated that expression of plac-lacZ in cya mutant was both lower and growth rate independent. In addition, ppGpp (guanosine tetraphosphate) was not involved in the mechanism of growth rate regulation of the lac promoter. Thus, the results of this study indicate that cAMP mediates the growth rate-dependent regulation of lac operon expression in Escherichia coli.

Bacteriophages↗
PubMed1984

Anomalous expression of the E. coli lac operon in Proteus mirabilis. I. Effects of L8 and L8 UV5.

The lac operon shows anomalous expression in Proteus mirabilis: the maximal induced level is 10% or less of that in E. coli, while repression reduces this by a factor of only 2-5. We have sought to determine whether this effect relates in any way to CRP-mediated activation of expression, by comparing expression in P. mirabilis of lac operons (introduced for technical reasons on IncP1 plasmids) either regulatorily wild-type or bearing L8 or L8UV5. Derivatives of RP1 bearing L8UV5 were obtained by homogenotisation of pGC9114 (RP1::Tn951) in a L8UV5 background; while derivatives of RP4 bearing lac+, L8 or L8UV5 were obtained by Mu-mediated translocation of chromosomal regions bearing these alleles, following partial heat-induction of Mucts62 on pGM14 (RP4::Mucts62) in the appropriate hosts. These plasmids could be readily transferred to, and stably maintained in, the P. mirabilis strains employed. It was found that L8 reduced the maximal level of beta-galactosidase activity, and L8UV5 restored this activity to around wild-type, in P. mirabilis quantitatively very much as in E. coli. Nevertheless, the low maximal level of expression and high basal level characteristic of the former host were unchanged. The simplest explanation of these results is that P. mirabilis contains a protein that mimics the E. coli CRP protein in interacting with the appropriate DNA binding site and thereby stimulating transcription; and that the anomalous regulation of lac in this host is unconnected with the CRP system.

Escherichia coli↗