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At least 109 records · Page 6Linked to original sources
PubMed1985

Effect of iso-propyl-thio-beta-D-galactoside concentration on the level of lac-operon induction in steady state Escherichia coli.

In steady state E. coli cells growing at their maximal rate in broth, maximum induction of beta-galactosidase occurs at 0.10 mM isopropyl-thio-beta-D-galactoside (IPTG). Although induction of lac is near zero in steady state cells that are growing in 0.01 mM IPTG, induction at mildly subdued levels persists down to at least 0.001 mM in post-steady state cultures. Meanwhile, thiogalactoside transacetylase remains uninduced over the full range in which the cells are in steady state.

Acetyltransferases↗
PubMed1983

Specific DNA binding of the cAMP receptor protein within the lac operon stabilizes double-stranded DNA in the presence of cAMP.

The effects of varying amounts of cAMP receptor protein (CRP) in the presence and absence of cAMP on the melting and differential melting curves of a 301-bp fragment containing the lac control region in 5 mM Na+ have been investigated. The native 301-bp fragment consists of three cooperatively melting thermalites. At 5 mM Na+, thermalite I (155 bp) has a Tm of 66.4 degrees C and the melting transitions of thermalites II (81 bp) and III (65 bp) are superimposed with a Tm of 61.9 degrees C. The specific DNA target site for CRP and the lac promotor are located within thermalite II. CRP alone exerts no specific effects on the melting of the 301-bp fragment, non-specific DNA binding of CRP resulting in a progressive stabilization of the double-stranded DNA by increasing the number of base pairs melting at a higher Tm in a non-cooperative transition. The cAMP-CRP complex, however, exerts a specific effect with a region of approximately 36 bp, comprising the specific CRP binding site and a neighbouring region of DNA, being stabilized. The appearance of this new cooperatively melting region, known as thermalite IV, is associated with a corresponding decrease in the area of thermalites II/III. The Tm of thermalite IV is 64.4 degrees C, 2.5 degrees C higher than that of thermalites II/III. With two or more cAMP-CRP complexes bound per 301-bp fragment, the stabilization also affects the remaining 110 bp now making up thermalites II/III whose Tm is increased by 1 degrees C to 62.9 degrees C. The implications of these findings for various models of the mode of action of the cAMP-CRP complex are discussed.

Cyclic AMP↗
PubMed1981

In vivo role of the relA+ gene in regulation of the lac operon.

Under conditions of amino acid limitation, beta-galactosidase was produced at a 70-fold higher rate in a relA+ strain than in an isogenic relA strain of Escherichia coli K-12. Under identical conditions with the relA+ and relA strains carrying various lac promoter mutations, rates of beta-galactosidase synthesis in relA+ (relative to relA) ranged from 26-fold higher (promoter mutant Pr 13) to only 5-fold higher (promoter mutant PrL8uv5). This promoter specificity was independent of strain background and the means of eliciting amino acid limitation. Addition of cyclic AMP to the growth medium altered the relA+/relA difference for beta-galactosidase synthesis from the wild-type lac promoter. The experiments suggest that the relA+/relA difference in lac expression arises primarily at the point of transcription initiation. The results are discussed in relation to recent in vitro data showing a promoter-specific guanosine 5'-diphosphate 3'-diphosphate stimulation of lac transcription (P. Primakoff and S. W. Artz, Proc. Natl. Acad. Sci. U.S.A. 76:1726-1730).

Cyclic AMP↗
PubMed1998

Evidence that gene amplification underlies adaptive mutability of the bacterial lac operon.

Adaptive mutability is the apparent alteration in specificity or rate of mutability seen in bacteria during stress. A model is proposed by which gene amplification during selective growth can give the appearance of adaptive mutability without requiring any change in mutability. The model is based on two assumptions, that a mutant lac locus with residual function allows growth if its copy number is increased, and that true reversion events are made more likely by replication of chromosomes with many copies of the locus. Apparent directed mutability, its recombination requirement, and its apparent independence of cell growth are all accounted for by the model. Evidence is provided for the required residual function and gene amplification.

Adaptation, Physiological↗
PubMed1980

Lactose carrier protein of Escherichia coli. Structure and expression of plasmids carrying the Y gene of the lac operon.

The previously described hybrid plasmid pC7 which carries lacI+O+delta(Z)Y+A+ on a 12.3 X 10(6)-Mr DNA fragment [Teather et al. (1978) Mol. Gen. Genet. 159, 239-248] was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to d(A-A-T-T) and ligated to the vector pB322. lac Y-carrying inserts of various sized (Mr 1.5-4.7 X 10(6)) were obtained. Hybrid plasmid pTE18 (2300-base-pair insert) carries part of the I (repressor) gene, the promotor-operator region, part of the Z (beta-galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18-harbouring strains the Y-gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12-16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.

Bacterial Proteins↗
PubMed1992

Inducible transformation of cells from transgenic mice expressing SV40 under lac operon control.

If it were possible to clone in vitro cells of any type, at any stage of differentiation, from an extensively characterized animal such as the mouse, many areas of cell biology would benefit. Indeed, it would be even more helpful if these cells could subsequently be restored to their normal in vivo phenotype whenever required. Here, we describe a step on the pathway to such an idealized "clonable" mouse. In principle, it seeks to link a "universal" transforming agent to a regulatory system that is relatively simple, yet quite foreign to the mouse. A plasmid containing the bacterial lac operator/promoter region linked to the SV40 large T antigen and a vector containing the lac repressor that can be expressed in mammalian cells were coinjected into fertilized mouse oocytes utilizing the standard techniques for generating transgenic mice. Two progeny were obtained that express large T antigen in the presence, but not the absence, of the nonmetabolizable lac inducer, isopropyl-beta-thio-D-galactoside. This report characterizes fibroblast cell lines established from these transgenics that are readily transformed in vitro with isopropyl-beta-thio-D-galactoside. A significant proportion of the cells are restored to their "normal" (nontransformed phenotype) when isopropyl-beta-thio-D-galactoside is removed.

Animals↗
PubMed1985

Regulatory region of the divergent Klebsiella pneumoniae lac operon.

The chromosomal DNA that lies between the lacI and lacZ genes of Klebsiella pneumoniae constitutes a 196-base pair intercistronic region that contains regulatory sequences for both genes. The probable locations of specific regulatory elements for both lacI and lacZ genes were determined by analogy with the corresponding Escherichia coli sequences. A recombinational event in ancestral DNA evidently has inverted the transcriptional direction of lacI in K. pneumoniae relative to the transcriptional direction of lacI in E. coli. One end of the inversion was located within a 19-base pair sequence in the K. pneumoniae regulatory region. Sequences partially homologous to these 19 base pairs were found in two locations on either side of the E. coli lacI gene. The nucleotide sequence of the lac regulatory region in K. pneumoniae exhibits more than one possibility for folded tertiary structures. The spatial relationships of transcriptional binding sites differ in two possible structures. Associations of regulatory and transcriptional proteins with the DNA might affect conformation of the regulatory sequences and, as a consequence, transcription of the lac genes.

Base Sequence↗
PubMed1979

[Effect of the dose of ptsI- and ptsH-genes on carbohydrate transport and regulation of lac-operon activity in Escherichia coli K-12].

Phage Mu-1 cts61 was used for transposition of pts1 and ptsH genes. The received F'-factors AUF2 and AUF3 carry short fragments of the bacterial chromosome. Merodiploid strains with double pts genes were selected in sexduction crosses with the appropriate recA recipients. Effect of the gene dose was not registered in pts+/pts+ strains in the case of accumulation of the substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and in the case of bacterial growth in the presence of these carbohydrates. This indicates that the enzyme (enzymes) II of the PTS is the limiting step in the transpost process. Induction of beta-galactosidase and the growth on carbohydrates not transported via the PTS (maltose, lactose) were greatly reduced in pts mutant. Introduction of the pts+ allele with episome lead to the restoration of the two above processes. These data show that the phospho approximately HPr generating system of the PTS is directly (or in indirect manner) involved in the regulation of catabolite-sensitive operons. Glucose repression was markedly increased in pts+/pts+ merodiploids as compared with pts+/pts- ones and with pts+ bacteria. Possible mechanisms of this effect are discussed.

Bacteriophage mu↗
PubMed1979

Positive control of lac operon expression in vitro by guanosine 5'-diphosphate 3'-diphosphate.

Maximal expression of the Escherichia coli lactose operon in a coupled in vitro transcription-translation system from a Salmonella typhimurium relA mutant was strongly dependent upon addition of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Without added ppGpp, at saturating 3',5'-cyclic AMP (cAMP) concentrations, synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was reproducibly only 5-7% of that which can be obtained with 0.5-0.8 mM ppGpp. Experiments in which transcription was uncoupled from translation indicated that this 14- to 20-fold stimulation by ppGpp occurred at the level of transcription. When coupled beta-galactosidase synthesis was primed with a template containing a well-characterized mutant lac promoter (lacP(r)L8UV5), the dependence on ppGpp was greatly reduced. This result provides an important experimental control previously unavailable for verifying the significance of ppGpp effects on gene regulation in vitro; it indicates that activation of lacP(+) expression by ppGpp is specifically an effect of increased transcription initiations. Furthermore, the large ppGpp stimulation of lacP(+) DNA enabled the level of expression of this template to approach that of lacP(r)L8UV5 DNA, an observation expected from results in vivo but not obtained with other transcription-translation systems in vitro. The importance of these results is considered with respect to previous ideas on the physiological role of ppGpp as a supercontrol molecule in bacterial regulation.

Alcohol Oxidoreductases↗
PubMed1993

Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains.

Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate.

Biological Transport↗
PubMed1995

Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP.

CRP-cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene (crp*). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of beta-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of beta-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS-PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp* mRNA levels. The level of CRP* protein correlates with beta-galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp* expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya- crp* cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose.

Adenylyl Cyclases↗
PubMed2003

Trp-999 of beta-galactosidase (Escherichia coli) is a key residue for binding, catalysis, and synthesis of allolactose, the natural lac operon inducer.

Trp-999 is a key residue for the action of beta-galactosidases (Escherichia coli). Several site specific substitutions (Phe, Gly, Tyr, Leu) for Trp-999 were made. Each substitution caused greatly decreased affinities for substrates and inhibitors that bind in the "shallow" mode, while the affinities of inhibitors that bind in the "deep" mode were not decreased nearly as much. This shows that Trp-999 is important for binding in the shallow mode. The residue is also very important for binding glucose to galactosyl-beta-galactosidase (as a transgalactosidic acceptor). Substitution greatly diminished the affinity for glucose. Substitutions also changed the activation thermodynamics and, subsequently, the rates of the catalytic reactions. The enthalpies of activation of the glycolytic bond cleavage step (galactosylation, k(2)) became less favorable while the entropies of activation of that step became more favorable as a result of the substitutions. Differing magnitudes of these enthalpic and entropic effects with ONPG as compared to PNPG caused the k(2) values for ONPG to decrease but to increase for PNPG. The enthalpies of activation for the common hydrolytic step (degalactosylation, k(3)) increased while the entropies of activation for this step did not change much. As a result, k(3) became small and rate determining for each substituted enzyme. The substitutions caused the rate constant (k(4)) of the transgalactosidic acceptor reactions with glucose (for the formation of allolactose) to become much larger and of the same order of magnitude as the normally large rate constants for transgalactosidic acceptor reactions with small alcohols. This is probably because glucose can approach with less restriction in the absence of Trp-999. However, since glucose binds very poorly to the galactosyl-beta-galactosidases with substitutions for Trp-999, the proportion of lactose molecules converted to allolactose is small. Thus, Trp-999 is also important for ensuring that an appropriate proportion of lactose is converted to allolactose.

Amino Acid Substitution↗
PubMed1971

Effect of point mutations in the lac promoter on transient and severe catabolite repression of the lac operon of Escherichia coli.

1. Experiments were devised to show whether the point mutations L8 and L29 in the lac promoter alleviate transient repression. 2. Several recombinants were picked from matings between a single F(-)p(+) strain and Hfr strains carrying mutations L8 and L29. All of the 19 p(-) recombinants tested proved to suffer no transient repression, whereas all of the eight p(+) recombinants tested suffered prolonged transient repression. 3. A diploid strain was constructed in which more than 90% of the thiogalactoside transacetylase is synthesized from the episome with a wild-type lac promoter, whereas 100% of the beta-galactosidase is synthesized from the chromosome with a promoter carrying mutation L8. In this diploid the synthesis of thiogalactoside transacetylase suffered transient repression but the synthesis of beta-galactosidase did not. 4. Exactly similar results were obtained with a diploid strain in which the chromosomal promoter carried mutation L29. 5. The same diploid strains were used in experiments to show whether mutations L8 and L29 alleviate the severe catabolite repression caused by growth in glucose plus gluconate. In both strains glucose+gluconate repressed the synthesis of beta-galactosidase much less than the synthesis of thiogalactoside transacetylase. 6. These and previously reported results can be explained by assuming (a) that both mutations L8 and L29 render the lac promoter partially, but not completely, insensitive to catabolite repression, and (b) that transient repression is an exceptionally severe form of catabolite repression.

Acyltransferases↗