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At least 181 records · Page 10Linked to original sources
PubMed1984

Isolation and analysis of two Escherichia coli K-12 ilv attenuator deletion mutants with high-level constitutive expression of an ilv-lac fusion operon.

A lysogenizing lambda phage, lambda dilv-lac11, was constructed to carry an ilvD-lac operon fusion. Expression from the phage of the ilvE and lacZ genes is controlled by an intact ilv control region also carried by this phage. Two spontaneous mutants of lambda dilv-lac11 that have high-level constitutive expression of the ilv-lac fusion operon were isolated by growth on a beta-chloroalanine selective medium. The mutants were shown by nucleotide sequence determination to contain large deletions (delta 2216, approximately 1.6 kilobases; delta 2219, approximately 1.9 kilobases), which in both cases remove the proposed ilv attenuator terminator. The rest of the ilv leader and promoter region DNA remains intact in these mutants. Deletion 2216 also removed part of the downstream ilvG gene, whereas delta 2219 extended through the entire ilvG gene into the ilvGE intercistronic region. A possible mechanism of deletion formation is discussed.

Bacteriophage lambda↗
PubMed1998

Non-genetic population heterogeneity studied by in situ polymerase chain reaction.

Expression of a lac operon in Salmonella typhimurium single cells was monitored using lac mRNA targeting in situ reverse transcription-polymerase chain reaction (RT-PCR). It is demonstrated that suboptimal induction of the lac operon in a culture of S. typhimuriuml/F'lac+ cells generates a subpopulation in which transcription of the lac operon occurs and another subpopulation in which transcription of the lac operon is repressed, whereas suboptimal induction of the lac operon in a culture of S. typhimuriuml/F'lacY cells generates a population with uniform levels of lac mRNA. The outcome of the single-cell lac mRNA detection assay was compared with the outcome of a single-cell beta-galactosidase assay. In cultures grown under different suboptimal lac induction conditions, the fraction of cells in which transcription of the lac operon occurred was concurrent with the fraction of cells showing beta-galactosidase activity. Besides supporting the hypothesis that the lactose permease has a role in generating non-genetic heterogeneity in suboptimally induced cultures of Lac+ cells, these results demonstrate the usefulness of in situ RT-PCR for the study of non-genetic population heterogeneities.

Escherichia coli Proteins↗
PubMed1983

Genetic mapping and transcriptional orientation of the fimD gene.

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation whereby individual cells can alternate between states of organelle expression and nonexpression. Strains carrying fim-lac operon fusions in which lac operon expression is under the control of a fim promoter undergo Lac+ in equilibrium Lac- phase variation. We have determined the genetic map location and direction of transcription of a fim-lac operon fusion which was obtained by insertion of lac into a locus we have named fimD. We found the gene order to be as follows: valS fimD uxuA serB. The direction of transcription of fimD was found to be clockwise on the E. coli chromosome.

Chromosome Mapping↗
PubMed1982

Gene dosage as a regulatory factor for gene expression. I. In lambda plac5-infected cells.

To study the effect of gene dosage on gene expression, lambda plac5cI857O29P3, a replication defective lambda phage carrying part of the lac operon (containing the lac promotor, operator and z gene) in the b2 region was studied in Escherichia coli strain JC6256 where the lac operon is deleted and at a temperature where the lambda repressor is inactive. In measuring the synthesis of beta-galactosidase, it was possible to separate the effects of the lac promoter from those of the phage promoter. When the synthesis of beta-galactosidase was initiated from the inserted lac promoter in JC6256(lambda +) in the presence of additional cyclic AMP, the rate and level of beta-galactosidase synthesis were directly proportional to the multiplicity of infection (gene dosage). Furthermore, beta-galactosidase synthesis was initiated about 5 min after infection, just as with isopropyl-beta-D-thiogalactoside (IPTG) induction. When the synthesis of beta-galactosidase was initiated from the phage promoter in JC6256 in the absence of additional cyclic AMP, the rate and level of beta-galactosidase synthesis were again linearly proportional to gene dosage. On the other hand, initiation of beta-galactosidase synthesis was delayed until 10 to 20 min after infection. These results suggest that: (i) in the absence of negative controlling factors, the extent of gene expression is proportional to gene dosage; (ii) varying the gene dosage can be used to regulate gene expression.

Bacteriophage lambda↗
PubMed1999

Decay of rplN and lacZ mRNA in Escherichia coli.

To examine the previously proposed retroregulation model of spc mRNA degradation, two strains of Escherichia coli B/r were used; one has wild-type spc and lac operons and the other has a lac operon deletion, a wild-type spc operon, and a Pspc-rplN-lacZ fusion operon lacking the normal control sites of the spc operon (rplN is the first gene in the spc operon of ribosomal proteins). The decay of rplN mRNA and of lacZ mRNA in these strains was determined during exponential growth at different rates and after transcript initiation was inhibited by the antibiotic rifampicin. Functional decay of lacZ mRNA was monitored by measurements of beta-galactosidase activity and chemical decay was monitored using probes complementary to rplN, rplX, and to the 5' and 3'-terminal sections of lacZ. Analysis of the data was based on the assumption that the decay involves an endonucleolytic cleavage that functionally inactivates the mRNA and that this is followed by exonucleolytic degradation of the cleavage products. The major conclusions were: (1) During exponential growth, lacZ mRNA of the lac operon was translated about twice as frequently as lacZ mRNA of the spc-lac fusion, and both kinds of lacZ mRNA were translated at an elevated rate in the presence of rifampicin. (2) For lacZ mRNA from the lac operon, the endonuclease inactivation reaction was not affected by rifampicin, but the exonuclease reaction was inhibited. (3) The decay of rplN mRNA from the spc operon was accelerated in the presence of rifampicin; the average life was estimated to be six minutes during exponential growth in LB medium, and 2.8 minutes in the presence of rifampicin. (4) The decay of the rplN section of mRNA from the spc-lac operon fusion was coupled to the decay of the downstream lacZ mRNA section and was strongly inhibited (i.e. partially blocked) in the presence of rifampicin. These results show that the decay of spc mRNA differs in some important aspects from the decay of lac mRNA and support the retroregulation model. Moreover, the results indicate that rifampicin can have a significant and selective impact on the kinetics of both mRNA translation and decay.

Escherichia coli↗
PubMed1973

Studies on beta-galactoside transport in a Proteus mirabilis merodiploid carrying an Escherichia coli lactose operon.

Merodiploid derivatives bearing an F-linked lac operon (i(+), o(+), z(+), y(+), a(+)) from Escherichia coli were prepared from a Proteus mirabilis strain unable to utilize lactose and from a lac deletion strain of E. coli. A suitable growth medium was found in which the episomal element in the P. mirabilis derivative was sufficiently stable to allow induction of the episome-borne lac operon and thus to permit a comparison of the activities and properties of E. coli lac products in the intracellular environments of P. mirabilis and E. coli. In both derivatives the episomal lac operon was shown to be repressed in the absence of inducer. Kinetics of induction with gratuitous inducer (isopropyl-1-thio-beta-d-galactoside) were similar for both beta-galactosidase activity (beta-d-galactoside galactohydrolase, EC 3.4.1.23) and beta-galactoside transport activity in both derivatives, although the ratio of galactoside transport to beta-galactosidase activity was approximately 1.6-fold higher in the E. coli derivative. Comparison of beta-galactosidase and M-protein (lac y gene product)-specific activities indicated coordinate expression of the induced lac operon in both derivatives. Quantitatively, the maximal beta-galactosidase specific activity was two or three times higher for the E. coli derivative. A significant sodium azide inhibition (65% inhibition by 10 mM sodium azide) of lactose permease-mediated transport of o-nitrophenyl-beta-galactoside from an outside region of high concentration to an inside region of very low concentration ("downhill transport") was observed for the P. mirabilis derivative. Identical conditions for the E. coli derivative yielded only about 15% inhibition. Active transport of thiomethyl-beta-galactoside was similar for both derivatives, the major difference being that active transport was more sensitive to azide poisoning in the P. mirabilis derivative. Preliminary examination of the thiomethyl-beta-galactoside derivatives following active transport did not demonstrate the accumulation of a phosphorylated product in either strain but did reveal an unidentified derivative present in the P. mirabilis merodiploid extract which was not detectable in the E. coli merodiploid.

Azides↗
PubMed1991

Inactivation of lacZ gene expression by UV light and bound DNA photolyase implies formation of extended complexes in the genomes of specific Escherichia coli strains.

In Escherichia coli strains WU and CS101, UV inactivation of lacZ gene expression is more effective when the cells contain amplified DNA photolyase, and flash photoreactivation (fPR) after 15 min of metabolism does not reverse inactivation by the photolyase-dimer complexes. In other strains, also studied with or without amplified DNA photolyase, there is no differential UV inactivation and fPR reverses inactivation by the complexes regardless of continued metabolism. The irreparable condition in strain WU is not due to dysfunction of photolyase: during post-UV metabolism, fPR still restores viability and dimers are removed from the region of the lac operon. When the wild-type lac promoter is replaced by the UV5 promoter, making expression insensitive to relaxed supercoiling and catabolite repression, inactivation by dimers alone becomes more resistant, i.e. requires higher fluences, but inactivation in WU and CS101 is still exceptionally sensitive to photolyase-dimer complexes. This indicates that dimers external to the wild-type lac operon may inhibit expression by altering supercoiling but that complexes must involve some other mechanism for their special effect in WU and CS101. The exceptionally efficient inactivation and irreparable condition are consistent with the idea that, in two specific laboratory strains, photolyase bound to dimers at a considerable distance from the lac operon may initiate an aggregation of DNA with other cellular molecules that extends to, and inactivates expression from, the operon.

DNA Damage↗
PubMed1999

[Effect of plasmid pKM101 on the expression of bacterial genes not related to DNa metabolism].

An experimental system ensuring fusion of bacterial genes to the lac operon of the Mu dl(Aplac) phage was used. Fusion operons in which the lac operon was under the control of promoters of the elt gene, responsible for synthesis of the LT toxin, of the tetracyclin-resistance tet gene, and sfiA gene encoding filament production, was studied. Using this experimental system, plasmid pKM101 was shown to be capable of activating the expression of the above Escherichia coli and Salmonella typhimurium genes, which is manifested as the activation of beta-galactosidase synthesis. The activation of the elt gene expression by the pKM101 plasmid was also confirmed in experiments on detecting the LT toxin synthesized by bacteria carrying this plasmid. Effect of the plasmid on the activation of elt operon expression, unlike the effect of this plasmid on mutability, does not depend on the functioning of the lexA and recA genes, i.e., this is not a SOS-regulated process. The mutant plasmid pGW12, a derivative of pKM101, deficient in the mucAB genes responsible for mutagenesis, causes a more pronounced activation of the elt gene than plasmid pKM101.

DNA, Bacterial↗
PubMed1996

Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.

The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-beta-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

Allosteric Regulation↗
PubMed1993

The use of bacterial luciferase genes as reporter genes in Lactococcus: regulation of the Lactococcus lactis subsp. lactis lactose genes.

Lactose metabolism is an important industrial trait in dairy lactococci. In Lactococcus lactis, lactose is taken up via the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS) and is subsequently metabolized via the glycolytic and tagatose 6-phosphate pathways. Genes for the lactose-specific PEP-PTS proteins, phospho-beta-galactosidase and tagatose 6-phosphate pathway enzymes are encoded by a single 8 kb operon, lacABCDFEGX, and there is a divergently transcribed lacR repressor gene. Transcriptional fusions of both the lac operon promoter and the lacR promoter to the luxAB genes of Vibrio fischeri were used to investigate the regulation of expression of both promoters. In vivo bioluminescence assays demonstrated that lacR negatively regulates the lac operon and also autoregulates itself. Induction of transcription occurred for both promoters during growth on lactose: sevenfold for lacR and fivefold for the lac operon. The lacR promoter was demonstrated to be a particularly strong promoter, being approximately four times more efficient than the lac operon promoter. Both promoters provide good potential for the inducible expression of foreign proteins in Lactococcus.

Base Sequence↗
PubMed1984

lon transcriptional regulation of genes necessary for capsular polysaccharide synthesis in Escherichia coli K-12.

It has previously been observed that Escherichia coli lon mutations increase the levels of enzymes involved in the synthesis of colanic acid capsular polysaccharide (A. Markovitz, p. 415-462, in I. Sutherland, ed., Surface Carbohydrates of the Prokaryotic Cell, 1977). To determine how lon regulates these enzymes, we have isolated, mapped, and characterized lac operon and lac protein fusions to genes necessary for capsule synthesis by the Mu d(lac Amp) in vivo fusion technique of Casadaban and Cohen (M. J. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1979). At least five genes have been identified which share a common pattern of regulation: they are transcribed at low levels in lon+ strains and at significantly higher levels in lon strains. These genes are located in a cluster close to udk at 45 min on the E. coli map; we have named these genes cpsA, B, C, D, and E. An additional locus, cpsF, located at 90 min, is regulated in a similar manner to cpsA to E but is not essential for colanic acid synthesis. Similar studies on the transcriptional regulation of fusions in the gal and manA operons, also necessary for colanic acid synthesis, do not show significant regulation by the lon locus. Therefore, the regulatory system described here does not extend to all genes in the colanic acid synthesis pathway.

DNA Restriction Enzymes↗
PubMed1986

2-Ketoglutarate as a possible regulatory metabolite involved in cyclic AMP-dependent catabolite repression in Escherichia coli K12.

2-Ketoglutarate--unlike any other derivative of the citric acid cycle--was found to strongly repress catabolite-sensitive genes, such as the lactose operon (lac) or the tryptophanase gene (tna), when added to cells grown in glycerol. 2-ketoglutarate affects the expression of these genes by decreasing cyclic AMP synthesis. Such inhibition of cyclic AMP synthesis requires the presence of enzyme III, a component of the phosphoenol pyruvate:sugar phosphotransferase transport system (PTS). Thus, it is proposed that 2-ketoglutarate is one of the catabolite repressors postulated by Magasanik in 1961. In addition, by studying the effect of 2-ketoglutarate in various mutants, we show the existence of a cyclic AMP-independent catabolite repression mechanism whose mediator is synthesized from 2-ketoglutarate.

Cyclic AMP↗
PubMedDate not supplied

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers.

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.

Bacteriophage mu↗
PubMed2004

Evolution of specialists in an experimental microcosm.

The impact of adaptation on the persistence of a balanced polymorphism was explored using the lactose operon of Escherichia coli as a model system. Competition in chemostats for two substitutable resources, methylgalactoside and lactulose, generates stabilizing frequency-dependent selection when two different naturally isolated lac operons (TD2 and TD10) are used. The fate of this balanced polymorphism was tracked over evolutionary time by monitoring the frequency of fhuA-, a linked neutral genetic marker that confers resistance to the bacteriophage T5. In four out of nine chemostats the lac polymorphism persisted for 400-600 generations when the experiments were terminated. In the other five chemostats the fhuA polymorphism, and consequently the lac operon polymorphism, was lost between 86 and 219 generations. Four of 13 chemostat cultures monomorphic for the lac operon retained the neutral fhuA polymorphism for 450-550 generations until they were terminated; the remainder became monomorphic at fhuA between 63 and 303 generations. Specialists on each galactoside were isolated from chemostats that maintained the fhuA polymorphism, whether polymorphic or monomorphic at the lac operon. Strains isolated from three of four chemostats in which the lac polymorphism was preserved had switched their galactoside preference. Most of the chemostats where the fhuA polymorphism was lost also contained specialists. These results demonstrate that the initial polymorphism at lac was of little consequence to the outcome of long-term adaptive evolution. Instead, the fitnesses of evolved strains were dominated by mutations arising elsewhere in the genome, a fact confirmed by showing that operons isolated from their evolved backgrounds were alone unable to explain the presence of both specialists. Our results suggest that, once stabilized, ecological specialization prevented selective sweeps through the entire population, thereby promoting the maintenance of linked neutral polymorphisms.

Bacterial Outer Membrane Proteins↗
PubMed1998

Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability.

E. coli recombinant expression systems that utilize lac operon control elements to modulate gene expression are known to produce some amount of uninduced (leaky) gene expression. Previously, we showed that high levels of uninduced gene expression was a major cause of instability in the pET expression system. We show here that the pET system, in which the phage T7 RNA polymerase gene is expressed via lac operon control elements, exhibits leaky expression that increases markedly as cells grown in complex medium enter stationary phase. Moreover, we found that this phenomenon occurs with the chromosomal lac operon as well. Further investigation revealed that stationary phase leaky expression requires cyclic AMP, and that substantial leaky expression could be effected in log phase cells by adding cyclic AMP and acetate at pH6.0. Finally, a comparison of otherwise isogenic cya and wild-type hosts showed that expression stability and plasmid maintenance in the cya host is greatly enhanced, even when cells are passaged repeatedly in non-selection medium. These findings both provide a method to enhance the stability of lac-based recombinant expression systems, and suggest that derepression of the lac operon in the absence of inducer may be part of a general cellular response to nutrient limitation.

Bacteriophage T7↗
PubMed1978

Altered mRNA metabolism in ribonuclease III-deficient strains of Escherichia coli.

The metabolism of mRNA from the lactose (lac) operon of Escherichia coli has been studied in ribonuclease (RNase) III-deficient strains (rnc-105). The induction lag for beta-galactosidase from the first gene was twice as long, and enzyme synthesis was reduced 10-fold in one such mutant compared with its isogenic rnc+ sister; in the original mutant strain AB301-105, synthesis of beta-galactosidase was not even detectable, although transduction analysis revealed the presence of a normal lac operon. This defect does not reflect a loss of all lac operon activity galactoside acetyltransferase from the last gene was synthesized even in strain AB301-105 but at a rate several times lower than normal. Hybridization analyses suggested that both the frequency of transcription initiation and the time to transcribe the entire operon are normal in rnc-105 strains. The long induction lag was caused by a longer translation time. This defect led to translational polarity with reduced amounts of distal mRNA to give a population of smaller-sized lac mRNA molecules. All these pleiotropic effects seem to result from RNase III deficiency, since it was possible to select revertants to rnc+ that grew and expressed the lac operon at normal rates. However, the rnc-105 isogenic strains (but not AB301-105) also changed very easily to give a more normal rate of beta-galactosidase synthesis without regaining RNase III activity or a faster growth rate. The basis for this reversion is not known; it may represent a "phenotypic suppression" rather than result from a stable genetic change. Such suppressor effects could account for earlier reports of a noninvolvement of RNase III in mRNA metabolism in deliberately selected lac+ rnc-105 strains. The ribosomes from rnc-105 strains were as competent as ribosomes from rnc+ strains to form translation initiation complexes in vitro. However, per mass, beta-galactosidase mRNA from AB301-105 was at least three times less competent to form initiation complexes than was A19 beta-galactosidase mRNA. RNase III may be important in the normal cell to prepare lac mRNA for translation initiation. A defect at this step could account for all the observed changes in lac expression. A potential target within a secondary structure at the start of the lac mRNA is considered. Expression of many operons may be affected by RNase III activity; gal and trp operon expressions were also abnormal in RNase III- strains.

Enzyme Induction↗
PubMed2002

The effect of genomic position on reversion of a lac frameshift mutation (lacIZ33) during non-lethal selection (adaptive mutation).

In a system described by Cairns and Foster, starvation of a particular leaky lac mutant (lacIZ33) in the presence of lactose appears to direct mutation in non-growing cells to sites that allow growth (adaptive mutation). This behaviour requires that the lac operon be located on an F' plasmid. This position effect was investigated by placing the mutant lac operon at many sites in the genome of Salmonella enterica (Typhimurium; LT2) and testing reversion behaviour. Genomic position did not affect reversion during non-selective growth. When lac was at any of 550 chromosomal sites, starvation caused little or no enhancement of reversion. In the 28 strains with the lac on Salmonella's conjugative plasmid (pSLT), selection enhanced reversion strongly, just as seen for strains with lac on an F' plasmid. In 46 strains, the lac operon was inserted within a small chromosomal duplication, and selection stimulated RecA-dependent partial reversion by simple amplification (about 8x) of the mutant lac region. The position of lac on a conjugative plasmid is important to reversion because it allows more frequent gene duplication and amplification. These events are central to growth and reversion under selection because they increase the number of replicating lac alleles within each developing revertant clone.

Adaptation, Biological↗
PubMed1983

The lactose carrier of Escherichia coli functionally incorporated in Rhodopseudomonas sphaeroides obeys the regulatory conditions of the phototrophic bacterium.

Rhodopseudomonas sphaeroides was provided with the ability to transport lactose via conjugation with a strain of Escherichia coli bearing a plasmid containing the lactose operon (including the lac Y gene, coding for the lactose carrier or M protein) and subsequent expression of the lac operon in Rps. sphaeroides (Nano, F.E. and Kaplan, S. submitted). The initial rate of lactose transport in Rps. sphaeroides was studied as a function of the light intensity and the magnitude of the proton-motive force. The results demonstrate that lactose transport is regulated by the rate of cyclic electron transfer in the same way as the endogenous transport systems.

Alanine↗