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At least 199 records · Page 11Linked to original sources
PubMed1971

Purification and DNA-binding properties of the catabolite gene activator protein.

A protein required for the activation of the lac operon has been extensively purified and partly characterized. This protein, called CGA protein (catabolite gene activator protein, sometimes named CAP), is a dimer with subunits of 22,000 daltons. Purified CGA protein has a substantial affinity for DNA; this affinity is greatly strengthened by cAMP and strongly inhibited by cGMP. Other studies have shown that these cyclic nucleotides compete for a binding site on CGA protein. The opposing effects of the two cyclic compounds in DNA-CGA protein binding show a parallel behavior to their effects on the expression of the lac operon. Thus cAMP, in addition to CGA protein, is required for expression of the lac operon, whereas cGMP inhibits the expression. The obvious inference is that CGA protein activates the lac operon by binding to the DNA under the influence of cAMP. Thus, CGA protein seems to be a new type of regulatory protein: a DNA-binding activator.

Bacterial Proteins↗
PubMed1987

The interaction of the recognition helix of lac repressor with lac operator.

We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.

Amino Acid Sequence↗
PubMed1981

Inducer and anti-inducer interactions with the lac repressor seen by nuclear magnetic resonance changes at tyrosines and tryptophans.

The effects of binding inducer and anti-inducer of the Escherichia coli lac operon to the lac repressor were examined by taking advantage of fluorine-19 NMR. The fluorine nucleus was biosynthetically incorporated into the lac repressor with either 5-fluorotryptophan or 3-fluorotyrosine. It is suggested that these small effector molecules influence the operator-binding properties of the tetrameric lac repressor by altering the intersubunit relationships in the protein.

Escherichia coli↗
PubMed1985

Deletion analysis of the Escherichia coli lactose promoter P2.

The Escherichia coli lactose (lac) operon transcription control region includes at least two sequences which are recognized by RNA polymerase holoenzyme in vitro, the normal lac promoter (termed P1) and an overlapping upstream promoter (termed P2). The structure of the P2 and the effect of RNA polymerase interaction at P2 on the association of RNA polymerase with P1 was analyzed by the isolation and characterization of various mutations at P2. A set of deletions with varying lengths of DNA between the lac P2 -10 region and a "-35 region" contributed by the vector DNA were constructed. In vitro studies indicate that as the spacing between the -10 region and "-35 region" is increased from 16 to 22 base pairs (bp), the steady state occupancy as measured by exonuclease III protection experiments and the ability to initiate transcripts from P2 decrease. Studies were also conducted using a single base pair insertion and a two base pair deletion between the natural -35 and -10 regions of P2. The mutation which decreases the in vitro occupancy and transcription initiation potential of P2 does not significantly affect the steady state in vitro occupancy of P1 nor the in vivo expression of the lac operon. These results are not consistent with the model that RNA polymerase occupancy at P2 competes with the P1 expression and therefore that this competition plays a role in cAMP bound catabolite gene activator protein (CAP-cAMP) control of the lac operon.

Base Sequence↗
PubMed1970

Catabolite-insensitive revertants of lac promoter mutants.

The maximum rate of expression of the lac operon is severely reduced in lac promoter mutants. Revertants of these mutations which produce higher levels of enzyme were isolated. Some of these revertants had lost sensitivity to catabolite repression and transient repression. The mutations responsible for these losses took place at sites very close to the original promoter mutations. From these results we conclude that the promoter itself is the target site for both catabolite and transient repression of the lac operon.

Chromosome Mapping↗
PubMed1972

Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli.

DNA from a transducing bacteriophage carrying a fusion of the tryptophan and lactose operons of E. coli (lambdadtrp-lac) has been used to direct cell-free synthesis of beta-galactosidase (EC 3.2.1.23). Whereas normal lac operon (lambdadlac) DNA requires adenosine-3':5'-cyclic monophosphate (cAMP) for beta-galactosidase synthesis, trp-lac DNA is unaffected by cAMP. This difference in cAMP dependence verifies the presence of a cAMP-requiring promoter in the lac operon that has been removed from the trp-lac DNA. Synthesis with trp-lac DNA is controlled by the protein product of the tryptophan repressor gene (trpR). Synthesis in extracts of trpR(-) (repressor-negative) cells is progressively reduced by increased additions of extract from trpR(+) cells. No trpR(-) product repression is seen when beta-galactosidase synthesis is programmed by normal lac DNA. This highly sensitive and specific assay has facilitated quantitation and partial purification of the trp repressor.

Bacterial Proteins↗
PubMed1985

Genetic analysis of the phase variation control of expression of type 1 fimbriae in Escherichia coli.

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation, whereby individual cells can alternate between states of organelle expression (Fim+) and nonexpression (Fim-). Strains with a fimD-lac operon fusion, in which lac, rather than fimD, expression is under the control of the fimD promoter, undergo Lac+ in equilibrium Lac- phase variation, instead. After positioning a lambda prophage adjacent to the operon fusion, we were able to isolate specialized lambda phage carrying both the fimD-lac fusion and the phase variation control region. Introduction of such phage into an Fim+ strain resulted in construction of a strain with a double, independently switching phenotype (Fim+ in equilibrium Fim- and Lac+ in equilibrium Lac-), demonstrating that the region controlling phase variation is contiguous with the fimD-lac operon fusion and is cis acting. When the specialized lambda phage was propagated on a delta lac delta fim strain, phase variation occurred within the plaques, confirming that the phase variation control region is carried on the specialized transducing phage. All lysogens acquired the Lac+ in equilibrium Lac- phenotype, except for two nonswitching Lac+ recombinants, which acquired Lac+ in equilibrium Lac- phase variation only by trans complementation with fim. Phase variation of type 1 fimbriae, therefore, appears to involve both a cis-active element, which is cloned on a specialized lambda phage, and a trans-active permissive factor, which is not present on the phage, but rather must be supplied by the recipient strain in the transduction.

Escherichia coli↗
PubMed1978

Tn951: a new transposon carrying a lactose operon.

A new transposon, Tn951, is described, which derives from plasmid pGC1, originally isolated from Yersinia enterocolitica. Tn951 is 16.6 kb long and presumably flanked by small inverted repeats. It carries the lac genes i, z and y. This lac system is homologous to the E. coli lac operon. However, homology is restricted to 5.6 kb. The DNA sequences surrounding the lac operons on Tn951 and E. coli are nonhomologous. This leads to speculations about the origin of the E. coli lac operon itself.

Base Sequence↗
PubMed1985

Coordinate expression of a small polypeptide with the lactose carrier of Escherichia coli.

Induction of the lac operon in wild type Escherichia coli strains results in synthesis of a 16-kDa inner membrane protein in addition to the known products of the lacZ, lacY, and lacA genes. Cells carrying the lacY gene on a multicopy plasmid overproduce this 16-kDa polypeptide as well as the Lac carrier, the membrane protein product of the lacY gene. However, [35S]methionine labeling of minicells carrying the lacY plasmid shows that the 16-kDa protein is not synthesized from the plasmid DNA. We have purified and partially characterized the 16-kDa protein. It is an acidic membrane protein of apparent Mr = 15,800 whose amino-terminal sequence (NH2-Met-Arg-Asn-Phe-Asp-Leu-) does not match any known lac operon DNA sequence. Using antibody prepared to the purified 16-kDa protein, we have quantitatively analyzed conditions under which this protein is made and have shown that the amount of 16-kDa protein which appears in the membrane is proportional to lac operon expression. Hybridization of a synthetic oligodeoxyribonucleotide probe complementary to the 5' end of 16-kDa protein mRNA shows that its synthesis is regulated at the level of transcription.

Amino Acid Sequence↗
PubMed1984

Anaerobiosis, formate, nitrate, and pyrA are involved in the regulation of formate hydrogenlyase in Salmonella typhimurium.

Three groups of mutants defective in the fermentative production of gas were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). One group consisted of strains which lacked hydrogenase. The mutation site for this group was located in the vicinity of the known hyd gene. A second group consisted of mutants which lacked the formate dehyrogenase associated with hydrogenase. The mutation site was located in four of them. It was not in the vicinity of the previously described fhlD gene but was instead located at 93 min on the Salmonella map. The third mutant group, which consisted of strains that produced gas in triple sugar iron agar but not in nutrient agar supplemented with glucose, appeared to be pyrA mutants. The insertion site was located in the vicinity of pyrA , and they required arginine and pyrimidines for growth. Expression of the lac operon in the hyd mutants was induced by anaerobiosis. It was only slightly increased by the addition of formate under anaerobic conditions and slightly decreased by the addition of nitrate. Nitrate had no effect in an hyd ::Mu d strain that also carried a chlC::Tn10 insertion. Full expression of the lac operon in the fhl mutants required both formate and anaerobic conditions. The presence of nitrate in addition to formate resulted in activities about half those obtained in its absence, even in the fhl ::Mu d chlC::Tn10 double mutant. In the absence of formate, nitrate reduced expression only in the fhl ::Mu d single mutants. Expression of the lac operon among the pyrA mutants was repressed by arginine and cytosine and also by anaerobiosis. An explanation for the involvement of pyrA in aerobic and anaerobic energy metabolism is proposed.

Aerobiosis↗
PubMed1969

A lipid requirement for induction of lactose transport in Escherichia coli.

The rate of derepressed synthesis of a membrane protein required for lactose transport (M protein) by Escherichia coli is increased in response to increased gene dosage to the same extent as the rates of synthesis of beta-galactosidase and galactoside acetylase. However, elevated gene dosage does not increase beta-galactoside transport to the same extent that it increases synthesis of M protein and of the soluble proteins of the lac operon. Though the factor or factors other than M protein which limit induction of the transport system at high levels of lac operon expression have not been identified, studies with Escherichia coli mutants blocked in the synthesis of unsaturated fatty acids indicate that unsaturated fatty acids must be supplied during the course of induction of the lac operon to permit synthesis of a functional lactose transport system, but not of beta-galactosidase or galactoside acetylase.

Biological Transport↗
PubMed1995

A basic tail increases repression by dimeric lac repressor.

Tetrameric Lac repressor achieves cooperative repression by binding simultaneously to O1 and to one of the auxiliary operators O2 or O3, thereby forcing the intervening DNA into a loop. Dimeric Lac repressor is not able to form DNA loops and consequently shows no cooperative repression. We constructed a dimeric Lac repressor mutant which exhibits increased repression to the lac operon that does not depend on specific operator-repressor-operator loops. This Lac repressor carries a synthetic tail of basic residues attached to its C terminus. With this construct, we observe an increase of the in vivo repression upon addition of auxiliary lac operators to a chromosomal lac operon controlled by O1. This suggests that the basic tail enables dimeric Lac repressor to enhance its repression by additional non-specific DNA contacts.

Amino Acid Sequence↗
PubMed1985

DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region.

The lac operon of Escherichia coli spans approximately 5300 base pairs and includes the lacZ, lacY, and lacA genes in addition to the operator, promoter, and transcription termination regions. We report here the sequence of the lacA gene and the region distal to it, confirming the sequence of thiogalactoside transacetylase and completing the sequence of the lac operon. The lacA gene is characterized by use of rare codons, suggesting an origin from a plasmid, transposon, or virus gene. UUG is the translation initiation codon. A preliminary examination of 3' end of the lac messenger in the region distal to the lacA gene indicates several endpoints. A predominant one is located at the 3' end of a G + C-rich hairpin structure, which may be involved in termination of transcription or in post-transcriptional processing. An open reading frame of 702 base pairs is present on the complementary strand downstream from lacA.

Acetyltransferases↗
PubMed1967

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.

Enzyme Induction↗
PubMed1992

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.

Journal Article↗
PubMed1999

The solution structure of Lac repressor headpiece 62 complexed to a symmetrical lac operator.

BACKGROUND: Lactose repressor protein (Lac) controls the expression of the lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the lac operon. Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator. Protein-protein and protein-DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail. RESULTS: We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized lac operator. Analysis of the structures reveals specific interactions between Lac repressor and DNA that were not found in previously investigated Lac repressor-DNA complexes. Important differences with the previously reported structures of the HP56-DNA complex were found in the loop following the helix-turn-helix (HTH) motif. The protein-protein and protein-DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail. The structures were also used for comparison with the available crystallographic data on the Lac and Pur repressor-DNA complexes. CONCLUSIONS: The structures of the HP62-DNA complex provide the basis for a better understanding of the specific recognition in the Lac repressor-operator complex. In addition, the structural features of the hinge region provide detailed insight into the protein-protein and protein-DNA interactions responsible for the high affinity of the repressor for operator DNA.

Bacterial Proteins↗
PubMed1975

Enzymatic adaptation by bacteria under pressure.

A study of enzymic adaptation under hydrostatic pressure by moderately barotolerant bacteria that can grow at pressure up to about 500 atm revealed that some adaptive processes are relatively insensitive to pressure, whereas others are sufficiently barosensitive to compromise survival capacity in situations requiring adaptation to new substrates under pressure. Examples of the former include adaptation of Escherichia coli to arabinose catabolism for growth and adaptation of Streptococcus faecalis to catabolism of lactose, ribose, or maltose. Examples of the latter include derepression of the lac operon in Escherichia coli and induction of penicillinase synthesis by Bacillus licheniformis. For both these barosensitive systems, pressure had little effect on enzyme levels in constitutive strains or in bacteria that had previously been induced at 1 atm. Moreover, it had no detectable effect on penicillinase secretion. However, pressures of 300 to 400 atm were found to reduce markedly rates and extents of enzyme synthesis by bacteria undergoing derepression or adaptation. This inhibitory effect of pressure was reflected in greater barosensitivity with extended lag and slower growth of initially unadapted Escherichia coli cells inoculated into minimal medium with lactose as sole source of carbon and fuel, and by major reductions in the minimal inhibitory concentrations of penicillin G for unadapted B. licheniformis cells inoculated into complex, antibiotic-containing media. Cyclic adenosine 5'-monophosphate did not reverse pressure inhibition of derepression of the lac operon, and catabolite repression was complete under pressure. However, derepression of the lac operon was more sensitive to pressure at low concentrations of inducer than at high concentrations. Apparent volume changes for derepression were 94 and 60 ml/mol at inducer concentrations of about 0.5 and 5 mM, respectively. Pressure was found not to be inhibitory for uptake of beta-galactosides; in fact, it was somewhat stimulatory. Therefore, results were interpreted in terms of inducer binding and subsequent conversion of an operator-inducer-repressor complex to inactive repressor and operator. Both reactions appeared to result in an increase in volume, the former more so than the latter. We found also that 200 atm was actually stimulatory for growth of Escherichia coli in minimal media, and the bacterium was in a sense barophilic.

Adaptation, Biological↗
PubMed1987

Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism.

A selection procedure using Mn2+ is described. A high percentage of the Mn2+ resistant mutants had constitutive iron transport systems. By P1 transduction, and complementation with the cloned fur gene it could be shown that nearly all the mutants constitutive in the expression of the operon fusion fiu::lambda placMu were only defective in fur. High concentrations of manganese inhibited the derepression of an iron-regulated lac operon fusion. In another iron-regulated lac operon fusion that was inducible by iron, manganese also induced the production of beta-galactosidase. Most of the fur mutants isolated (80%) were not able to grow on succinate, fumarate or acetate. After transformation with a fur+ plasmid all 39 mutants tested were able to grow on succinate. In fur mutants the presence of succinate in the growth medium reduced succinate uptake rates by 50%-70%. Succinate dehydrogenase activity was reduced to 10% of that of the parent strain.

Biological Transport, Active↗