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PubMed1980

Feedback regulation of ribosomal protein gene expression in Escherichia coli.

The structural genes for Escherichia coli ribosomal protein (r-protein) genes L1, S4, and S11 were inserted into a plasmid vector containing the lac operator and promoter such that the synthesis of L1, S4, and S11 was controlled by lac regulatory elements. Synthesis of L1, S4, and S11 was stimulated by addition of an inducer of the lac operon (isopropyl thiogalactoside) to exponentially growing cells. Elevated synthesis of L1 caused a specific decrease in L11 synthesis, whereas overproduction of S4 resulted in lowered synthesis of S13 and L17. Stimulation of L1 or S4 synthesis also inhibited cell growth. Overproduction of S11 did not affect synthesis of other r-proteins or alter growth. These results confirm previous in vitro studies [Yates, J. L., Arfsten, A. E. & Nomura, M. (1980) Proc. Natl. Acad. Sci. USA 77, 1837-1841] and support the hypothesis that certain r-proteins have the capacity to selectively inhibit synthesis of r-proteins whose genes are in the same operon as their own.

Bacteriophage lambda↗

PubMed1985

Measurement of the proton motive force in Rhizobium meliloti with the Escherichia coli lacY gene product.

An Escherichia coli lac operon constitutive for lacY was subcloned into the EcoRI site of a wide-host-range plasmid of the Q incompatibility group, and the resulting recombinant plasmid was introduced into Tn5-generated Lac- mutants of Rhizobium meliloti. The R. meliloti transconjugants accumulated lactose about 1,000-fold, equivalent to a proton motive force of -170 to -180 mV, not significantly different from the values calculated from the distributions of weak acids and lipophilic cations.

Cloning, Molecular↗

PubMed2006

Plasticity of the cis-regulatory input function of a gene.

The transcription rate of a gene is often controlled by several regulators that bind specific sites in the gene's cis-regulatory region. The combined effect of these regulators is described by a cis-regulatory input function. What determines the form of an input function, and how variable is it with respect to mutations? To address this, we employ the well-characterized lac operon of Escherichia coli, which has an elaborate input function, intermediate between Boolean AND-gate and OR-gate logic. We mapped in detail the input function of 12 variants of the lac promoter, each with different point mutations in the regulator binding sites, by means of accurate expression measurements from living cells. We find that even a few mutations can significantly change the input function, resulting in functions that resemble Pure AND gates, OR gates, or single-input switches. Other types of gates were not found. The variant input functions can be described in a unified manner by a mathematical model. The model also lets us predict which functions cannot be reached by point mutations. The input function that we studied thus appears to be plastic, in the sense that many of the mutations do not ruin the regulation completely but rather result in new ways to integrate the inputs.

Base Sequence↗

PubMed2004

Constructing biological pathway models with hybrid functional Petri nets.

In many research projects on modeling and analyzing biological pathways, the Petri net has been recognized as a promising method for representing biological pathways. From the pioneering works by Reddy et al., 1993, and Hofestädt, 1994, that model metabolic pathways by traditional Petri net, several enhanced Petri nets such as colored Petri net, stochastic Petri net, and hybrid Petri net have been used for modeling biological phenomena. Recently, Matsuno et al., 2003b, introduced the hybrid functional Petri net (HFPN) in order to give a more intuitive and natural modeling method for biological pathways than these existing Petri nets. Although the paper demonstrates the effectiveness of HFPN with two examples of gene regulation mechanism for circadian rhythms and apoptosis signaling pathway, there has been no detailed explanation about the method of HFPN construction for these examples. The purpose of this paper is to describe method to construct biological pathways with the HFPN step-by-step. The method is demonstrated by the well-known glycolytic pathway controlled by the lac operon gene regulatory mechanism.

Adenosine Diphosphate↗

PubMed1994

[UV-induction of the LT-toxin operon depending on genes lexA, recA, and umuD].

UV induction of the elt operon (the LT-toxin operon in Escherichia coli) was demonstrated in experiments using fusion of elt::lac operons with the help of Mud1(Ap lac) phage. UV induction of the elt operon is lexA-dependent; thus, the possibility of SOS regulation of this process may be assumed. However, UV induction of the elt operon turned out to be recA-independent, which makes it impossible to consider this induction as a typical SOS response. UV induction of the elt operon is also observed in Salmonella typhimurium, which differs from E. coli in the product of umuD, which suggests that the UV induction of the elt operon is umuD independent.

Bacterial Proteins↗

PubMed1995

Transcriptional control of the nuo operon which encodes the energy-conserving NADH dehydrogenase of Salmonella typhimurium.

The 14 nuo genes encode the subunits of the type I (energy-conserving) NADH dehydrogenase, a key component of the respiratory chain. Salmonella typhimurium, like Escherichia coli, has two enzymes that can oxidize NADH and transfer electrons to ubiquinone, but only the type I enzyme translocates protons across the membrane to generate a proton motive force. Cells with the type I enzyme are energetically more efficient; the role of the type II enzyme (encoded by ndh) is not established, but it may function like a relief valve to allow more rapid NADH recycling. Here, we have investigated transcription of the nuo gene cluster, primarily in S. typhimurium. Studies with polar insertion mutants demonstrate that these genes are arranged as a single, large operon that is expressed from a complex promoter region upstream of nuoA. The DNA sequence of the promoter region was determined, and primer extension analysis of nuo transcripts was used to map four major RNA 5' ends to this region. A set of lac operon fusions to various DNA segments from the nuo promoter region was also constructed. Analysis of these fusions confirmed the presence of at least two nuo promoters. Mutations in the global regulatory genes arcA, oxrA (fnr), crp, cya, and katF were tested for effects on expression of the nuo operon. However, none of the mutations tested had a large effect on expression of type I NADH dehydrogenase.

Base Sequence↗

PubMed1987

The functional stability of the lacZ transcript is sensitive towards sequence alterations immediately downstream of the ribosome binding site.

Various synthetic DNA sequences were inserted downstream of the fourth codon of the Escherichia coli lacZ gene on plasmids containing a hybrid lacZ-galK operon. Several different sequences, one as short as 10 bp, reduced the functional stability of the lacZ message three- to fourfold, whereas others had little or no effect. Introduction of synthetic sequences into a plasmid containing the intact lac operon resulted in similar reductions of mRNA stability. The sequence alterations also reduced the translational efficiency and transcription through lacZ as monitored by measurements of galactokinase synthesis from the downstream galK gene. There was no correlation between the average translational frequency and the stability of the lacZ message indicating that some of the inserted sequences reduced mRNA stability directly and not as a consequence of their effect on translation. The reduction of transcription through the lacZ gene correlated with the reduction of translation in agreement with current models of transcriptional polarity.

Amino Acid Sequence↗

PubMed2003

The art and design of genetic screens: Escherichia coli.

This article summarizes the general principles of selections and screens in Escherichia coli. The focus is on the lac operon, owing to its inherent simplicity and versatility. Examples of different strategies for mutagenesis and mutant discovery are described. In particular, the usefulness and effectiveness of simple colour-based screens are illustrated. The power of lac genetics can be applied to almost any bacterial system with gene fusions that hook any gene of interest to lacZ, which is the structural gene that encodes beta-galactosidase. The diversity of biological processes that can be studied with lac genetics is remarkable and includes DNA metabolism, gene regulation and signal transduction, protein localization and folding, and even electron transport.

DNA Transposable Elements↗

PubMed1995

Plasmid mutagenesis by PCR for high-level expression of para-hydroxybenzoate hydroxylase.

We report a PCR deletion mutagenesis method for the exact positioning of a foreign gene (pobA) in the lac operon of an expression plasmid in place of the lacZ protein code. This method requires the synthesis of four oligonucleotides and three PCR reactions to delete unwanted bases and retain the nucleotide sequence naturally found between the lac promoter and the protein code. The engineered plasmid results in the production of at least 40% of the cellular protein as the foreign polypeptide. In the example presented the expression of the protein is high even with a substantial difference in codon usage between the host (Escherichia coli) and a foreign gene from Pseudomonas aeruginosa. Some of the polypeptide produced has the ame properties as native protein and is easily purified. The remainder is present as insoluble inclusion bodies. This method of plasmid refinement may be applicable to the expression of many proteins.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1980

The phage promoter responsible for the expression of the inserted beta-galactosidase gene in bacteriophage lambda plac5.

The lac transducing phage, lambda plac5, carries a segment of the E. coli lac operon on the left side of the b2 region of the lambda phage. In the absence of additional cyclic AMP, beta-galactosidase can only be expressed from the phage promoter, and the expression of the inserted lac promoter is suppressed. This phage promoter responsible for beta-galactosidase synthesis is shown to be under the control of the cI and N gene products; however, the repressive action of the cro gene product at high multiplicity of infection is not observed although some turn off at very late time is detected. To pin down this phage promoter, results described in this communication and those described elsewhere can rule out the promoter PI, PR, P'R, and the promoter PL also looks rather unlikely. No firm identification of this phage promoter has been made, but the promoter(s) in the b2 region (the b2 promoter) is proposed. The phage promoter responsible for beta-galacrosidase synthesis is shown to be a weak promoter, requires the Q gene product or one (or more) of the late gene products for activation, and the time of expression is very late.

Bacteriophage lambda↗

PubMed1981

Growth-rate-dependent regulation of ribosome synthesis in E. coli: expression of the lacZ and galK genes fused to ribosomal promoters.

Hybrid transducing phages were constructed in vitro that carry the galK gene fused to each of three ribosomal promoters: the promotor for an rRNA operon (rrnE); the promoter for the spec r protein operon and the promotor for the alpha r protein operon. We also constructed hybrid transducing phages that carry the IacZ gene fused to the promoter for the rrnE operon or to the promoter for the spc r protein operon. The amounts of galactokinase (or beta-galactosidase) were analyzed in lysogens carrying these various transducing phages grown in several different growth media. The synthesis rate of galactokinase (or beta-galactosidase) from the fused rrn-gal (or rrn-lac) operon relative to the total protein synthesis rate increased with increasing growth rate, as expected from the transcriptional activity of rRNA operons. In contrast, the relative synthesis rate of galactokinase (or beta-galactosidase) from the operon fused to alpha or spc r protein promoter remained approximately constant with increasing growth rate. These results were interpreted to mean that the characteristic increase in the relative synthesis rate of r protein with increasing growth rate is determined not by transcription regulatory mechanisms, but by posttranscriptional mechanisms, which presumably involve the feedback inhibition of r protein mRNA translation by free r proteins.

Escherichia coli↗

PubMed1993

Transport of 5-aminolevulinic acid by the dipeptide permease in Salmonella typhimurium.

In a previous search for mutants of Salmonella typhimurium that are defective in heme synthesis, one class that is apparently defective in 5-aminolevulinic acid (ALA) uptake (alu) was found. Here, I describe the characterization of these mutations. The mutations all map to a single locus near 77.5 min on the genetic map, which is transcribed counterclockwise. Nutritional tests, genetic and physical mapping, and partial DNA sequence analysis revealed that alu mutants are defective in a periplasmic binding protein-dependent permease that also transports dipeptides, encoded by the dpp operon. The uptake of labeled ALA is defective in dpp mutants and is markedly increased in a strain that has elevated transcription of the dpp locus. Unlabeled L-leucyl-glycine competes with labeled ALA for uptake. In a strain carrying both a dpp-lac operon fusion and a functional copy of the dpp locus, the expression of beta-galactosidase is not induced by ALA, nor, in a hemL mutant, does expression of dpp change substantially during starvation for ALA. The dipeptide permease displays a relaxed substrate specificity that allows transport of the important nonpeptide nutrient ALA, whose structure is closely related to that of glycyl-glycine.

Aminolevulinic Acid↗

PubMed1983

Effect of ptsI and ptsH mutations on initiation of transcription of the Escherichia coli lactose operon.

Mutations in the pts genes (which code for the enzyme I and HPr protein - the general components of the phosphoenolypyruvate-dependent phosphotransferase system) lead to decreases in enzyme-inducible synthesis at the level of transcription. The intracellular content of cyclic AMP in the ptsIH mutant was severely diminished, while the ptsH bacteria contain the same amounts of this nucleotide as the wild-type cells. Nevertheless expression of the lac operon was diminished in the ptsH as well as in the ptsIH mutant. The exogenous cyclic AMP did not prevent repression of beta-galactosidase synthesis in a delta cya ptsI mutant in a wide range of concentrations in the growth medium (from 0.05 mM to 5 mM). The combination of ptsI or ptsH mutations with rpoC1 (synthesis of thermosensitive beta' subunit of RNA polymerase) leads to greater disturbance of beta-galactosidase production at the nonpermissive temperature than demonstrated in the pts+ rpoC1 strain. The stimulatory effect of exogenous cyclic AMP was more pronounced in pts rpoC1 than in pts+ rpoC1 bacteria. The data presented confirm the hypothesis that pts mutations alter the function of CRP in initiation of transcription.

Cyclic AMP↗

PubMed1984

Cellular control models with linked positive and negative feedback and delays. I. The models.

Basic techniques from biochemical kinetics are used to develop models for a cellular control system with linked positive and negative feedback. The models are represented by a system of nonlinear differential equations with delays. The lac operon provides an example of a control system where the transcription of the operon is controlled by induction or positive feedback control and catabolite repression or negative feedback control. These processes are linked through the metabolism of lactose.

Escherichia coli↗

PubMed2001

SOS mutator DNA polymerase IV functions in adaptive mutation and not adaptive amplification.

Adaptive point mutation and amplification are induced responses to environmental stress, promoting genetic changes that can enhance survival. A specialized adaptive mutation mechanism has been documented in one Escherichia coli assay, but its enzymatic basis remained unclear. We report that the SOS-inducible, error-prone DNA polymerase (pol) IV, encoded by dinB, is required for adaptive point mutation in the E. coli lac operon. A nonpolar dinB mutation reduces adaptive mutation frequencies by 85% but does not affect adaptive amplification, growth-dependent mutation, or survival after oxidative or UV damage. We show that pol IV, together with the major replicase, pol III, can account for all adaptive point mutations at lac. The results identify a role for pol IV in inducible genetic change.

Adaptation, Physiological↗

PubMed2001

A structural view of the action of Escherichia coli (lacZ) beta-galactosidase.

The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for beta-galactosidase are presented. These complexes clarify and enhance previous proposals regarding the catalytic mechanism. The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety. Of the two potential acids, Mg(2+) and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role. A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl. The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket. For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794-804 move up to 10 A closer to the site of binding. In some cases this can be inhibited by the binding of additional ligands. The resulting restricted access to the intermediate helps to explain why allolactose, the natural inducer for the lac operon, is the preferred product of transglycosylation.

Binding Sites↗

PubMed2003

Response to culture aeration mediated by the nitrate and nitrite sensor NarQ of Escherichia coli K-12.

Respiratory enzyme synthesis in enterobacteria is controlled in response to electron acceptor availability. The iron-sulphur protein Fnr and the sensor-regulator proteins ArcB-ArcA control respiratory gene transcription in response to oxygen and quinone pool redox status respectively. The sensor-regulator proteins NarX-NarL and NarQ-NarP control anaerobic respiratory gene expression in response to nitrate and nitrite. Our laboratory recently engineered the lac operon to replace the primary operator O1-lac with the NarL and NarP protein binding site from the nirB operon. Expression of the lacZ gene from this construct is repressed by nitrate in Nar+ strains. Here, we found that lacZ gene expression was repressed in aerated cultures of narQ+narX null strains. This repression was not observed in narX+narQ+ or narX+narQ null strains. Thus, the NarQ sensor responds to aeration as well as to nitrate and nitrite. The NarX and NarQ sensors are composed of three distinct modules: an amino-terminal sensory module, a carboxyl-terminal transmitter module and a central module of unknown function. Experiments with NarX-NarQ hybrid proteins suggest that the NarQ protein central module is necessary for response to aeration. The physiological significance of this additional sensory role for the NarQ sensor remains obscure.

Binding Sites↗

PubMed1984

New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition.

We describe below several new variants of the tetracycline-resistance transposon Tn10 which are more useful than the wild-type transposon for many types of genetic and physical analysis of bacteria. These derivatives have one or more of the following new properties: (i) new drug resistance markers; (ii) high transposition frequencies; (iii) removal of the transposase gene to a position outside of the transposing segment; (iv) internal deletions which eliminate the ability of Tn10 to make adjacent deletion/inversions; or (v) addition of a trp-lac operon fusion segment just inside one terminus such that insertion can automatically generate a transcriptional fusion to the interrupted operon. Phage and plasmid vehicles carrying these new elements are described.

DNA Transposable Elements↗