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PubMed1989

[Regulation in the expression of alpha-galactosidase gene in raf operon in Escherichia coli].

The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme. In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers. The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc.. The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth. The structure and regulation of raf operon is similar to those of lac operon. The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation. When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold. Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression. Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively. The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP. The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1987

Improved single and multicopy lac-based cloning vectors for protein and operon fusions.

We describe several new vectors for the construction of operon and protein fusions to the Escherichia coli lacZ gene. In vitro constructions utilize multicopy plasmids containing suitable cloning sites located between upstream transcription terminators and downstream lac operon segments whose lacZ genes retain or lack translational start signals. Single-copy lambda prophage versions of multicopy constructs can be made genetically, without in vitro manipulation. The new vectors, both single and multicopy, are improved in that they have very low levels of background lac gene expression, which makes possible the easy detection and accurate quantitation of very weak transcriptional and translational signals. These vectors were developed for analysis of the expression of IS10's transposase gene, which is transcribed less than, once per generation, and whose transcripts are translated on average less than once each. Both single and multicopy constructs can also be used to select mutations affecting fusion expression, and mutations isolated in single-copy constructs can be crossed genetically back onto multicopy plasmids for further analysis.

Gene Expression Regulation↗
PubMed1988

The cpx proteins of Escherichia coli K12. Structure of the cpxA polypeptide as an inner membrane component.

Gene cpxA of Escherichia coli K12 encodes the 52,000 Mr CpxA polypeptide. The complete cpxA nucleotide sequence, reported here, predicted that CpxA contains two extended, hydrophobic segments in its amino-terminal half and could therefore be a membrane protein. Using a lac-cpxA operon fusion plasmid to overproduce CpxA and an immunochemical assay to detect the polypeptide, we show that CpxA fractionated with the bacterial inner membrane during differential and isopycnic sedimentation. Moreover, the protein could be solubilized by extraction of crude membranes with non-ionic detergents but not with KCl or NaOH, indicating that Cpx is an intrinsic membrane component. Analysis of TnphoA insertions in cpxA indicated that the region between the hydrophobic segments of CpxA is periplasmic, whereas the region carboxy-terminal to the second such segment is cytoplasmic. Based on these structural data, we propose that CpxA functions as a trans-membrane sensory protein. The DNA sequence data also indicate that cpxA is the 3' gene of an operon.

Base Sequence↗
PubMed2004

Mutational analysis of the Myxococcus xanthus Omicron4499 promoter region reveals shared and unique properties in comparison with other C-signal-dependent promoters.

The bacterium Myxococcus xanthus undergoes multicellular development during times of nutritional stress and uses extracellular signals to coordinate cell behavior. C-signal affects gene expression late in development, including that of Omega4499, an operon identified by insertion of Tn5 lac into the M. xanthus chromosome. The Omega4499 promoter region has several sequences in common with those found previously to be important for expression of other C-signal-dependent promoters. To determine if these sequences are important for Omega4499 promoter activity, the effects of mutations on expression of a downstream reporter gene were tested in M. xanthus. Although the promoter resembles those recognized by Escherichia coli sigma(54), mutational analysis implied that a sigma(70)-type sigma factor likely recognizes the promoter. A 7-bp sequence known as a C box and a 5-bp element located 6 bp upstream of the C box have been shown to be important for expression of other C-signal-dependent promoters. The Omega4499 promoter region has C boxes centered at -33 and -55 bp, with 5-bp elements located 7 and 8 bp upstream, respectively. A multiple-base-pair mutation in any of these sequences reduced Omega4499 promoter activity more than twofold. Single base-pair mutations in the C box centered at -33 bp yielded a different pattern of effects on expression than similar mutations in other C boxes, indicating that each functions somewhat differently. An element from about -81 to -77 bp exerted a twofold positive effect on expression but did not appear to be responsible for the C-signal dependence of the Omega4499 promoter. Mutations in sigD and sigE, which are genes that encode sigma factors, reduced expression from the Omega4499 promoter. The results provide further insight into the regulation of C-signal-dependent genes, demonstrating both shared and unique properties among the promoter regions so far examined.

Base Sequence↗
PubMed1985

Growth of the Escherichia coli cell envelope.

The growth pattern of the Escherichia coli envelope was studied by immunoelectron microscopy, using the outer membrane protein LamB specifically labelled by a double antibody gold particle technique. An operon fusion placing the lamB gene under lac promoter control permitted rapid turn-off of LamB synthesis. In the generation following turn-off no lamB-free regions appeared, strongly suggesting that bulk outer membrane material is not inserted in restricted growth zones.

Bacterial Outer Membrane Proteins↗
PubMed2007

The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding.

In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli. This proposal was followed by an explication of allosteric behavior by Monod and colleagues. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function.

Allosteric Regulation↗
PubMed1998

Lac and lambda repressors relieve silencing of the Escherichia coli bgl promoter. Activation by alteration of a repressing nucleoprotein complex.

The Escherichia coli bgl promoter is kept in a repressed state by silencer sequences which flank the promoter and by the histone-like protein H-NS. Silencing of the bgl promoter is likely due to the formation of a repressing nucleoprotein complex of which H-NS is an essential component. Here, we show that silencing is abolished by the binding of Lac or lambda repressors to their respective operators that were inserted within the bgl upstream silencer. Efficient activation of bgl operon transcription by Lac and lambda repressors was independent of the position and phasing of the operators with respect to the promoter. Activation by Lac and lambda repressors as shown here is unprecedented. We conclude that the activation of bgl transcription by both repressors is achieved by a novel mechanism, that is by alteration of the repressing nucleoprotein complex rather than by protein-protein interactions with RNA polymerase and the catabolite activator protein, CAP.

Bacterial Proteins↗
PubMed1985

Identification of osmoresponsive genes in Escherichia coli: evidence for participation of potassium and proline transport systems in osmoregulation.

Mu d1(Ap lac)-generated operon fusions were used in the identification of genes in Escherichia coli whose transcriptional expression is altered by changes in the osmolarity of the growth medium. One such osmoresponsive gene, designated osrA, was induced 400-fold when the osmolarity of the medium was increased with the addition of either ionic or neutral impermeable solutes but was not induced with glycerol, which is freely permeable across the cell membrane. osrA was mapped to 57.5 min and was shown to be transcribed clockwise on the E. coli chromosome. The ability of small concentrations of L-proline to promote the growth of E. coli in high-osmolar medium was shown to have been specifically lost in osrA mutants; other lines of evidence were also obtained to support the notion that osrA codes for an osmoresponsive L-proline transport system and is homologus to proU in Salmonella typhimurium. A second osmoresponsive operon identified was kdp, which codes for an inducible K+-transport system in E. coli. kdp expression was elevated 12-fold when the osmolarity of the growth medium was increased with the addition of impermeable ionic solutes but not neutral solutes; furthermore, osmoresponsivity of kdp expression was demonstrable only in K+-limiting media. kdp mutants were able to grow normally in high-osmolar media, but strains defective in both kdp and trkA (a gene for a second major K+-transport system) displayed an osmosensitive phenotype. The results suggest that transport systems for L-proline and K+, specified by osrA (proU) and kdp, respectively, play independent and important roles in osmoregulation in E. coli. A third osmoresponsive gene that was identified was lamB, which codes for an outer membrane protein for maltodextrin transport and lambda phage adsorption; its expression was reduced fourfold with increase in the osmolarity of the growth medium.

Biological Transport↗
PubMed1997

The inducible lactose operator-repressor system is functional in the whole animal.

Mouse liver cell lines that bear a stably integrated lactose operon repressor (lacI) gene and a Ha-ras gene linked to a lactose operator-containing SV40 early promoter were generated. When grown in medium containing more than 0.1 mM isopropyl beta-D-thiogalactoside (IPTG), the Ha-ras gene was induced up to 20-fold. Maximum induction of Ha-ras gene expression occurred after 12 h of exposure. The tumorigenicity of these cell lines in syngeneic mice was enhanced when the mice were maintained on drinking water containing 12.5 mM IPTG. Ha-ras gene expression in tumors was strongly induced in the presence of IPTG in vivo. Induction of Ha-ras gene expression in mice was consistently observed after 48 hr of exposure to drinking water containing IPTG. This system provides an approach for studying the function of oncogene in vivo as well as other genes of interest.

Animals↗
PubMed1985

Entry exclusion determinant(s) of IncN plasmid pKM101.

pKM101 renders its host a poor recipient in conjugal matings with genetically distinguishable derivatives of itself. The gene(s) primarily responsible for this, denoted eex, is located in between genes required for both conjugal transfer and sensitivity to donor-specific bacteriophage, although it itself is not necessary for transfer. A gene linked to, or coincident with, the region needed for vegetative plasmid replication also inhibited establishment of related plasmids under certain conditions. Construction of an operon fusion between eex and the Escherichia coli lac promoter has shown that this gene is transcribed in a clockwise fashion on the circular map of pKM101. To date, we have not been able to visualize a protein product(s) of the eex gene(s).

Bacteriophages↗
PubMed1987

Isolation and characterization of Salmonella typhimurium glyoxylate shunt mutants.

Growth of Salmonella typhimurium on acetate as a sole carbon source requires expression of the glyoxylate shunt; however, the genes for the glyoxylate shunt enzymes have not been previously identified in S. typhimurium. In this study, we isolated transposon insertions in the genes for the two unique enzymes of this pathway, aceA (isocitrate lyase) and aceB (malate synthase). The aceA and aceB genes were located at 89.5 min on the S. typhimurium genetic map. Genetic linkage to nearby loci indicated that the relative gene order is purDJ metA aceB aceA. Transposon insertions in aceB were polar on aceA, suggesting that the genes form an operon transcribed from aceB to aceA. Transcriptional regulation of the aceBA operon was studied by constructing mini-Mu d(lac Kan) operon fusions. Analysis of these fusions indicated that expression of the aceBA operon is regulated at the level of transcription; the aceBA genes were induced when acetate was present and repressing carbon sources were absent. Although glucose represses expression of the aceBA operon, repression does not seem to be mediated solely by cyclic AMP-cyclic AMP receptor protein complex. Mutants with altered regulation of the aceBA operon were isolated.

Acetates↗
PubMed1985

Ultraviolet radiation-induced derepression of the lactose operon of E. coli.

The effect of ultraviolet irradiation of a regulatory protein, the lac repressor, on its interactions with operator DNA is investigated by spectroscopic and electrophoresis methods. A second set of experiments is performed to assay the capacity of the system containing the irradiated repressor to be induced by IPTG. The protein-nucleic acid interactions are modified upon ultraviolet irradiation of the repressor. The inducer becomes ineffective and repressor stays "locked" to DNA in conditions in which the native repressor is released from the system. These facts are discussed in terms of genes repression and of promotion step in ultraviolet induced carcinogenesis.

Circular Dichroism↗
PubMed2001

Salmonella enterica serotype typhimurium elicits cross-immunity against a Salmonella enterica serotype enteritidis strain expressing LP fimbriae from the lac promoter.

The biological significance of fimbrial phase variation in Salmonella serotypes is currently unknown. Exposure to long polar (LP) fimbriae of Salmonella enterica serotype Typhimurium results in selection against lpf phase ON cells of serotype Enteritidis during a subsequent challenge, suggesting that fimbrial phase variation may be a mechanism to evade cross-immunity between Salmonella serotypes. This notion was tested by assessing the effect of an immune response against serotype Typhimurium LP fimbriae on colonization of mice with a serotype Enteritidis mutant in which the lpf promoter region was replaced with the Escherichia coli lac promoter. During a challenge with a serotype Enteritidis mutant carrying the lac promoter in front of the lpf operon, significantly lower numbers were recovered from organs and feces of mice previously immunized with an lpf phase ON culture of serotype Typhimurium than from mice not previously exposed to LP fimbriae. Immunization with the lpf phase ON culture of serotype Typhimurium elicited antibodies that cross-reacted with a purified gluthathione-S-transferase-LpfA fusion protein of serotype Enteritidis. These data suggested that cross-immunity against LP fimbrial proteins cannot be evaded if phase variation on the transcriptional level is prevented by expressing the lpf operon from the lac promoter. These data hence support the idea that phase variation of LP fimbriae is a mechanism to evade cross-immunity between serotypes Enteritidis and Typhimurium.

Animals↗
PubMed1992

Repression of the E. coli lactose operon by cooperation between two individually unproductive "half-operator" sites.

Two remote and weak lac regressor binding sites can be used jointly to repress the synthesis of beta-galactosidase in E. coli, while they cannot separately. When this result is discussed in reference to the various modes of cooperation between the sites, it supports with a new approach a model implying the simultaneous binding of lac repressor to both sites with the formation of a DNA loop. In connection with this point, we present a new strategy to detect cooperative interactions in vivo, based on the asymmetry of the DNA binding site, formally equivalent here to a half-site, heterodimerization of the protein, and influence of orientation of the sites on repression at short and long distance.

DNA, Bacterial↗
PubMed2002

Galactose and lactose genes from the galactose-positive bacterium Streptococcus salivarius and the phylogenetically related galactose-negative bacterium Streptococcus thermophilus: organization, sequence, transcription, and activity of the gal gene products.

Streptococcus salivarius is a lactose- and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (beta-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3' end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities.

Base Sequence↗
PubMed1981

Operon fusions in the nitrate reductase operon and study of the control gene nir R in Escherichia coli.

Strains carrying operon fusions between the promotor of the chl I gene and the lac structural genes were constructed. From these strains in which the expression of the lac genes is under the control of both nitrate and oxygen, spontaneous regulatory mutants were selected: (i) mutants which synthesize beta-galactosidase constitutively in anaerobiosis; (ii) mutants in which beta-galactosidase synthesis is no longer repressed by oxygen. Introduction of the nir R mutated allele into strains carrying these fusions resulted in the total loss of beta-galactosidase synthesis, confirming that nir R is a regulatory gene controlling the expression of the biosynthesis of the nitrate reductase.

Anaerobiosis↗
PubMed1989

Outreading promoters are located at both ends of the gamma-delta transposon.

Two plasmids were isolated containing oppositely oriented gamma-delta insertions between the wild-type transcription initiation site of the nifHDKY operon and the nifH coding sequence. The nifHDKY promoter of Klebsiella pneumoniae, similar to other nitrogen fixation (nif) promoters, normally requires the products of ntrA and nifA for activity. Mutations that allowed constitutive expression of the nifHDKY operon were searched for by transforming a plasmid, containing the regulatory region of this operon followed by an in-frame nifH'-'lacZ fusion, into a Lac- Escherichia coli strain (which contains no nifA) and screening for Lac+ derivatives. The plasmids described here were isolated from such derivatives and directed the constitutive expression of beta-galactosidase. Deletion analysis indicated that gamma-delta promoters other than those transcribing tnpA and tnpR were involved in this expression. Nuclease S1 mapping revealed outward-reading transcription initiation sites in both the gamma end and the delta end of the transposon. Most interestingly, one initiation site on each end was located in corresponding positions within the terminal inverted repeats. The sites were in the center of the longest sequence, of 12 bp, contiguously conserved between the terminal inverted repeats of gamma-delta and the related transposon Tn3. In gamma-delta and Tn3, this sequence has been recently implicated in transposase binding. These results suggest a possible interrelationship between transcription from the "end" promoters and transposition.

Base Sequence↗