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PubMed2002

Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA-mediated regulation.

Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars. Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA). CcpA is required for induction of the gapA operon by glucose. Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region. Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS). A functional CcpA is needed for efficient transport of these sugars. This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS. In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser-46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer. A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon. We conclude that a low-molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.

Adenosine Triphosphate↗

PubMed2003

Inducible bacteriocin production in Lactobacillus is regulated by differential expression of the pln operons and by two antagonizing response regulators, the activity of which is enhanced upon phosphorylation.

Expression of the five (pln) operons involved in the bacteriocin production of Lactobacillus plantarum C11 is regulated by a so-called pheromone-based signal-transducing network, in which the peptide pheromone (PlnA) induces bacteriocin production through the action of a histidine protein kinase (PlnB) and two antagonizing response regulators (PlnC as an activator and PlnD as a negative regulator). All pln-regulated promoters contain a conserved pair of direct repeats that serve as binding sites for PlnC and PlnD. In the present work, we show that the five PlnA-responsive operons are differentially expressed with regard to both timing and strength, and that the pheromone triggers a strong autoactivating loop of the regulatory unit (plnABCD) during an early stage of induction that gradually leads to enhanced activation of the other operons. The transport operon (plnGHSTUV), which is involved in the secretion of the pheromone and bacteriocins, is also expressed relatively early upon induction, but is quickly turned off soon after peak expression. Further investigation of the various promoters revealed that, although subtle differences within the promoter regions could account for the observed differential regulation, the presence of a downstream promoter-proximal sequence in one promoter was found to cause delayed peak activity. How phosphorylation regulates the activity of the pln response regulators was also accessed by direct mutagenesis at their phosphorylation sites. It was found that the two response regulators exert activity at two different levels: a low level when they are not phosphorylated and an elevated level when they are phosphorylated. The present data demonstrate that bacteriocin production in L. plantarum C11 is a highly regulated process, in which different regulatory mechanisms are applied to fine tune the timing and strength of expression of the five pln operons.

Amino Acid Sequence↗

PubMed1994

The psaA operon pre-mRNA of the Euglena gracilis chloroplast is processed into photosystem I and II mRNAs that accumulate differentially depending on the conditions of cell growth.

The chloroplast genome of Euglena gracilis contains a psaA operon which encodes a lysine tRNA gene, trnK; psaA and psaB photosystem I genes, and psbE, psbF, psbL and psbJ photosystem II genes. The pre-mRNA of the psaA operon undergoes a complex processing pathway of 5' and 3' tRNA processing, splicing of 11 group II introns and one group II twintron, plus three intercistronic RNA cleavage events. The accumulated transcripts of the psaA operon have been characterized by Northern hybridization, S1 nuclease analysis and primer extension RNA sequencing. The mature 5' end of the psaA-psaB-psbE-psbF-psbL-psbJ hexacistronic transcript lies 8 nt downstream of the trnK gene, and is the result of intercistronic trnK-psaA cleavage. Other intercistronic processing events occur between the psaA and psaB genes and the psaB and psbE genes. Processing at the latter site produces a dicistronic mRNA of PSI genes and a tetracistronic mRNA of PSII genes. The PSI dicistronic transcript is further processed to monocistronic psaA and psaB mRNAs. Secondary structural motifs within the intercistronic regions may be recognition sites for processing. The steady-state levels of psaA operon mRNAs from Euglena grown under several different conditions have been determined. Accumulated transcripts from all growth conditions are spliced, and a proportion are also processed at the intercistronic sites. The products of intercistronic processing increase from heterotrophic dark- to heterotrophic light-grown Euglena, and from heterotrophic light- to photoautotrophic light-grown Euglena. The differential accumulation of psaA operon mRNAs may be a means of chloroplast gene regulation or, alternatively, a consequence of gene expression during chloroplast development.

Animals↗

PubMed2003

Dynamic model of Escherichia coli tryptophan operon shows an optimal structural design.

A mathematical model has been developed to study the effect of external tryptophan on the trp operon. The model accounts for the effect of feedback repression by tryptophan through the Hill equation. We demonstrate that the trp operon maintains an intracellular steady-state concentration in a fivefold range irrespective of extracellular conditions. Dynamic behavior of the trp operon corresponding to varying levels of extracellular tryptophan illustrates the adaptive nature of regulation. Depending on the external tryptophan level in the medium, the transient response ranges from a rapid and underdamped to a sluggish and highly overdamped response. To test model fidelity, simulation results are compared with experimental data available in the literature. We further demonstrate the significance of the biological structure of the operon on the overall performance. Our analysis suggests that the tryptophan operon has evolved to a truly optimal design.

Escherichia coli↗

PubMed1999

Methanobacterium thermoautotrophicum encodes two multisubunit membrane-bound [NiFe] hydrogenases. Transcription of the operons and sequence analysis of the deduced proteins.

Two gene groups, designated energy converting hydrogenase A (eha) and energy converting hydrogenase B (ehb), each encoding a putative multisubunit membrane-bound [NiFe] hydrogenase, were identified in the genome of Methanobacterium thermoautotrophicum. The length of the transcription units was determined using reverse transcription (RT)-PCR. The eha operon (12.5 kb) and the ehb operon (9.6 kb) were found to be composed of 20 and 17 open reading frames, respectively. Competitive RT-PCR was used to compare the amounts of eha and ehb transcripts with the amounts of transcripts of genes encoding the M. thermoautotrophicum catabolic enzymes cyclohydrolase (mch) and a subunit of heterodisulfide reductase (hdrC). In cells grown under conditions in which H2 was nonlimiting, the eha transcripts were 250-fold and 125-fold less abundant and the ehb transcripts were approximately sixfold and threefold less abundant than the hdrC and mch transcripts, respectively. In cells grown under H2 limitation, the amounts of eha and ehb transcripts were about threefold higher than in cells grown with sufficient H2 when compared to the amounts of hdrC and mch transcripts. Sequence analysis of the deduced proteins indicated that the eha and ehb operons each encode a [NiFe] hydrogenase large subunit, a [NiFe] hydrogenase small subunit, and two conserved integral membrane proteins. These proteins show high sequence similarity to subunits of the Ech hydrogenase from Methanosarcina barkeri, Escherichia coli hydrogenases 3 and 4, and CO-induced hydrogenase from Rhodospirillum rubrum, all of which form a distinct group of multisubunit membrane-bound [NiFe] hydrogenases and show high sequence similarity to the energy-conserving NADH:quinone oxidoreductase (complex I) from various organisms. In addition to these four subunits, the eha operon encodes a 6[4Fe-4S] polyferredoxin, a 10[4F-4S] polyferredoxin, four nonconserved hydrophilic subunits, and 10 nonconserved integral membrane proteins; the ehb operon encodes a 2[4Fe-4S] ferredoxin, a 14[4Fe-4S] polyferredoxin, two nonconserved hydrophilic subunits, and nine nonconserved integral membrane proteins. A function of these putative membrane-bound [NiFe] hydrogenases as proton pumps involved in endergonic reactions, such as the synthesis of formylmethanofuran from CO2, H2 and methanofuran, is discussed.

Amino Acid Sequence↗

PubMed1999

Signalling by the fungus Pythium ultimum represses expression of two ribosomal RNA operons with key roles in the rhizosphere ecology of Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 produces antifungal metabolites that protect the roots of sugarbeet from the fungus Pythium ultimum. The phytopathogen, in turn, has the ability to downregulate the expression of genes fundamental to the rhizosphere competence of the bacterial strain. This paper describes the characterization of two of these genes, which were isolated by screening a mini-Tn5::lacZ mutant bank for differential expression of beta-galactosidase in the presence of P. ultimum. In order to identify the genes affected in reporter mutants SF3 and SF5, the transposons and flanking regions were cloned. Sequence analysis of the regions flanking the transposons in SF3 revealed that mini-Tn5::lacZ had inserted into a tRNA(Ile) gene, which maps within a ribosomal RNA (rrn) operon. In SF5, the transposon inserted between the promoter of a second rrn operon and a gene encoding a 16S rRNA. Southern blot analysis demonstrated that there are five rrn operons in P. fluorescens F113 and that the transposons in SF3 and SF5 had inserted into two different operons. Further characterization of these mutants suggests that their reduced rhizosphere competence is not the result of reduced viability in the short term but may be accounted for partly by reduced growth rates under conditions that support rapid growth. Analysis of lacZ expression in the reporter mutants indicate that the marked rrn operons are regulated differently, suggesting different physiological roles.

Base Sequence↗

PubMed1969

DNA-directed peptide synthesis. VI. Regulating the expression of the lac operon in a cell-free system.

The operon model was proposed by Jacob and Monod in 1961 to explain the regulation of enzyme synthesis in bacteria. The model requires two genetic elements: a locus i which directs the synthesis of a repressor, and a locus called the operon which contains an operator gene and associated structural genes. The repressor is believed to stop RNA and enzyme synthesis by combining with the operator site. The inhibiting effect of the repressor is reversed by an inducer which interacts directly with the repressor. A DNA-dependent cell-free system has been developed for studying the regulation of the operon containing the gene for beta-galactosidase enzyme. In this cell-free system, gene activity is indirectly measured by the synthesis of part of the enzyme. Qualitatively, the regulation of the operon in the cell-free system is similar to its regulation in intact cells. The cell-free system is used to advantage in experiments where it is desirable to use predetermined levels of repressor, operon, and inducer. Such quantitative studies suggest that two inducer molecules are most effective in removing the repressor from the gene.

Cell-Free System↗

PubMed1973

Isolation of the self-regulated repressor protein of the Hut operons of Salmonella typhimurium.

In Salmonella typhimurium the structural genes of the enzymes responsible for histidine utilization (hut) are clustered in two adjacent operons. A single repressor regulates both operons. The repressor itself is a member of one of the hut operons and, thus, regulates its own synthesis. We have assayed the hut repressor by its ability to bind radioactive DNA to nitrocellulose filters. The binding is specific for DNA bearing the hut operons, and the binding is abolished by the inducer, urocanate. As a member of one of the hut operons, the repressor is inducible, subject to catabolite repression, and affected by a promoter mutation.

Acrylates↗

PubMed1981

Osmotic control of kdp operon expression in Escherichia coli.

Turgor pressure, the difference in osmotic pressure across the inner membrane, has been found to regulate expression of the kdp operon in Escherichia coli. The kdp operon codes for a high-affinity repressible transport system for the uptake of potassium. We have studied the regulation of Kdp expression in a strain in which the gene for beta-galactosidase, lacZ, was placed under control of the kdp promotor. Neither internal nor external K+ concentrations directly controlled Kdp expression. Only when the external K+ concentration was reduced to the point of limiting growth was the kdp operon expressed. An increase in external osmolarity at constant K+ concentration, a procedure that reduces turgor pressure, caused expression of the kdp operon. As the magnitude of the osmotic shift was increased, corresponding to greater decreases in turgor pressure, the amount of Kdp expression also increased. The kdp operon thus appears to be controlled by changes in a physical force, the turgor pressure.

Biological Transport, Active↗

PubMed1982

Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli.

There are seven ribosomal RNA operons (rrn operons) in Escherichia coli. A single rrn operon was amplified by use of a multicopy recombinant plasmid containing a complete rrnH operon. rrnH thereby has the potential to contribute a greater fraction of the rRNA found in ribosomes. Erythromycin-resistant mutants were isolated from cells containing the plasmid, and at least one mutation to resistance was shown to reside in rrnH on the plasmid. Erythromycin resistance was retained when a major deletion was introduced into the 16S rRNA gene and was abolished by deletions that affect the 16S and 23S rRNA genes but do not alter the 5S rRNA gene or non-rrnH DNA. Cell-free S30 protein-synthesizing extracts from cells containing the mutant plasmid have an increased resistance to erythromycin. The selection procedure used to isolate erythromycin-resistance mutations in rrnH may allow, with minor modifications, the isolation of mutations in rrn operons that change resistance of the ribosome to other antibiotics or that alter other properties of ribosomes.

DNA, Recombinant↗

PubMed1985

Electron microscopic visualization of trp operon expression in Salmonella typhimurium.

Transcriptional activity of plasmids carrying wild-type and mutant trp operons was visualized in cell lysates of Salmonella typhimurium. Plasmid and transcription-unit sizes varied with the size of the cloned operon. Following 3-(3-indolyl)acrylic acid derepression, all operons of a particular type exhibited the same high level of transcriptional activity. An estimated 11-14 transcripts must be initiated each minute to maintain the 190-base-pair spacing of RNA polymerases observed on the promoter-proximal half of the wild-type trp operon. A decline in RNA polymerase density was observed on promoter-distal portions of cloned trp operons, which may be attributable to premature transcription termination accompanying translation inhibition due to indolylacrylic acid's interference with normal tryptophanyl-tRNA synthetase activity.

Chromosome Deletion↗

PubMed1986

Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida.

The in vivo transcription start sites of the nah and sal operons of the NAH7 plasmid were determined by S1 nuclease mapping and the nucleotide sequence surrounding these transcription start sites was determined. Since expression of both of these operons is coordinately controlled by the product of the transcriptional activator gene nahR, the sequences were compared to locate potential sites involved in common regulation. In the 100-base-pair region preceding transcription start sites of both operons, three regions of extensive homology were found and may be involved in nahR-mediated transcriptional control: between -80 and -60 with 81% homology; between -40 and -28 with 75% homology; between -1 and +15 with 70% homology. Comparison of the promoter sequences of nah and sal with the analogous sequences of the xylABC and xylDEFG operons of the TOL plasmid showed little homology between the 5' regions of these two sets of positively regulated hydrocarbon degradation operons. In addition, the transcription start site of the nahR regulatory gene was located and its promoter sequence was determined. The nahR promoter overlapped at the -35 position of the sal promoter; however, the nahR gene is transcribed in the opposite direction. Sequences similar to the consensus sequences of Escherichia coli promoters (at -35 and -10) were found in nah, sal, and nahR at the appropriate positions.

Chromosome Mapping↗

PubMed1987

The gluconate operon gnt of Bacillus subtilis encodes its own transcriptional negative regulator.

The gluconate (gnt) operon of Bacillus subtilis consists of four gnt genes; the second and third genes code for gluconate kinase (gluconokinase, EC 2.7.1.12) and gluconate permease, respectively. A fragment carrying the promoter of this operon (gnt promoter) and the first gene (gntR) was subcloned into a promoter probe vector (pPL603B). Repression of the expression of cat-86 gene, encoded in the vector portion of a constructed plasmid (pgnt21), that is under the control of the gnt promoter was removed by gluconate. The results of deletion analysis and of insertional inactivation of the gntR gene cloned in pgnt21 suggested that the product of the gntR gene, actually synthesized as a 29-kDa protein in vivo, is involved in repression of the gnt promoter. A 4-base-pair insertional mutation within the gntR gene constructed in vitro was introduced into the B. subtilis chromosomal gnt operon by use of linkage of the 4 base pairs to gntK10 in transformation. The introduced mutation gntR1 caused the constitutive expression of the gluconate kinase and gluconate permease genes. S1 nuclease analysis indicated that the mRNA of this operon is synthesized in the gntR1 strain and amounts of mRNA are not changed very much by gluconate, which acts as an inducer in the wild-type gene. These results strongly indicate that the gntR gene codes for a transcriptional negative regulator for the gnt operon.

Bacillus subtilis↗

PubMed1995

Effect of the FruR regulator on transcription of the pts operon in Escherichia coli.

The promoters of the pts operon of Escherichia coli are controlled by the cyclic AMP receptor protein (CRP) complexed with cAMP (CRP.cAMP). In addition, glucose stimulates pts operon expression in vivo. The pts promoter region has a fructose repressor (FruR)-binding site (the FruR box) that partially overlaps with one of the CRP.cAMP-binding sites. The effects of the pleiotropic transcriptional regulator FruR on pts operon expression were studied to determine whether the in vivo glucose effect on pts operon expression is mediated by FruR. In vitro, FruR can repress P1b transcription, which is activated by CRP.cAMP, and restore P1a transcription, which is repressed by CRP.cAMP. FruR can displace CRP.cAMP from its binding site in the presence of RNA polymerase even though FruR and CRP.cAMP can bind simultaneously to their partially overlapping binding sites in the absence of RNA polymerase. FruR had very little effect on the transcription of the P0 promoter, which is most important for regulation by glucose. Consistent with the in vitro results, pts P0 transcription did not increase as much in cells grown in the presence of fructose or in fruR- mutant cells as in cells grown in the presence of glucose. These results suggest that FruR alone does not mediate the in vivo glucose effect on pts operon expression.

Bacterial Proteins↗

PubMed1996

Leucine-responsive regulatory protein-DNA interactions in the leader region of the ilvGMEDA operon of Escherichia coli.

The leucine-responsive regulatory protein (Lrp) regulates the expression of many operons in Escherichia coli including several involved in the metabolism of the branched-chain amino acids, L-isoleucine, L-valine, and L-leucine. The ilvGMEDA operon contains the genes for four of the five enzymes of the common pathway for the biosynthesis of these amino acids. A high affinity, consensus-like Lrp-DNA binding site has been identified at an unusual position in the leader region of this operon 226 base pairs downstream of the transcriptional initiation site between the attenuator and the ilvG gene. Binding to this site facilitates the cooperative binding of a second Lrp protomer to an adjacent, upstream, secondary site. At higher Lrp concentrations, binding to a third site is observed. Chemical, enzymatic, and alkylation protection and interference footprinting experiments demonstrate that the Lrp homodimer contacts the DNA helix at symmetrical half-sites present in adjacent major grooves and that the primary and secondary binding sites are separated by one helical turn and aligned along the same face of the DNA helix. In vivo, Lrp represses transcription through the leader-attenuator region of the ilvGMEDA operon. Lrp-dependent production of attenuated RNA transcripts is also observed in vitro. No transcriptional effects are observed, in vivo or in vitro, in the absence of an intact Lrp primary binding site. A possible physiological role for Lrp in the regulation of ilvGMEDA operon expression is discussed.

Bacterial Proteins↗

PubMed1999

Transcriptional inhibition of the operon for the spermidine uptake system by the substrate-binding protein PotD.

Inhibition of spermidine uptake in Escherichia coli, which occurs in the presence of accumulated polyamines, has been studied using the spermidine uptake operon consisting of the potA, -B, -C, and -D genes. Transcription of the potABCD operon was inhibited by PotD, a spermidine-binding protein usually found in the periplasm, and the inhibitory effect of PotD was increased by spermidine. Transcription was not affected by bovine serum albumin, PotA, or PotF, suggesting that the effects of PotD are specific to the PotD protein. In the presence of 8 mM spermidine, a 50% inhibition of transcription was observed with a molar ratio of approximately 1:500 of template DNA:PotD. It was found that PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene. Binding of PotD to the downstream site was stimulated by spermidine. Overexpression of PotD in Escherichia coli DH5alpha inhibited the uptake of spermidine, the synthesis of PotABCD mRNA, and expression of a lacZ reporter gene fused downstream of a potA gene containing the PotD binding sites. In cells overexpressing PotD, a large amount of PotD existed as PotD precursor in spheroplasts. Our results indicate that PotD precursor can also inhibit spermidine transport. The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using mutated PotD proteins. Thr-35 and Ser-85 of PotD were found to be important for this interaction. These results suggest that transcription of the spermidine transport (potABCD) operon is inhibited in vivo by PotD precursor rather than PotD through its binding to two regions close to the transcriptional initiation site of the operon.

Amino Acid Sequence↗

PubMed2003

Interactions of the cbbII promoter-operator region with CbbR and RegA (PrrA) regulators indicate distinct mechanisms to control expression of the two cbb operons of Rhodobacter sphaeroides.

In a previous study (Dubbs, J. M., Bird, T. H., Bauer, C. E., and Tabita, F. R. (2000) J. Biol. Chem. 275, 19224-19230), it was demonstrated that the regulators CbbR and RegA (PrrA) interacted with both promoter proximal and promoter distal regions of the form I (cbb(I)) promoter operon specifying genes of the Calvin-Benson-Bassham cycle of Rhodobacter sphaeroides. To determine how these regulators interact with the form II (cbb(II)) promoter, three cbbF(II)::lacZ translational fusion plasmids were constructed containing various lengths of sequence 5' to the cbb(II) operon of R. sphaeroides CAC. Expression of beta-galactosidase was monitored under a variety of growth conditions in both the parental strain and knock-out strains that contain mutations that affect synthesis of CbbR and RegA. The binding sites for both CbbR and RegA were determined by DNase I footprinting. A region of the cbb(II) promoter from +38 to -227 bp contained a CbbR binding site and conferred low level regulated cbb(II) expression. The region from -227 to -1025 bp contained six RegA binding sites and conferred enhanced cbb(II) expression under all growth conditions. Unlike the cbb(I) operon, the region between -227 and -545 bp that contains one RegA binding site, was responsible for the majority of the observed enhancement. Both RegA and CbbR were required for maximal cbb(II) expression. Two potentially novel and specific cbb(II) promoter-binding proteins that did not interact with the cbb(I) promoter region were detected in crude extracts of R. sphaeroides. These results, combined with the observation that chemoautotrophic expression of the cbb(I) operon is RegA independent, indicated that the mechanisms controlling cbb(I) and cbb(II) operon expression during chemoautotrophic growth are quite different.

Bacterial Proteins↗

PubMed2002

A powerful non-homology method for the prediction of operons in prokaryotes.

MOTIVATION: The prediction of the transcription unit organization of genomes is an important clue in the inference of functional relationships of genes, the interpretation and evaluation of transcriptome experiments, and the overall inference of the regulatory networks governing the expression of genes in response to the environment. Though several methods have been devised to predict operons, most need a high characterization of the genome analysed. Log-likelihoods derived from inter-genic distance distributions work surprisingly well to predict operons in Escherichia coli and are available for any genome as soon as the gene sets are predicted. RESULTS: Here we provide evidence that the very same method is applicable to any prokaryotic genome. First, the method has the same efficiency when evaluated using a collection of experimentally known operons of Bacillus subtilis. Second, operons among most if not all prokaryotes seem to have the same tendencies to keep short distances between their genes, the most frequent distances being the overlaps of four and one base pairs. The universality of this structural feature allows us to predict the organization of transcription units in all prokaryotes. Third, predicted operons contain a higher proportion of genes with related phylogenetic profiles and conservation of adjacency than predicted borders of transcription units.

Algorithms↗