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At least 343 records · Page 19Linked to original sources

PubMed2000

TfdR, the LysR-type transcriptional activator, is responsible for the activation of the tfdCB operon of Pseudomonas putida 2, 4-dichlorophenoxyacetic acid degradative plasmid pEST4011.

In Pseudomonas putida EST4021, the tfdCB operon of plasmid pEST4011 encodes enzymes involved in 2,4-dichlorophenoxyacetic acid degradation. We have identified a gene, tfdR, important for the regulation of the tfdCB operon. Sequence analysis of the tfdR gene revealed an open reading frame with amino acid sequence similar to the LysR family of transcriptional activators. The tfdR gene is located upstream and transcribed divergently from the tfdCB operon. Utilizing primer extension analysis, the transcription initiation sites of the gene tfdR and the tfdCB operon were localized 85 (84)bp and 292bp upstream from the coding sequences of these genes, respectively. Multiple sequence analysis revealed that the genes tfdR, tfdC and tfdB of plasmid pEST4011 are most similar to the regulatory gene tfdR and the module 2 genes tfdC(II) and tfdB(II) of pJP4, respectively. The promoter-operator sequences of tfdR and its target tfdCB operon of pEST4011 have regions with highly conserved nucleotides characteristic for the catechol-subgroup LysR-type transcriptional activators. We showed that the pEST4011 tfdR gene product activates the expression of the tfdCB operon and the effector molecule for TfdR is 2,4-dichloro-cis,cis-muconate. Our data indicate that the structure and the mode of regulation of tfd genes are similar, despite the bacteria being isolated from different geographical regions.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2000

Fructose utilization and pathogenicity of Spiroplasma citri: characterization of the fructose operon.

Transposon Tn4001 mutagenesis of Spiroplasma citri wild-type (wt) strain GII-3 led to the isolation and characterization of non-phytopathogenic mutant GMT 553. In this mutant, transposon Tn4001 is inserted within the first gene of the fructose operon. This operon comprises three genes. The first gene (fruR) codes for a putative transcriptional regulator protein belonging to the deoxyribonucleoside repressor (DeoR) family. Sequence similarities and functional complementation of mutant GMT 553 with different combinations of the wt genes of the fructose operon showed that the second gene (fruA) codes for the permease of the phosphoenolpyruvate:fructose phosphotransferase system (fructose PTS), and the third, fruK, for the 1-phosphofructokinase (1-PFK). Transcription of the fructose operon in wt strain GII-3 resulted in two messenger RNAs, one of 2.8kb and one of 3.8kb. Insertion of Tn4001 in the genome of mutant GMT 553 abolished transcription of the fructose operon, and resulted in the inability of this mutant to use fructose. Functional complementation experiments demonstrated that fructose utilization was restored with fruR-fruA-fruK, fruA-fruK or fruA only, but not with fruR or fruR-fruA. This is the first time that an operon for sugar utilization has been functionally characterized in the mollicutes.

Amino Acid Sequence↗

PubMed1992

Interactions between DNA-bound transcriptional regulators of the Escherichia coli gal operon.

Regulation of the initiation of gene transcription from the gal operon of Escherichia coli is activated by the binding of CAP (catabolite activator protein) to a site centered at base pair -41.5 relative to the S1 start site of transcription. This operon is repressed by the specific binding of Gal repressor (GalR) to two operators, OE and OI, centered at -60.5 and +53.5, respectively. It has been proposed that this negative regulation results from the interaction of GalR dimers bound to OE and OI to form a protein-mediated "looped complex" [cf. Adhya, S. (1989) Annu. Rev. Genet. 23, 207-230]. In order to test whether DNA-bound CAP would facilitate or inhibit the binding of GalR, the simultaneous binding of these proteins was studied by quantitative DNase I footprint titration analysis. These studies demonstrate that GalR binding is noncooperative in the presence and in the absence of CAP and that GalR and CAP bind to the gal operon independently. No evidence was found that CAP stabilizes a putative Gal repressor-mediated protein-DNA looped complex. It has been shown that the gal operon can be negatively regulated by the binding of Lac repressor (LacI) to a gal operon in which OE and OI were both modified to be recognized by LacI [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683-9687]. In contrast to GalR, LacI binds to the chimeric gal operon with moderate cooperativity via the formation of a stable protein-DNA looped complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMedDate not supplied

Nonlinear dynamics of regulation of bacterial trp operon: model analysis of integrated effects of repression, feedback inhibition, and attenuation.

The trp operon encodes the five genes for the enzymes required to convert chorismate to tryptophan, and its switching on and off is controlled by both feedback repression and attenuation in response to different levels of tryptophan in the cell. Repression of the operon occurs when tryptophan concentration is high, and attenuation fine-tunes the transcription level at a lower cellular concentration of tryptophan. An extended mathematical model is established in this study to describe the switching on and off of the trp operon by considering the integrated effects of repression and attenuation. The influences of cell growth rate on the biosynthesis of tryptophan, stability and dynamic behavior of the trp operon are investigated. Sustained oscillations of tryptophan levels are predicted from the regulated turning on and off of the trp operon. It is interesting to note that during such oscillations the regulation of transcription displays a kind of "on" and "off" state in terms of gene expression, indicating the existence of a genetic circuit or switch in the regulation of the trp operon. Time lags between transcription and translation are also predicted and may explain the occurrence of such oscillation phenomenon.

Down-Regulation↗

PubMed1989

Overexpression and site-directed mutagenesis of the succinyl-CoA synthetase of Escherichia coli and nucleotide sequence of a gene (g30) that is adjacent to the suc operon.

The succinyl-CoA synthetase of Escherichia coli is encoded by two genes, sucC (beta subunit) and sucD (alpha subunit), which are distal genes in the sucABCD operon. They are expressed from the suc promoter, which also expresses the dehydrogenase and dihydrolipoyl succinyl-transferase subunits of the 2-oxoglutarate dehydrogenase complex. Strategies have now been devised for the site-directed mutagenesis and independent expression of the succinyl-CoA synthetase (alpha 2 beta 2 tetramer) and the individual subunits. These involve (1) subcloning a promoterless sucCD fragment downstream of the lac promoter in M13mp10, and (2) precise splicing of the suc coding regions with the efficient atpE ribosome-binding site and expression from the thermoinducible lambda promoters in the pJLA503 vector. Succinyl-CoA synthetase specific activities were amplified 40-60-fold within 5 h of thermoinduction of the lambda promoters, and the alpha and beta subunits accounted for almost 30% of the protein in supernatant fractions of the cell-free extracts. Site-directed mutagenesis of potential CoA binding-site residues indicated that Trp-43 beta and His-50 beta are essential residues in the beta-subunit, whereas Cys-47 beta could be replaced by serine without inactivating the enzyme. No activity was detected after the histidine residue at the phosphorylation site of the alpha-subunit was replaced by aspartate (His-246 alpha----Asp), but this alteration seemed to have a deleterious effect on the accumulation of the enzyme in cell-free supernatant extracts. The nucleotide sequence of an unidentified gene (g30) that is adjacent to the sucABCD operon was defined by extending the sequence of the citric acid cycle gene cluster by 818 bp to 13379 bp: gltA-sdhCDAB-sucABCD-g30. This gene converges on the suc operon and encodes a product (P30) that contains 230 amino acids (Mr 27,251). Highly significant similarities were detected between the N-terminal region of P30 and those of GENA [the product of another unidentified gene (geneA) located upstream of the aceEF-lpd operon], and GNTR (a putative transcriptional repressor of the gluconate operon of Bacillus subtilis). Possible roles for GENA and P30 as transcriptional regulators of the adjacent operons encoding the pyruvate and 2-oxoglutarate dehydrogenase complexes are discussed.

Amino Acid Sequence↗

PubMed1996

Suppression of transcription polarity in the Escherichia coli haemolysin operon by a short upstream element shared by polysaccharide and DNA transfer determinants.

Expression of the Escherichia coli hlyCABD operon encoding synthesis, maturation and export of haemolysin toxin was strongly dependent upon a 35 bp DNA sequence, spanning the element GGCGGTAG, located 2 kbp upstream. When the hly operon was placed under the control of the inducible tac promoter, expression remained dependent upon this element, when transcribed in its native orientation 3' of the promoter. The increase in ptac-directed transcription was strongest for the distal, export genes of the hly operon, and was particularly striking when ptac and the element were placed far upstream. The element did not influence transcript stability, and we suggest that it is a key component of a novel regulatory mechanism may suppresses transcription polarity within operons. The mechanism that be of widespread importance in bacterial gene expression because the 8 bp element is present in many Gram-negative species as an upstream component of operons encoding the production of toxins and the surface assembly of polysaccharides and components required for the conjugal transfer of DNA. We name it the ops element for operon polarity suppressor.

Bacterial Proteins↗

PubMed1996

Analysis of nitrate regulatory protein NarL-binding sites in the fdnG and narG operon control regions of Escherichia coli K-12.

During anaerobic growth, expression of the fdnGHI and narGHJI operons of Escherichia coli is induced by the NarL protein in response to nitrate. The fdnG operon control region contains four NarL-binding sites (termed NarL heptamers) between positions -70 and -130. The two central NarL heptamers of fdnG are arranged as an inverted repeat and are essential for regulation by NarL. We used mutational analysis of these central heptamers to investigate the precise sequence requirements for NarL-dependent induction. Mutations were examined for their effects on NarL-dependent expression in vivo. Substitutions at position 1 of either heptamer had the strongest effect whereas substitutions at position 7 had the weakest effect. For some positions, alterations in both heptamers had a stronger effect than either of the single changes. The 2 bp spacing between these NarL heptamers was also important for normal nitrate induction. The narG operon control region has at least eight NarL heptamers arranged in two groups. Previous work has shown that nucleotide substitutions in two of these heptamers, centred at positions -195 and -89, severely reduce nitrate induction of narG operon expression in vivo and significantly interfere with NarL-DNA interactions in vitro. Substitutions in heptamers -185 and -101 affected narG operon induction only when the concentration of phospho-NarL was low (during growth in the presence of nitrite). Changes in each of the other four NarL heptamers studied had little or no effect on nitrate or nitrite induction of narG operon expression or on NarL-DNA interactions in vitro.

Bacterial Proteins↗

PubMed1997

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT.

Glucose is the preferred carbon and energy source of Bacillus subtilis. It is transported into the cell by the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI locus. We show here that these three genes (ptsG, ptsH, and ptsI) form an operon, the expression of which is inducible by glucose. In addition, ptsH and ptsl form a constitutive ptsHI operon. The promoter of the ptsGHI operon was mapped and expression from this promoter was found to be constitutive. Deletion mapping of the promoter region revealed the presence of a transcriptional terminator as a regulatory element between the promoter and coding region of the ptsG gene. Mutations within the ptsG gene were characterized and their consequences on the expression of ptsG studied. The results suggest that expression of the ptsGHI operon is subject to negative autoregulation by the glucose permease, which is the ptsG gene product. A regulatory gene located upstream of the ptsGHI operon, termed glcT, was also identified. The GlcT protein is a novel member of the BglG family of transcriptional antiterminators and is essential for the expression of the ptsGHI operon. A deletion of the terminator alleviates the need for GlcT. The activity of GlcT is negatively regulated by the glucose permease.

Amino Acid Sequence↗

PubMed2006

Eukaryotic operon-like transcription of functionally related genes in Drosophila.

Complex biological processes require coordinated function of many genes. One evolutionary solution to the problem of coordinately expressing functionally related genes in bacteria and nematodes is organization of genes in operons. Surprisingly, eukaryotic operons are considered rare outside the nematode lineage. In Drosophila melanogaster, we found lounge lizard (llz), which encodes a degenerin/ENaC cation channel, cotranscribed with CheB42a, a nonhomologous gene of unknown function residing <100 bp upstream. These two genes were transcribed from a single promoter as one primary transcript and were processed posttranscriptionally to generate individual mRNAs. The mechanism did not involve alternative splicing, and it differed from the trans splicing used in nematode operons. Both genes were expressed in the same tissues, and previous work suggested that both may be involved in courtship behavior. A bioinformatic approach identified numerous additional loci as potential Drosophila operons. These data reveal eukaryotic operon-like transcription of functionally related genes in Drosophila. The results also suggest that operon-based transcription may be more common in eukaryotes than previously appreciated.

Animals↗

PubMed1982

Regulatory regions of two transport operons under nitrogen control: nucleotide sequences.

We have determined the nucleotide sequences of the regulatory regions from two amino acid transport operons from Salmonella typhimurium: dhuA, which regulates the histidine transport operon, and argTr, which regulates argT, the gene encoding the lysine-arginine-ornithine-binding protein, LAO. The promoter for the histidine transport operon has been identified from the sequence change in the promoter-up mutation dhuA1. Neither regulatory region has any of the features typical of the regulatory regions of the amino acid biosynthetic operons, indicating that regulation of at least these transport genes does not involve a transcription attenuation mechanism. We have identified three interesting features, present in both of these sequences, which may be of importance in the regulation of these and other operons: a "stem-loop-foot" structure, a region of specific homology, and a mirror symmetry. The region of mirror symmetry may be a protein recognition site important is regulating expression of these and other operons in response to nitrogen availability. Mirror symmetry as a structure for DNA-protein interaction sites has not been proposed previously.

Amino Acids↗

PubMed1983

Synthesis of aspartate transcarbamoylase in Escherichia coli: transcriptional regulation of the pyrB-pyrI operon.

The first committed reaction in pyrimidine biosynthesis in Escherichia coli and Salmonella typhimurium is catalyzed by the allosteric enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), the product of the pyrB-pyrI operon. Regulation of the pyrimidine pathway is achieved in part by changes in the enzyme's catalytic activity as a function of the concentration of substrates and other metabolites as well as by variations in enzyme synthesis in response to changes in cellular levels of pyrimidine nucleotides. Although there is substantial evidence that UTP concentration has a marked influence on expression of the pyrB-pyrI operon, the mechanism of this control is not known. We have cloned the operon and determined the nucleotide sequence of the region preceding the first structural gene (pyrB). These studies show two regions sharing considerable homology with the consensus sequence of E. coli promoters, a segment that can code for a 44-amino-acid leader peptide, and a sequence very similar to that of the attenuator of the trp operon. RNA transcripts from several bacterial strains were studied by S1 nuclease mapping. Under conditions leading to extensive enzyme synthesis there was a large production of transcript whose 5' end correlated with the putative promoter closer to the structural genes. At low levels of operon expression there was little transcript in the extracts and both promoters appeared to serve as initiation sites. The results are interpreted in terms of transcriptional control of the pyrB-pyrI operon according to an attenuation model that differs in novel ways from the mechanisms proposed for the regulation of amino acid biosynthesis.

Amino Acid Sequence↗

PubMed1990

Ribosomal protein L4 stimulates in vitro termination of transcription at a NusA-dependent terminator in the S10 operon leader.

The 11-gene S10 ribosomal protein operon of Escherichia coli is under the autogenous control of L4, the product of the third gene of the operon. Ribosomal protein L4 inhibits both transcription and translation of the operon. Our in vivo studies indicated that L4 regulates transcription by causing premature termination within the untranslated S10 operon leader. We have now used an in vitro transcription system to study the effect of purified L4 on expression of the S10 operon. We find that the cell-free system reproduces the in vivo observations. Namely, in the absence of L4, most of the RNA polymerases read through the termination site in the S10 attenuator; the addition of L4 results in increased termination at this site. However, RNA polymerase does not terminate at the S10 attenuator, with or without L4, unless an additional factor, protein NusA, is added to the transcription reaction. These results suggest that the attenuator in the S10 operon is a NusA-dependent terminator whose efficiency is regulated by ribosomal protein L4.

Bacterial Proteins↗

PubMed1997

Wild-type Escherichia coli grows on the chitin disaccharide, N,N'-diacetylchitobiose, by expressing the cel operon.

We report here that wild-type Escherichia coli can grow on the chitin disaccharide, N,N'-diacetylchitobiose (GlcNAc)2, as the sole source of carbon. Transposon mutants were isolated that were unable to ferment (GlcNAc)2 but grew normally on the monosaccharide GlcNAc. One such mutant was used to screen a wild-type E. coli genomic cosmid library for restoration of (GlcNAc)2 fermentation. A partial sequence analysis of the isolated fragment mapped the clone to the (previously sequenced) E. coli genome between 39.0 and 39.2 min. The nucleotide ORFs at this region had been previously assigned to code for a "cryptic" cellobiose utilization (cel) operon. We report here, however, that functional analysis of the operon, including growth and chemotaxis, reveal that it encodes a set of proteins that are not cryptic, but are induced by (GlcNAc)2 and catabolize the disaccharide. We therefore propose to rename the cel operon as the chb (N,N'-diacetylchitobiose) operon, with the letter designation of the genes of the operon to be reassigned consistent with the nomenclature based on functional characterization of the gene products as follows: celA to chbB, celB to chbC, celC to chbA, celD to chbR, and celF to chbF. Furthermore, sequencing evidence indicates that the operon contains an additional gene of unknown function to be designated as chbG. Thus, the overall gene sequence is to be named chbBCARFG.

Cellulase↗

PubMed1999

Hypermutation in derepressed operons of Escherichia coli K12.

This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation. Derepression of the leu operon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response). A quantitative correlation was established between leuB mRNA abundance and leuB- reversion rates. To further demonstrate that derepression increased mutation rates, the chromosomal leu operon was placed under the control of the inducible tac promoter. When the leu operon was induced by isopropyl-D-thiogalactoside, both leuB mRNA abundance and leuB- reversion rates increased. These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulating leu operon expression and that higher rates of mutation are specifically associated with the derepressed leu operon.

Escherichia coli↗

PubMed2004

Comparative analysis of the four rRNA operons in Finegoldia magna ATCC29328.

There are four rRNA operons rrnA, rrnB, rrnC and rrnD on the genome of Finegoldia magna (formerly Peptostreptococcus magnus) ATCC29328, which, in contrast to those of Clostridia, are dispersed around the chromosome. Using a BAC library we determined the nucleotide sequences and structures of all four operons, including their flanking regions, and performed comparative analyses. We identified putative boxA sequences in the operons, which should be required for rRNA transcription antitermination, as well as their respective tandem promoters, AT-rich UP elements in the upstream region and Rho-independent terminators in the downstream region. The mosaic features of the operons were revealed. Multiple tRNAs were identified in the downstream region of two operons, 18 in rrnC and 11 in rrnD. They were presumed to form transcription units together with rRNAs. rrnA and rrnB had repeat units with Rho-independent terminators instead of tRNAs in the downstream region. rrnB and rrnC were the most similar in rrn upstream promoter region. Focusing on the sequence variations of rRNA genes, rrnB alone was heterogeneous. In light of previous reports, we also assessed the correlation between intercistronic rRNA sequence differences and distances between the operons, but no positive correlation was seen in this strain.

Base Sequence↗

PubMed1990

Characterization and nucleotide sequence of the cryptic cel operon of Escherichia coli K12.

Wild-type Escherichia coli are not able to utilize beta-glucoside sugars because the genes for utilization of these sugars are cryptic. Spontaneous mutations in the cel operon allow its expression and enable the organism to ferment cellobiose, arbutin and salicin. In this report we describe the structure and nucleotide sequence of the cel operon. The cel operon consists of five genes: celA, whose function is unknown; celB and celC which encode phosphoenolpyruvate-dependent phosphotransferase system enzyme IIcel and enzyme IIIcel, respectively, for the transport and phosphorylation of beta-glucoside sugars; celD, which encodes a negative regulatory protein; and celF, which encodes a phospho-beta-glucosidase that acts on phosphorylated cellobiose, arbutin and salicin. The mutationally activated cel operon is induced in the presence of its substrates, and is repressed in their absence. A comparison of proteins encoded by the cel operon with functionally equivalent proteins of the bgl operon, another cryptic E. coli gene system responsible for the catabolism of beta-glucoside sugars, revealed no significant homology between these two systems despite common functional characteristics. The celD and celF encoded repressor and phospho-beta-glucosidase proteins are homologous to the melibiose regulatory protein and to the melA encoded alpha-galactosidase of E. coli, respectively. Furthermore, the celC encoded PEP-dependent phosphotransferase system enzyme IIIcel is strikingly homologous to an enzyme IIIlac of the Gram-positive organism Staphylococcus aureus. We conclude that the genes for these two enzyme IIIs diverged much more recently than did their hosts, indicating that E. coli and S. aureus have undergone relatively recent exchange of chromosomal genes.

Amino Acid Sequence↗

PubMed2000

The genes encoding endonuclease VIII and endonuclease III in Escherichia coli are transcribed as the terminal genes in operons.

Escherichia coli endonuclease VIII and endo-nuclease III are oxidative base excision repair DNA glycosylases that remove oxidized pyrimidines from DNA. The genes encoding these proteins, nei and nth, are both co-transcribed as the terminal genes in operons. nei is the terminal gene in an operon with four open reading frames that encode proteins of unknown function. This operon has two confirmed transcription initiation sites upstream of the first open reading frame and two transcript termination sites downstream of nei. nth is the terminal gene in an operon with seven open reading frames that encode proteins of unknown function. The six open reading frames immediately upstream of nth show homology to the genes rnfA, rnfB, rnfC, rnfD, rnfG and rnfE from Rhodobacter capsulatis. The rnf genes are required for nitrogen fixation in R.capsulatis and have been predicted to make up a membrane complex involved in electron transport to nitrogenase. The nth operon has transcription initiation sites upstream of the first and second open reading frames and a single transcript termination site downstream of nth. The order of genes in these operons has been conserved or partially conserved in other bacteria, although it is not known whether the genes are co-transcribed in these other organisms.

Conserved Sequence↗

PubMed2005

A novel method for accurate operon predictions in all sequenced prokaryotes.

We combine comparative genomic measures and the distance separating adjacent genes to predict operons in 124 completely sequenced prokaryotic genomes. Our method automatically tailors itself to each genome using sequence information alone, and thus can be applied to any prokaryote. For Escherichia coli K12 and Bacillus subtilis, our method is 85 and 83% accurate, respectively, which is similar to the accuracy of methods that use the same features but are trained on experimentally characterized transcripts. In Halobacterium NRC-1 and in Helicobacter pylori, our method correctly infers that genes in operons are separated by shorter distances than they are in E.coli, and its predictions using distance alone are more accurate than distance-only predictions trained on a database of E.coli transcripts. We use microarray data from six phylogenetically diverse prokaryotes to show that combining intergenic distance with comparative genomic measures further improves accuracy and that our method is broadly effective. Finally, we survey operon structure across 124 genomes, and find several surprises: H.pylori has many operons, contrary to previous reports; Bacillus anthracis has an unusual number of pseudogenes within conserved operons; and Synechocystis PCC 6803 has many operons even though it has unusually wide spacings between conserved adjacent genes.

Base Sequence↗