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A Bayesian network approach to operon prediction.

MOTIVATION: In order to understand transcription regulation in a given prokaryotic genome, it is critical to identify operons, the fundamental units of transcription, in such species. While there are a growing number of organisms whose sequence and gene coordinates are known, by and large their operons are not known. RESULTS: We present a probabilistic approach to predicting operons using Bayesian networks. Our approach exploits diverse evidence sources such as sequence and expression data. We evaluate our approach on the Escherichia coli K-12 genome where our results indicate we are able to identify over 78% of its operons at a 10% false positive rate. Also, empirical evaluation using a reduced set of data sources suggests that our approach may have significant value for organisms that do not have as rich of evidence sources as E.coli. AVAILABILITY: Our E.coli K-12 operon predictions are available at http://www.biostat.wisc.edu/gene-regulation.

Algorithms↗

Regulation of the put operon in Salmonella typhimurium: characterization of promoter and operator mutations.

The two genes required for proline utilization by S. typhimurium form a divergent operon. Expression of the put operon is induced by proline and subject to catabolite repression. Genetic evidence suggests that putA protein autogenously represses transcription of the putA and putP genes. In order to establish the molecular mechanism of put operon regulation we isolated regulatory mutations in the put control region. These mutants were selected using two phenotypes: the ability to degrade a toxic proline analogue, dehydroproline, due to overexpression of putA enzyme activity, or overexpression of lacZ from put::Mud operon fusions. The effect of each mutation on transcription in both directions was determined by measuring lacZ expression from putA and putP operon fusions. These regulatory mutations were cis-dominant when the putA protein was provided in trans, and they map in a region between the two genes. The phenotypes of the mutants suggest that the put regulatory region has a single operator site where the putA protein binds to repress transcription in both directions, and the putA and putP promoters overlap.

Genes, Bacterial↗

Exchange of spacer regions between rRNA operons in Escherichia coli.

The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala1B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala1B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids.

Base Sequence↗

A mutation in a new gene, bglJ, activates the bgl operon in Escherichia coli K-12.

A new mutation, bglJ4, has been characterized that results in the expression of the silent bgl operon. The bgl operon encodes proteins necessary for the transport and utilization of the aromatic beta-glucosides arbutin and salicin. A variety of mutations activate the operon and result in a Bgl+ phenotype. Activating mutations are located upstream of the bgl promoter and in genes located elsewhere on the chromosome. Mutations outside of the bgl operon occur in the genes encoding DNA gyrase and in the gene encoding the nucleoid associated protein H-NS. The mutation described here, bglJ4, has been mapped to a new locus at min 99 on the Escherichia coli K-12 genetic map. The putative protein encoded by the bglJ gene has homolgy to a family of transcriptional activators. Evidence is presented that increased expression of the bglJ product is needed for activation of the bgl operon.

Amino Acid Sequence↗

Divergent operons and the genetic structure of the maltose B region in Escherichia coli K12.

Complementation and polarity suppression data are interpreted in terms of the genetic structure of the maltose B region. It is proposed that this region comprises two divergent operons. One operon includes malK, a cistron involved in maltose permeation, and lamB the only known cistron specifically involved in lambda receptor synthesis. The other operon includes malJ(1) and malJ(2) which are most probably two different cistrons, both involved in maltose permeation*. It is further assumed that expression of the two operons is controlled by malT, the positive regulatory gene of the maltose system, located in the malA region. The target(s) for the action of the malT product is (are) most likely to be located between malJ(1) and malK. There is an indication that the two operons might overlap in the region of their promoters. The structure of such an overlap as well as the possible function of the products of the different cistrons in malB are briefly discussed.

Chromosome Mapping↗

Enterococcus faecium N03-0072 carries a new VanD-type vancomycin resistance determinant: characterization of the VanD5 operon.

OBJECTIVES: To genotypically characterize the vancomycin resistance mechanism of Enterococcus faecium N03-0072, which was negative by PCR for the currently known van genotypes. METHODS: PCR was used to amplify the entire vancomycin resistance operon and the complete nucleotide sequence was determined by dideoxy cycle sequencing. RESULTS: Analysis revealed a VanD-type operon with 94% nucleotide identity to the VanD4 operon and 90% nucleotide identity to the VanD1/D3 operons. A set of universal primers was designed in order to identify all current vanD variants by PCR. CONCLUSIONS: E. faecium N03-0072 carries a new VanD-type operon, designated VanD5.

Bacterial Proteins↗

Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC.

The product of the korA gene of broad host range plasmid RK2 is a key transcriptional repressor which regulates not only the expression of the essential replication gene trfA but also its own expression and that of the kilA operon. It has previously been proposed that korA also encodes a positive activator of transcription of the korC gene, which may act as a transcriptional antiterminator. Here we show that the action of korA in relation to korC can be explained entirely through the korA protein's property as a transcriptional repressor. The limited ability of the previously cloned korC gene to suppress kilC on its own is shown to be due to the fact that korC in RK2 is transcribed from the bla promoter of Tn1 which was deleted in the original korC clones. We demonstrate that korA is a second repressor along with korC of three operons, one of which encodes kilC, the other two not having been described previously and serving an as yet unknown function. We have designated these operons kcrA, B and C for KorC-regulated. Putative kilC is designated kcrC. The homology between the expression signals of these operons suggests that they have arisen by duplication. This is confirmed in the case of kcrA and B by the existence of considerable homology between the products of the first ORFs in each of these operons.

Amino Acid Sequence↗

The elongation factor EF-Tu from E. coli binds to the upstream activator region of the tRNA-tufB operon.

The polypeptide chain elongation factor EF-Tu of Escherichia coli is encoded by two genes, tufA and tufB, located in two different operons. Experiments in which either tufA or tufB was inactivated demonstrated that expression of the tRNA-tufB operon is dependent on a functioning tufA and thus on EF-Tu (1, to be published). In order to study a possible role of EF-Tu as trans-activator of the tRNA-tufB operon, we have investigated in vitro binding of an EF-Tu. GDP preparation to various DNA fragments of the operon. We demonstrate that specific binding occurs to a cis-acting region delimited from position -134 to the promoter, previously shown to enhance tufB transcription. Electrophoretic retardation assays reveal the formation of maximally three protein/DNA complexes, indicating that more than one protein molecule can bind to the DNA. The EF-Tu preparation used was obtained by affinity chromatography and appeared to be 95% pure. It lost its DNA binding activity upon further purification. That EF-Tu is nonetheless involved in the DNA binding is suggested by the observation that none of the three complexes is formed in the presence of kirromycin, an antibiotic that binds EF-Tu with high specificity. If so, EF-Tu.GDP most likely binds to the activator region of the tRNA-tufB operon in combination with another non-identified protein or component.

DNA, Bacterial↗

Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus.

The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.

Bacillus subtilis↗

Glucose repression of the Escherichia coli sdhCDAB operon, revisited: regulation by the CRP*cAMP complex.

Expression of the Escherichia coli sdhCDAB operon encoding the succinate dehydrogenase complex is regulated in response to growth conditions, such as anaerobiosis and carbon sources. An anaerobic repression of sdhCDAB is known to be mediated by the ArcB/A two-component system and the global Fnr anaerobic regulator. While the cAMP receptor protein (CRP) and Cra (formerly FruR) are known as key mediators of catabolite repression, they have been excluded from the glucose repression of the sdhCDAB operon. Although the glucose repression of sdhCDAB was reported to involve a mechanism dependent on the ptsG expression, the molecular mechanism underlying the glucose repression has never been clarified. In this study, we re-examined the mechanism of the sdhCDAB repression by glucose and found that CRP directly regulates expression of the sdhCDAB operon and that the glucose repression of this operon occurs in a cAMP-dependent manner. The levels of phosphorylated enzyme IIA(Glc) and intracellular cAMP on various carbon sources were proportional to the expression levels of sdhC-lacZ. Disruption of crp or cya completely abolished the glucose repression of sdhC-lacZ expression. Together with data showing correlation between the intracellular cAMP concentrations and the sdhC-lacZ expression levels in several mutants and wild type, in vitro transcription assays suggest that the decrease in the CRP.cAMP level in the presence of glucose is the major determinant of the glucose repression of the sdhCDAB operon.

Bacterial Proteins↗

Effects of carriage and expression of the Tn10 tetracycline-resistance operon on the fitness of Escherichia coli K12.

We have been examining the consequences of alternative modes of regulation of plasmid-borne, Tn10-encoded tetracycline resistance for the fitness of Escherichia coli. In a tetracycline-free environment, we measured the effects on fitness that were caused by (1) maximally induced expression of the resistance operon, (2) low-level constitutive expression of the resistance protein, (3) residual expression of the repressed resistance operon, (4) carriage of the resistance operon, (5) the remainder of the plasmid genome, and (6) hyperexpression of the repressor protein. We observed large reductions in fitness that were associated with induction and with constitutive expression of the tetracycline-resistance protein, but there was no discernible effect of hyperexpression of the repressor protein. We also observed a small reduction in fitness associated with the remainder of the plasmid genome. However, any reductions in fitness that were caused by residual expression and by carriage of the repressed operon were not more than 0.3%. We conclude that tight gene regulation has eliminated antagonistic pleiotropic effects of the resistance gene on fitness, so that possession of an inducible Tn10-encoded tetracycline-resistance operon imposes essentially no burden in the absence of antibiotic.

DNA Transposable Elements↗

Dual control of the gua operon of Escherichia coli K12 by adenine and guanine nucleotides.

The gua operon of Escherichia coli K12 comprises structural genes for the two enzymes, IMP dehydrogenase and GMP synthetase, required for the biosynthesis of GMP from IMP. The specific activities of these enzymes were measured in various purine auxotrophs. GuaA and guaB mutants (guanine-specific) were depressed under conditions of growth limitation by guanine but were repressed by excess guanine. This suggests that formation of the enzymes is normally controlled by a guanine nucleotide. Derepression of the operon in purine-starved pur mutants depended on the type of mutant and on whether adenine or guanine was provided. A purA strain (adenine-specific) and strains with early blocks in purine biosynthesis (purF and purD) did not derepress. PurE or purC strains [5'-phosphoribosyl-5-aminoimidazole (AIR)-accumulating] derepressed only 4-fold. The operon was repressed in purH strains [5'-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR)-accumulating] grown with limiting guanine or hypoxanthine, but derepressed by growth with limiting adenine. Two mutants (purA guaA and purA guaB) which can neither synthesize AMP and GMP de novo, nor interconvert them, were isolated. The specific activity of IMP dehydrogenase in one of these strains grown with different concentrations of guanine and adenine revealed that adenine induces tha gua operon whereas guanine represses it. Intracellular purine nucleotide pools wee measured in a purH mutant repressed (guanine-grown) and derepressed (adenine-grown) for IMP dehydrogenase. The guanylate pool was similar under the two growth conditions; however the adenylate pool of the adenine-grown bacteria was two to three times greater than that of the guanine-grown cells. A dual mechanism for regulating expression of the gua operon, involving induction by AMP and repression by GMP, is proposed.

Adenine↗

The presence of two complete homologous meta pathway operons on TOL plasmid pWW53.

pWW53 is a 110 kbp catabolic plasmid which encodes the complete pathway for the utilization of toluene and the xylenes. The upper pathway operon xylCAB is located between two homologous but distinct meta pathway operons, xylDLEGF(I,J,K)H, which are in direct repeat. These have each been cloned on large HindIII restriction fragments HA (17.5 kbp) and HB (15.6 kbp), the restriction sites of which have been mapped. During growth of MT53 on benzoate, mutants which have lost the ability to grow on hydrocarbons such as m-xylene (Mxy-) but which retain the ability to grow on their carboxylic acid metabolites such as m-toluate (Mtol+) take over the culture before ultimately being displaced by plasmid-free strains which are Mxy- Mtol-. The plasmids in the Mxy- Mtol+ mutants are formed by a large deletion between homologous regions of the two duplicate meta pathway operons. This causes the loss of the intervening xylCAB operon and the formation of a hybrid xylDLEGF(I, J, K)H operon, starting with the genes originally on HA and terminating with the genes originally on HB.

Cloning, Molecular↗

Duplication of both xyl catabolic operons on TOL plasmid pWW15.

Pseudomonas fluorescens MT15 is the host of the large (250 kbp) TOL plasmid pWW15. We have shown by a combination of hybridization, molecular cloning and enzyme assay that pWW15 carries two distinct regions which share homology with the upper pathway operons (xylCMABN) of other TOL plasmids and two distinct regions which are homologous to the meta pathway operons (xylXYZLTEGFJQKIH) of other TOL plasmids. Both the areas of homology to the upper pathway operons appear to carry all of the structural genes for the three catabolic enzymes of the operon. One of the regions of meta pathway operon homology encodes a complete functional pathway, but the second is incomplete and appears to carry only the genes from xylF downstream.

Catechol 2,3-Dioxygenase↗

Role of the colicin A lysis protein in the expression of the colicin A operon.

The involvement of the cal gene, which encodes the colicin A lysis protein, in the expression of the colicin A operon is demonstrated. Colicin A synthesis by Escherichia coli was studied at various temperatures in cells containing either the wild-type colicin A operon or the colicin A operon with the cal gene deleted. The amount of colicin A produced was lower in cells containing the colicin A operon devoid of the cal gene than in wild-type cells. In cells treated with the antibiotic globomycin, the synthesis of colicin A was blocked in null cal mutants at all temperatures. It was blocked only at low temperature in cells containing the wild-type colicin A operon, but not in cells subjected to heat shock or azide treatment. The cal gene product may be an activator of colicin A expression and of its own expression. An unidentified product, possibly a heat-shock protein, may also be involved and could complement the cal gene product in some situations.

Bacterial Proteins↗

The kdgRKAT operon of Bacillus subtilis: detection of the transcript and regulation by the kdgR and ccpA genes.

Transcription of a new catabolic operon in Bacillus subtilis, involved in the late stages of galacturonic acid utilization, has been studied. The operon consists of four genes: kdgR, encoding the putative regulator protein; kdgK, encoding 2-keto-3-deoxygluconate kinase; kdgA, encoding 2-keto-3-deoxygluconate-6-phosphate aldolase; and kdgT, encoding a transporter. These four genes are organized in one transcriptional unit and map at 198 degrees of the B. subtilis chromosome. Primer extension experiments and Northern blot analysis show that an active sigmaA-dependent promoter precedes kdgR and transcription is terminated at the putative p-independent terminator downstream of kdgT. The operon is negatively regulated by the kdgR and ccpA gene products, which belong to the LacI family of transcription regulators. The expression of the genes in this operon can be induced by galacturonate and strongly repressed when glucose is present in the growth medium. Knockout mutations in genes kdgR and ccpA remove, respectively, the effects of galacturonate and glucose on the transcription of this operon.

Bacillus subtilis↗

The genes for erythritol catabolism are organized as an inducible operon in Brucella abortus.

Erythritol utilization is a characteristic of pathogenic Brucella abortus strains. The attenuated vaccine strain B19 is the only Brucella strain that is inhibited by erythritol, so a role for erythritol metabolism in virulence is suspected. A chromosomal fragment from the pathogenic strain B. abortus 2308 containing genes for the utilization of erythritol was cloned taking advantage of an erythritol-sensitive Tn5 insertion mutant. The nucleotide sequence of the complete 7714 bp fragment was determined. Four ORFs were identified in the sequence. The four genes were closely spaced, suggesting that they were organized as a single operon (the ery operon). The first gene (eryA) encoded a 519 aa putative erythritol kinase. The second gene (eryB) encoded an erythritol phosphate dehydrogenase. The function of the third gene (eryC) product was tentatively assigned as D-erythrulose-1-phosphate dehydrogenase and the fourth gene (eryD) encoded a regulator of ery operon expression. The operon promoter was located 5' to eryA, and contained an IHF (integration host factor) binding site. Transcription from this promoter was repressed by EryD, and stimulated by erythritol. Functional IHF was required for expression of the operon in Escherichia coli, suggesting a role for IHF in its regulation in B. abortus. The results obtained will be helpful in clarifying the role of erythritol metabolism in the virulence of Brucella spp.

Animals↗

Analysis of the expression and regulation of the gerB spore germination operon of Bacillus subtilis 168.

The gerB spore germination operon of Bacillus subtilis 168 is a homologue of the gerA spore germination operon. The expression and regulation of the gerB operon has been examined using a lacZ transcriptional fusion and the transcriptional start defined. The gerB operon is expressed during sporulation under the control of RNA polymerase containing the forespore-specific sigma factor, delta G. This is a further homology to the gerA operon, which is similarly regulated. It is predicted from the localization of expression and the encoded primary sequences that the GerB proteins are located at the inner spore membrane.

Bacillus subtilis↗