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Operon prediction using both genome-specific and general genomic information.

We have carried out a systematic analysis of the contribution of a set of selected features that include three new features to the accuracy of operon prediction. Our analyses have led to a number of new insights about operon prediction, including that (i) different features have different levels of discerning power when used on adjacent gene pairs with different ranges of intergenic distance, (ii) certain features are universally useful for operon prediction while others are more genome-specific and (iii) the prediction reliability of operons is dependent on intergenic distances. Based on these new insights, our newly developed operon-prediction program achieves more accurate operon prediction than the previous ones, and it uses features that are most readily available from genomic sequences. Our prediction results indicate that our (non-linear) decision tree-based classifier can predict operons in a prokaryotic genome very accurately when a substantial number of operons in the genome are already known. For example, the prediction accuracy of our program can reach 90.2 and 93.7% on Bacillus subtilis and Escherichia coli genomes, respectively. When no such information is available, our (linear) logistic function-based classifier can reach the prediction accuracy at 84.6 and 83.3% for E.coli and B.subtilis, respectively.

Bacillus subtilis↗

The distinctive signatures of promoter regions and operon junctions across prokaryotes.

Here we show that regions upstream of first transcribed genes have oligonucleotide signatures that distinguish them from regions upstream of genes in the middle of operons. Databases of experimentally confirmed transcription units do not exist for most genomes. Thus, to expand the analyses into genomes with no experimentally confirmed data, we used genes conserved adjacent in evolutionarily distant genomes as representatives of genes inside operons. Likewise, we used divergently transcribed genes as representative examples of first transcribed genes. In model organisms, the trinucleotide signatures of regions upstream of these representative genes allow for operon predictions with accuracies close to those obtained with known operon data (0.8). Signature-based operon predictions have more similar phylogenetic profiles and higher proportions of genes in the same pathways than predicted transcription unit boundaries (TUBs). These results confirm that we are separating genes with related functions, as expected for operons, from genes not necessarily related, as expected for genes in different transcription units. We also test the quality of the predictions using microarray data in six genomes and show that the signature-predicted operons tend to have high correlations of expression. Oligonucleotide signatures should expand the number of tools available to identify operons even in poorly characterized genomes.

Bacillus subtilis↗

Evolutionary instability of operon structures disclosed by sequence comparisons of complete microbial genomes.

Gene orders have been shown to be generally unstable by comprehensive analyses in several complete genomes. In this study, we examined instability of genome structures within operons, where functionally related genes are clustered. We compared gene orders of known operons obtained from Escherichia coli and Bacillus subtilis with corresponding those of operons in 11 complete genome sequences. We found that in many cases, gene orders within operons could be shuffled frequently during evolution, although several operon structures, such as ribosomal protein operons, were well conserved. This suggests that shuffling of a genome structure is virtually neutral in long-term evolution. Moreover, degrees of instability of the operon structures depended on the genomes examined. Variation in degrees of instability of the genome structures was likely to be related to differences in amounts of insertion sequences. Effects on transcription regulation are also discussed in association with operon destruction.

Bacillus subtilis↗

Unique organization and regulation of the mrx fimbrial operon in Xenorhabdus nematophila.

Xenorhabdus nematophila, a Gram-negative bacterium belonging to the Proteus clade of the family Enterobacteriaceae, forms a mutualistic association with the soil nematode Steinernema carpocapsae. The nematode invades insects and releases Xenorhabdus into the haemolymph, where it participates in insect killing. To begin to understand the role of fimbriae in the unique life cycle of Xenorhabdus, the organization and expression of the mrx fimbrial operon was analysed. The mrx operon contained only five structural genes (mrxACDGH), making it one of the smallest chaperone-usher fimbrial operons studied to date. Unlike the mrp operon of Proteus mirabilis, a site-specific recombinase was not linked to the mrx operon. The intergenic region between the major fimbrial gene (mrxA) and the usher gene (mrxC) lacked a mrpB-like gene, but contained three tandem inverted repeat sequences located downstream of mrxA. A 940 nt mrxA-containing mRNA was the major transcript produced in cells growing on agar, while an mrx polycistronic mRNA was produced at low levels. A canonical sigma(70) promoter, identified upstream of mrxA, was not subject to promoter inversion. Fimbriae were not produced in an lrp-mutant strain, suggesting that the leucine-responsive regulatory protein, Lrp, plays a role in the regulation of the mrx operon. These findings show that the genetic organization and regulation of the mrx operon is in several respects distinct from other chaperone-usher fimbrial operons.

Amino Acid Sequence↗

Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP.

The chitobiose operon, chbBCARFG, encodes genes for the transport and degradation of the N-acetylglucosamine disaccharide, chitobiose. Chitobiose is transported by the phosphotransferase system (PTS) producing chitobiose-6P which is hydrolysed to GlcNAc-6P by the chbF gene product and then further degraded by the nagBA gene products. Expression of the chb operon is repressed by NagC, which regulates genes involved in amino sugar metabolism. The inducer for NagC is GlcNAc-6P. NagC binds to two sites separated by 115 bp and the transcription start point of the chb operon lies within the downstream NagC operator. In addition the chb operon encodes its own specific regulator, ChbR, an AraC-type dual repressor-activator, which binds to two direct repeats of 19 bp located between the two NagC sites. ChbR is necessary for transcription activation in the presence of chitobiose in vivo. Induction of the operon also requires CAP, which binds to a site upstream of the ChbR repeats. In the absence of chitobiose both NagC and ChbR act as repressors. Together these three factors cooperate in switching chb expression from the repressed to the activated state. The need for two specific inducing signals, one for ChbR to activate the expression of the operon and a second for NagC to relieve its repression, ensure that the chb operon is only induced when there is sufficient flux through the combined chb-nag metabolic pathway to activate expression of both the chb and nag operons.

Chitin↗

Control of the tRNA-tufB operon in Escherichia coli. 2. Mechanisms of the feedback inhibition of tufB expression studied in vivo and in vitro.

The mechanism underlying feedback inhibition of tufB expression has been studied in vivo by gene-dosage experiments and by gene and operon fusions involving lacZ. Raising the cellular EF-Tu content, by introducing a multicopy plasmid encoding EF-TuA into the cell, repressed the level of EF-TuB but left the content of tRNA(Thr)3, encoded by the tRNA-tufB operon, unaffected. This indicates that autoregulation of chromosomal tufB expression does not occur by modulating transcription initiation at the promoter of the tRNA-tufB operon. This conclusion is further substantiated by experiments with a tRNA':lacZ operon fusion. The molecular ratio of chromosome-borne tufA and tufB transcripts also remained unaltered under conditions of excess EF-Tu, though experiments with a tRNA-tufB':lacZ operon fusion showed a decrease of tufB transcripts. Our data further exclude drastic effects of the autogenous repressor on processing of the contranscript of the operon into monocistronic tufB RNA and on alteration of EF-TuB turnover. Two possible mechanisms remain, which cannot yet be decided between. One is modulation of EF-Tu by transcription termination either directly or indirectly by affecting antitermination. The second is translational repression. In vitro translation of transcripts derived from SP6 clones did not reveal any feedback inhibition of EF-TuB synthesis. Surprisingly, addition of EF-Tu to a coupled transcription/translation systems was found to block transcription initiation at the primary promoter of the tRNA-tufB operon by over 90%. Although this in vitro effect of EF-Tu could not be demonstrated in vivo, possibly because of a difference in higher-order structure between plasmid-borne and chromosome-borne DNA, it indicates that under certain conditions EF-Tu binds very specifically to the tRNA-tufB operon promoter or its upstream region.

Cloning, Molecular↗

Regulation and organization of the groE and dnaK operons in Eubacteria.

groEL and dnaK are the most highly conserved protein-coding genes known. Most groEL operons and several dnaK and dnaJ operons contain a highly conserved inverted repeat (IR) sequence in their regulatory region. So far, this IR has been found only as part of the groE, dnaK and dnaJ operons and genes. In most cases, the IR is part of the operon transcript, and is involved in the regulation of expression at both the DNA and mRNA levels. A detailed analysis of groE and dnaK operons indicates that the organization of the groE operons is highly conserved. They contain only the groES and groEL genes and always in the same order. In contrast, the organization of the dnaK operons has changed during evolution: genes have been added and deleted from it, and the gene order within the operon is variable.

Amino Acid Sequence↗

Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon.

Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have evolved mechanisms of resistance to several of these different chemical forms, and play a major role in the global cycling of mercury in the natural environment. Five mechanisms of resistance to mercury compounds have been identified, of which resistance to inorganic mercury (HgR) is the best understood, both in terms of the mechanisms of resistance to mercury and of resistance to heavy metals in general. Resistance to inorganic mercury is encoded by the genes of the mer operon, and can be located on transposons, plasmids and the bacterial chromosome. Such systems have a worldwide geographical distribution, and furthermore, are found across a wide range of both Gram-negative and Gram-positive bacteria from both natural and clinical environments. The presence of mer genes in bacteria from sediment cores suggest that mer is an ancient system. Analysis of DNA sequences from mer operons and genes has revealed genetic variation both in operon structure and between individual genes from different mer operons, whilst analysis of bacteria which are sensitive to inorganic mercury has identified a number of vestigial non-functional operons. It is hypothesised that mer, due to its ubiquity with respect to geographical location, environment and species range, is an ancient system, and that ancient bacteria carried genes conferring resistance to mercury in response to increased levels of mercury in natural environments, perhaps resulting from volcanic activity. Models for the evolution of both a basic mer operon and for the Tn21-related family of mer operons and transposons are suggested. The study of evolution in bacteria has recently become dominated by the generation of phylogenies based on 16S rRNA genes. However, it is important not to underestimate the roles of horizontal gene transfer and recombinational events in evolution. In this respect mer is a suitable system for evaluating phylogenetic methods which incorporate the effects of horizontal gene transfer. In addition, the mer operon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.

Biological Evolution↗

Analysis of expression profile of mammalian cell entry (mce) operons of Mycobacterium tuberculosis.

The sequencing of the complete genome of M. tuberculosis H37Rv has resulted in the recognition of four mce operons in its genome by in silico analysis. In an attempt to understand the significance of the redundancy of mce operons, we analyzed the expression profile of mce operons after different periods of growth in culture as well as during in vivo infection. Our results strongly suggest that mce1 is expressed as a polycistronic message. In culture from day 8 to day 12, expression of only mce1 was observed, but as the cultures progress towards stationary phase the expression profile of mce operons was altered; the transcripts of the mce1 operon were barely detected while those of the mce4 operon were prominent. In an analysis of the expression of mce operons in tubercle material collected from infected animal tissues, we detected the expression of mce1, -3 and -4. Our results imply that mce operons other than mce1 are also expressed during infection and that it is necessary to examine their role in pathogenesis.

Animals↗

Effect of tryptophan analogs on derepression of the Escherichia coli tryptophan operon by indole-3-propionic acid.

The abilities of 14 tryptophan analogs to repress the tryptophan (trp) operon have been studied in Escherichia coli cells derepressed by incubation with 0.25 mM indole-3-propionic acid (IPA). trp operon expression was monitored by measuring the specific activities of anthranilate synthase (EC 4.1.3.27) and the tryptophan synthase (EC 4.2.1.20) beta subunit. Analogs characterized by modification or removal of the alpha-amino group or the alpha-carboxyl group did not repress the trp operon. The only analogs among this group that appeared to interact with the trp aporepressor were IPA, which derepressed the trp operon, and d-tryptophan. Analogs with modifications of the indole ring repressed the trp operon to various degrees. 7-Methyl-tryptophan inhibited anthranilate synthase activity and consequently derepressed the trp operon. Additionally, 7-methyltryptophan prevented IPA-mediated derepression but, unlike tryptophan, did so in a non-coordinate manner, with the later enzymes of the operon being relatively more repressed than the early enzymes. The effect of 7-methyltryptophan on IPA-mediated derepression was likely not due to the interaction of IPA with the allosteric site of anthranilate synthase, even though feedback-resistant mutants of anthranilate synthase were partially resistant to derepression by IPA. The effect of 7-methyltryptophan on derepression by IPA was probably due to the effect of the analog-aporepressor complex on trp operon expression.

Anthranilate Synthase↗

Tn9 and IS1 inserts in a ribosomal ribonucleic acid operon of Escherichia coli are incompletely polar.

Transcription is known to be coupled to translation in many or all bacterial operons which code for proteins. In these operons, nonsense codons which prevent normal translation often result in premature termination of transcription (polarity). However, efficient transcription of ribosomal ribonucleic acid operons (rrn operons) occurs, although rrn transcripts are not translated. It therefore seemed possible that insertion sequences and transposable elements which are polar in protein-coding operons might not be polar in rrn operons. Previously, it has been shown (E. A. Morgan, Cell 21:257-265, 1980) that Tn10 is incompletely polar in the rrnX operon. Here we show that the transposon Tn9 and the insertion sequence IS1 also incompletely polar in rrnX. In normal cells expression of sequences distal to the insertions can be detected by genetic methods. In ultraviolet-irradiated cells expression of distal sequences is about 80% of that observed in uninterrupted rrnX operons. These observations provide evidence that ribonucleic acid polymerase molecules beginning at rrnX promoters can read through Tn9 and IS1 and that, at least in ultraviolet-irradiated cells, read-through is very efficient.

Bacteriophage lambda↗

cis-acting regulatory elements involved in oxygen and light control of puc operon transcription in Rhodobacter sphaeroides.

Transcriptional expression of the puc operon in Rhodobacter sphaeroides is highly regulated by both oxygen and light. The approximately 600 bp of DNA upstream of the 5' ends of the two puc-specific transcripts encompasses two functionally separable cis-acting domains. The upstream regulatory region (URS) (-629 to -150) is responsible for enhanced transcriptional regulation of puc operon expression by oxygen and light. The more proximal upstream region (downstream regulatory region [DRS]), containing putative promoter(s), operator(s), and factor binding sites (-150 to -1), is involved in unenhanced transcriptional expression of the puc operon under aerobic and anaerobic conditions. Thus, the DRS shows normal derepression of puc operon expression when cells are shifted from aerobic to photosynthetic growth conditions in terms of percent change but does not show the potential range of expression that is only observed when elements of the URS are present. Because of these observations, we have made a distinction between anaerobic control (describing the shift) and oxygen control (describing the magnitude of derepression). Promoter(s) and/or activator function(s) of the puc operon is associated with a 35-bp DNA region between -92 and -57. Homologous sequences at -10 to -27 and -35 to -52 appear to involve additional regulatory elements: mutations at -12 (A to C) and -26 (G to A) result in partial derepression of puc operon expression under conditions of high aeration. Both point mutations require the upstream regulatory region (-629 to -150) to be present in cis for partial derepression of puc operon transcription under aerobic conditions. Immediately upstream of the promoter and/or activator region are overlapping consensus sequences for IHF (integratin host factor) and FNR (fumarate nitrate reductase) (-105 to -129). This region appears to be essential for enhanced expression of the puc operon. Thus, these two regulatory domains (URS and DRS) appear to involve approximately seven unique regulatory elements. In addition, the data reveal a direct interaction between the URS (-629 to -150) and the DRS (-150 to -1).

Anaerobiosis↗

The products of the kdpDE operon are required for expression of the Kdp ATPase of Escherichia coli.

The expression of the Kdp system for K+ uptake in Escherichia coli requires the products of two genes, kdpD and kdpE. These genes constitute an operon adjacent to the kdpABC operon that encodes the three membrane protein subunits of Kdp. Both operons are transcribed in the same direction and overlap; the kdpDE promoter is in kdpC, the last gene of the kdpABC operon. Transcription of the kdpDE operon is at a low level when Kdp is not expressed; transcription increases about 10-fold when kdpABC is turned on, indicating significant read-through of the kdpDE operon by transcripts beginning at the promoter of kdpABC operon. The proximal region of the kdpD gene is the site of most mutations that lead to constitutive expression of the kdpABC operon.

Adenosine Triphosphatases↗

In vivo regulatory responses of four Escherichia coli operons which encode leucyl-tRNAs.

Four Escherichia coli operons, the leuV operon which encodes tRNA(1Leu), the leuX operon which encodes tRNA(6Leu), the metT operon which encodes tRNA(3Leu), and the argT operon which encodes tRNA(1Leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation. In nuclease protection assays, the leuV operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-tRNA synthetase mutant at nonpermissive temperatures. The leuV operon also exhibited the stringent response in multicopy plasmids. The promoters of all four leucyl operons were fused to the gene for beta-galactosidase and inserted into the chromosome by using bacteriophage lambda. All except the leuX promoter displayed growth rate-dependent regulation, consistent with the recent report that the concentration of tRNA(6Leu) actually decreases as growth rate increases. The leuV promoter fused to the beta-galactosidase gene showed a decrease in efficiency in the presence of extrachromosomal copies of rRNA genes. All chromosomal tRNA genes examined showed decreased transcriptional activity following a stringent response, but the leuX gene responded to a lesser extent (3-fold versus 10-fold or more) than the others. Primer extension analysis of this promoter showed little if any response to serine hydroxamate treatment, suggesting that multiple levels of control may exist or that promoter context effects are important in regulation.

Base Sequence↗

Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon.

Amino Acid Sequence↗

The dnaKJ operon of Agrobacterium tumefaciens: transcriptional analysis and evidence for a new heat shock promoter.

The dnaKJ operon of Agrobacterium tumefaciens was cloned and sequenced and was found to be highly homologous to previously analyzed dnaKJ operons. Transcription of this operon in A. tumefaciens was stimulated by heat shock as well as by exposure to ethanol and hydrogen peroxide. There were two transcripts representing the dnaKJ operon: one containing the dnaK and dnaJ genes and the second containing only the dnaK gene. Primer extension analysis indicated that transcription started from the same site in heat-shocked cells and in untreated cells. The upstream regulatory region of the dnaKJ operon of A. tumefaciens does not contain the highly conserved inverted repeat sequence previously found in the groESL operon of this bacterium, as well as in many other groE and dnaK operons. Sequence analysis of the promoter region of several groESL and dnaK operons from alpha-purple proteobacteria indicates the existence of a putative promoter sequence different from the known consensus promoter sequences recognized by the Escherichia coli vegetative or heat shock sigma factor. This promoter may constitute the heat shock promoter of these alpha-purple proteobacteria.

Agrobacterium tumefaciens↗

Regulation of the dnaK operon of Streptomyces coelicolor A3(2) is governed by HspR, an autoregulatory repressor protein.

The dnaK operon of Streptomyces coelicolor contains four genes (5'-dnaK-grpE-dnaJ-hspR). The fourth gene encodes a novel heat shock protein, HspR, which appears so far to be unique to the high-G+C actinomycete group of bacteria. HspR binds with high specificity to three inverted repeat sequences in the promoter region of the S. coelicolor dnaK operon, strongly suggesting a direct role for HspR in heat shock gene regulation. Here we present genetic and biochemical evidence that HspR is the repressor of the dnaK operon. Disruption of hspR leads to high-level constitutive transcription of the dnaK operon. Parallel transcriptional analyses of groESL1 and groEL2 expression demonstrated that heat shock regulation of the groE genes was essentially unaffected in an hspR null mutant, although the basal (uninduced) level of groEL2 transcription was slightly elevated compared with the wild type. The results of HspR titration experiments, where the dnaK operon promoter region was cloned at ca. 50 copies per chromosome, were consistent with the prediction that HspR functions as a negative autoregulator. His-tagged HspR, overproduced and purified from Escherichia coli, was shown to repress transcription from the dnaK operon promoter in vitro, providing additional evidence for the proposal that HspR directly regulates transcription of the dnaK operon. These studies indicate that there are at least two transcriptional mechanisms for controlling heat shock genes in S. coelicolor--one controlling the dnaK operon and another controlling the groE genes.

Bacterial Proteins↗

Transcription analysis of two disparate rRNA operons in the halophilic archaeon Haloarcula marismortui.

The genome of the halophilic archaeon Haloarcula marismortui contains two rRNA operons designated rrnA and rrnB. Genomic clones of the two operons and their flanking regions have been sequenced, and primary transcripts and processing intermediates derived from each operon have been characterized. The 16S, 23S, and 5S genes from the two operons were found to differ at 74 of 1,472 positions, 39 of 2,922 positions, and 2 of 122 positions, respectively. This degree of sequence divergence for multicopy (paralogous) rRNA genes was 10- to 50-fold or more higher than anticipated. The two operons exhibit other profound differences that include (i) the presence in rrnA and the absence in rrnB of tRNAAla and tRNACys genes in the intergenic and distal regions, respectively, (ii) divergent 5' flanking sequences, and (iii) distinct pathways for processing and maturation of 16S rRNA. Processing and maturation of 16S and 23S rRNA from rrnA operon transcripts and of 23S rRNA from rrnB operon transcripts follow the canonical halophilic pathway, whereas maturation of 16S rRNA from rrnB operon transcripts follows an unusual and different pathway that is apparently devoid of any 5' processing intermediate.

Base Sequence↗