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PubMed1998

Sequence diversity in the 16S-23S intergenic spacer region (ISR) of the rRNA operons in representatives of the Escherichia coli ECOR collection.

The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S-23S intergenic spacer region (ISR) for tRNA(Glu-2) and three (rrnA, D, and H) contain genes for tRNA(Ile-1) and tRNA(Ala-1B). To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions.

Base Sequence↗

PubMed2003

Improved vectors for transcriptional/translational signal screening in corynebacteria using the melC operon from Streptomyces glaucescens as reporter.

The tyrosinase operon ( melC) from Streptomyces glaucescens was cloned and functionally expressed in Brevibacterium lactofermentum and Corynebacterium glutamicum under the control of the promoter of the kan gene from Tn 5. Recombinant corynebacterial cells containing the tyrosinase operon produced melanin on agar plates and in liquid culture when supplemented with copper and tyrosine. A conjugative bifunctional replacement vector for transcriptional/translational signal screening (pEMel-1) was constructed using expression of the melC operon from S. glaucescens, which can be used for cloning promoter sequences as EcoRI- NdeI fragments. When the DNA fragments with promoter activity such as cspBp or trpp were inserted into pEMel-1, B. lactofermentum harboring the chimeric plasmids produced melanin at different stages of growth, allowing temporal detection of promoter activity. The vector was also used to detect the activity of a Streptomyces promoter ( xysAp), which was inactive in B. lactofermentum, after PCR mutagenesis. The melC operon can be used for the visual, inexpensive (compared to the high price of starch azure for amylase detection), and non-selective (in contrast to the kan or cat genes) screening of several thousand clones at high colony density without killing of the transformants due to the presence of iodine (as in the case of amylase assay).

Bacterial Proteins↗

PubMed2005

The puf operon of the purple sulfur bacterium Amoebobacter purpureus: structure, transcription and phylogenetic analysis.

The puf operon, encoding photosynthetic reaction center and light-harvesting genes, of the purple sulfur phototrophic bacterium Amoebobacter purpureus was cloned and sequenced. This revealed an unusual operon structure of the genes pufB1 A1 LMCB2 A2 B3 A3. The sequence represents the second complete puf operon available for Chromatiaceae. So far, additional sets of light-harvesting 1 (LH1) genes, pufB2 A2 and pufB3 A3 in the region downstream of pufC have only been described for Allochromatium vinosum. Along with reports of multiple LH1 polypeptides found in some Ectothiorhodospiraceae by direct protein sequencing, our results indicate that multiple LH1 genes may occur frequently in phototrophic gamma-proteobacteria. Phylogenetic analyses suggested a coevolution of the core puf genes pufB1 A1 LM. Separate analysis of the LH1 alpha and beta polypeptides revealed a high intraspecies relatedness for the secondary LH1beta polypeptides, possibly caused by functional constraints. In contrast, LH1alpha subunits of Amb. purpureus and Alc. vinosum are closely related (85% sequence identity) which could reflect horizontal gene transfer. RNA analyses suggested co-transcription of all puf genes in Amb. purpureus as a 5.5 kb primary transcript which appears to be more stable than the puf operon primary transcripts of purple non-sulfur bacteria. The 5' end of the transcript mapped to a putative promoter, which contains a -35 region located in an inverted repeat DNA sequence.

Amino Acid Sequence↗

PubMed2005

Global gene expression analysis revealed an unsuspected deo operon under the control of molybdate sensor, ModE protein, in Escherichia coli.

ModE protein, a molybdate sensor/regulator, controls the transcription of genes coding for molybdate uptake (mod), molybdopterin synthesis (moa), molybdoenzymes nitrate reductase (nap) and dimethylsulfoxide reductase (dms), as well as fermentative dihydrogen production (fdhF and hyc) and respiratory nitrate reductase (narXL) in Escherichia coli. The catalytic product of a second protein, MoeA, is also required for molybdate-dependent positive regulation of hyc and nar operons. To explore the potential role of ModE and MoeA in the regulation of other E. coli genes, the global gene expression profile of a wild type and a modE, moeA double mutant grown in glucose-minimal medium under anaerobic conditions were compared. Expression of 67 genes was affected by the modE and moeA mutations (P value <0.01). Of these, 17 differed by at least 2-fold or higher. Fourteen genes were expressed at a higher level in the mutant (2.4- to 23.9-fold) (notably, mod-molybdate transport, deo-nucleoside catabolism and opp-oligopeptide transport operons) and dmsA and yli operon were expressed at a higher level in the wild type parent (2.6- to 5.7-fold). One of the unexpected findings was repression of the deo operon by ModE. This was confirmed by quantitative RT-PCR and by the analysis of a deoC-lacZ fusion. The deo promoter/operator region contains a putative ModE-consensus sequence centered at -35 in which the adenines are replaced by guanines (TGTGT-N7-TGTGT). The ModE protein did bind to the deo upstream DNA and shifted its electrophoretic mobility. Bioinformatics analysis of the E. coli genome for ModE-consensus motif (TATAT-N7-TAYAT) identified 21 additional genes/operons including the moa as potential targets for Mo-control. The physiological role of many of the genes identified solely by bioinformatics (19/21) is unknown. Expression levels of these genes were similar in the parent and the isogenic modE, moeA mutant when cultured anaerobically in glucose-minimal medium. This study identified additional targets, such as deo and opp, for the Mo-dependent control in E. coli.

Escherichia coli↗

PubMed1997

Deletion of two downstream genes alters expression of the hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough.

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough consists of six genes (hmcA to hmcF) that encode structural components of the high-molecular-mass cytochrome redox protein complex (the Hmc complex). Two genes (rrf1 and rrf2) encoding regulatory proteins are present downstream of hmcF. Expression of the hmc operon, monitored by incubating protein blots with HmcA-specific or HmcF-specific antibodies, was found to be highest when hydrogen was the sole electron donor for sulfate reduction. Use of lactate or pyruvate as electron donor reduced expression of the hmc operon. A mutant with a deletion of the rrf1 and rrf2 genes was generated with the sacB mutagenesis method. This mutant overexpressed the hmc operon approximately threefold. It grew more rapidly than the wild type when hydrogen was used as the electron donor for sulfate reduction, but more slowly than the wild type when lactate was used. The results indicate that a physiological function of the Hmc complex is in electron flow from hydrogen to sulfate. At least one redox carrier is shared competitively by the hydrogen and lactate oxidation pathways in D. vulgaris.

Bacterial Proteins↗

PubMed2001

Transcriptional regulation of the moe (molybdate metabolism) operon of Escherichia coli.

Regulation of transcription of the Escherichia coli moe operon, which codes for proteins connecting molybdate metabolism, molybdopterin synthesis, and apomolybdoenzyme synthesis, was investigated. Expression of the moe operon was independent of genes coding for molybdate transport and Mo-cofactor biosynthesis. Expression of moeA-lacZ increased during anaerobic growth (2.5-fold over the aerobic value) and in the presence of nitrate and trimethylamine N-oxide (3.5- and 1.5-fold, respectively). The nitrate-dependent increase in moe expression required the NarL protein, while the anaerobiosis-dependent increase in moeA-lacZ expression required Arc proteins. ArcA-phosphate and not ArcA bound to the DNA upstream of moe, shifted the electrophoretic mobility of moe promoter DNA, and protected the DNA from DNase I hydrolysis. Nitrate-independent transcription of moeA-lacZ was repressed by the FNR protein, which also protected moe operator DNA from DNase I hydrolysis. These results show that ArcA-phosphate and FNR have opposite effects on the transcriptional regulation of the moe operon, and the combined action of the two redox regulators modulate the level of Mo-cofactor in the cell. Apparently, the control of synthesis of Mo-cofactor and the apomolybdoenzymes nitrate reductase and trimethylamine N-oxide reductase are coupled at the level of the moe operon.

Base Sequence↗

PubMed2005

Horizontal transfer of two operons coding for hydrogenases between bacteria and archaea.

Using a phylogenetic approach, we discovered three putative horizontal transfers between bacterial and archaeal species involving large clusters of genes. One transfer involves an operon of 13 genes, called mbx, which probably was transferred into the genome of Thermotoga maritima from a species belonging or close to the Pyrococcus genus. The two others implied an operon of six genes, called ech, transferred independently to the genomes of Thermoanaerobacter tengcongensis and Desulfovibrio gigas, from a species belonging or close to the Methanosarcina genus. All these transfers affected operons coding for multisubunit membrane-bound (NiFe) hydrogenases involved in the energy metabolism of the donor genomes. The functionality of the transferred operons has not been experimentally demonstrated for T. maritima, whereas in D. gigas and T. tengcongensis the encoded multisubunit hydrogenase could have a role in energy conservation. This report adds several cases of horizontal gene transfers among hydrogenases already described.

Archaea↗

PubMed2006

On the paucity of duplicated genes in Caenorhabditis elegans operons.

Spliced leader trans-splicing is an mRNA maturation process used by a small set of eukaryotes, including the nematode C. elegans, to cap the downstream genes of operons. We analyzed the frequency of duplication of operonic genes in C. elegans and confirmed that they are duplicated less often in the genome than monocistronic genes. Because operons account for about 15% of the genes in C. elegans, this lower duplication frequency might place a large constraint on the plasticity of the genome. Further analyses suggest that this paucity of duplicated genes results from operon organization hindering specific types of gene duplication.

Animals↗

PubMed2004

Comparative genomic analysis of the sigB operon in Listeria monocytogenes and in other Gram-positive bacteria.

The stress-responsive, alternative sigma factor sigmaB has been described in members of three Gram-positive genera, Bacillus, Listeria, and Staphylococcus. In these bacteria, sigmaB appears to play an important role in facilitating rapid adaptation to and survival in stressful environments. sigmaB activity is regulated through a complex system of phosphatases and kinases encoded by rsb (regulator of sigma B) genes. We describe the sigB operon structure for the facultative intracellular pathogen Listeria monocytogenes and apply this sequence as well as other previously described sigB operon sequences to probe the evolution and functional conservation of the sigmaB stress response system among different Gram-positive bacteria. While sigmaB as well as two Rsbs (RsbS and RsbT) are highly conserved (73%, 84%, and 79% average amino acid [aa] identities, respectively), the predicted aa sequences of the other Rsb proteins showed less conservation (62-71% aa identities). Furthermore, the sigB operon structure varies among bacterial species. Bacterial species differ in the numbers and identities of rsb genes encoded in their genomes. We thus conclude that the sigmaB stress-response system as represented by the sigB operon has diverged in both its overall components as well as in the sequences of its individual proteins, even among closely related bacterial species. Differential evolution of this stress response system among various genera may represent a strategy that enables bacteria to adapt cellular response and survival systems to a variety of stress conditions.

Amino Acid Sequence↗

PubMed2004

Factors affecting interpretation of restriction fragment length polymorphism (RFLP) patterns from PCR-amplified bacterial 16S rRNA genes: operon number and primer mismatching.

PCR methods have been shown to be biased by several factors. In the present study, we have developed a theoretic and practical approximation to elucidate how the presence of mismatches at the primers annealing regions and the different number of rDNA operons per cell can influence PCR and subsequent restriction fragment length polymorphism (RFLP) analyses from bacterial populations. We have performed RFLP analyses of 16S rRNA genes amplified by PCR from mixed bacterial cultures showing different primer identities and number of rDNA operons. Our results clearly corroborate that both factors, number of rDNA operons and primers identity, clearly influence the 16S rDNA-RFLP estimations. It has been demonstrated that a higher number of operons leads to a higher efficiency of detection, but a lower degree of primer complementarity implies a decrease in such efficiency.

Bacteria↗

PubMed2001

Comparison of psbK operon organization and group III intron content in chloroplast genomes of 12 Euglenoid species.

A novel mixed operon has been identified in the photosynthetic protist E. gracilis. The genes for psbK, ycf12, psaM, and trnR are co-transcribed. The resulting tetracistronic transcripts are processed through endonucleolytic cleavage of the intergenic spacers and intron splicing to form three mature monocistronic mRNAs and a tRNA. A group III twintron and a group III intron are located in psbK. Another group III intron is found in ycf12. The psbK operon has been cloned by PCR amplification from nine related Euglenoid species. In each species, the gene order and content of the psbK operon is conserved. The psbK operons contain phylogenetically conserved eubacterial promoter, translational, and 3' processing elements. Intron content varies significantly from species to species. Based on a comparison of the intron content with the results of phylogenetic analysis, group III intron evolution within the Euglenoid lineage is much more complex than previously believed.

Algal Proteins↗

PubMed1998

Transcript analysis of the tobacco plastid operon rps2/atpI/H/F/A reveals the existence of a non-consensus type II (NCII) promoter upstream of the atpI coding sequence.

The plastid ATP synthase complex is composed of nine subunits, of which six are encoded in the plastome. The plastid-encoded genes are arranged in two transcriptional units: atpB/E and atpI/H/F/A. We have recently reported that besides containing four -10 and -35 consensus-type (CT) promoters, the atpB/E operon also contains a non-consensus type (NCII) promoter that alone is responsible for its expression in non-photosynthetic plastids. As the functionality of ATP synthase requires expression of all nine subunits, NCII promoter-driven transcription of the atpI/H/F/A operon is to be expected in non-photosynthetic plastids. Therefore, a detailed transcriptional analysis of this operon was carried out using RNA samples from tobacco leaf, cultured cells (BY-2) and seedlings grown on streptomycin and spectinomycin; which contain chloroplasts, translationally active non-photosynthetic plastids and translationally inactive plastids, respectively. We identified a total of three transcription initiation sites (TIS) and four transcript processing sites in the non-coding regions of this operon. Our results also demonstrate that rps2 is co-transcribed with the atpI/H/F/A genes. One of the TIS (-208 atpI) is characterized by an NCII type promoter, while other two primary transcripts (-131 atpI and -384 atpH) initiate from CT promoters. In non-photosynthetic plastids the atpI/H/F/A-specific transcript pool seems to be solely contributed by initiation at the -208 atpI (NCII type) promoter, because transcripts from CT promoters do not accumulate in these plastid types.

Base Sequence↗

PubMed2006

Identification and characterization of a germination operon from Bacillus thuringiensis.

Spore cortex-lytic enzymes are essential for germination in Bacillus. A homologue of the cwlJ gene involved in spore germination was isolated from Bacillus thuringiensis. The deduced product of this gene exhibits striking sequence similarity to CwlJ of Bacillus subtilis. Another open reading frame (ORF) was found 27 bp downstream of cwlJ and its deduced product shows high similarity to YwdL of B. subtilis. Reverse transcription polymerase chain reaction analysis indicated that cwlJ and ywdL formed a bicistronic operon in B. thuringiensis. Disruption of this operon did not affect sporulation and the spores of corresponding mutant showed the same refractility as the wild-type strain. In contrast, the fall of optical density at 600 nm in the mutant culture was slower than that of the wild-type strain during the spore germination response to different germinants such as L: -alanine, inosine or CaDPA. The spore germination of the cwlJ mutant was restored by introducing this operon into the disruption mutant. These results suggest this operon is essential for normal spore germination in B. thuringiensis. The expression of cwlJ was observed in the sporulating cells through Western blot experiments.

Bacillus thuringiensis↗

PubMed1987

Dual level control of the Escherichia coli pheST-himA operon expression. tRNA(Phe)-dependent attenuation and transcriptional operator-repressor control by himA and the SOS network.

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli have established that the pheST operon transcription is controlled by a Phe-tRNA(Phe)-mediated attenuation mechanism. More recently, the himA gene, encoding the alpha-subunit of integration host factor, was recognized immediately downstream from pheT, possibly forming part of the same transcriptional unit. By using the in-vitro transcription and S1 mapping techniques, transcription termination after pheT could be excluded, indicating that himA can be expressed from polycistronic messenger RNAs encompassing the pheST region. However, the presence of a secondary promoter able to express himA and located within pheT is demonstrated. To further investigate the regulation of the pheST-himA operon expression, genetic fusions between various parts of this operon and the lacZ gene were constructed and studied. Our results confirm the autoregulation of himA previously described, and demonstrate that it occurs through the modulation of the secondary promoter activity within pheT. Surprisingly, it is found that the pheST promoter is also submitted to the same control. Consistent with this, DNA sequences homologous to the integration host factor binding site consensus are present at the level of both promoters. However, evidence in favor of two different repressor complexes is provided. Previously observed SOS induction of the himA expression is shown to occur through the modulation of both promoter activities. Contrasting with the other genes under SOS control, the LexA protein binding site consensus sequence could not be found in the two promoter regions. This suggests that either the LexA protein directly participates in the formation of an active holorepressor, or that the product of an SOS gene is able to inhibit the formation or the binding of such a repressor. Finally, our results indicate that the pheST-himA operon expression is controlled by two different mechanisms acting independently. (1) The phenylalanyl-tRNA synthetase and the himA product expressions are controlled by an operator-repressor type mechanism, in which the himA product and the SOS network are involved. (2) Through its partial cotranscription with pheST, himA expression is also under attenuation control. The latter control may provide a way to couple the intracellular concentration of the himA product to the functional state of the translational apparatus.

DNA Repair↗

PubMed1979

DNA sequences of promoter regions for rRNA operons rrnE and rrnA in E. coli.

The nucleotide sequences have been determined for the promoter regions of two ribosomal RNA operons, rrnA and rrnE, in E. coli. The sequences cover the two in vitro transcription start sites identified for each operon (Gilbert, der Boer and Nomura, 1979). The first two start sites are 283 and 291 bp preceding the mature 16S rRNA (m16S rNA) coding regions for rrnE and rrnA, respectively; the second start sites are 174 and 174 +/- 1 bp preceding the m16S rRNA coding regions for rrnE and rrnA, respectively. Each of these start sites has an identifiable "Pribnow box" sequence 6-7 bp upstream from the start site. The nucleotide sequences of the two operons have nearly complete homology from the m16S rRNA coding regions to positions 145 bp upstream from those regions, and at the regions surrounding the Pribnow boxes preceding the first start sites. The DNA sequences indicate that the RNAs transcribed from the first start sites of rrnE and rrnA are quite different in their first 150 nucleotides. These heterogeneous regions, however, precede the RNAse III cleavage sites (deduced previously by Young and Steitz, 1978), and the "precursor 16S rRNA" molecules are largely homogeneous. The nucleotide sequences of the promoter regions of the two rRNA operons are also compared with those or rrnD and rrnX, determined by Young and Steitz (1979), and some common features are discussed.

Base Sequence↗

PubMed1980

Protein L4 of the E. coli ribosome regulates an eleven gene r protein operon.

We have previously reported autogenous regulation of the S10 operon encoding eleven ribosomal proteins. By measuring the synthesis of individual r proteins after specific oversynthesis of nine different ribosomal proteins from the S10 operon, we now find that one, L4, affects the expression of the operon. Moreover, the induction of L4 synthesis results in a strong reduction of the synthesis of mRNA from at least four genes of the S10 operon.

Bacterial Proteins↗

PubMed1987

Open reading frames in the control regions of the phenylalanyl-tRNA synthetase operon of E. coli.

The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and it expression is controlled by attenuation in a way similar to many amino acid biosynthetic operons. The nucleotide sequence of the control regions of the operon indicates the presence of several open reading frames besides that of the leader peptide. One of these open reading frames, called the alternative leader peptide, starts at about the same place as the leader peptide and ends after the terminator of the attenuator. Another open reading frame, called the terminator peptide, starts after the terminator and covers about half the distance to pheS, the first structural gene of the operon. The present report shows that, in fact, the only open reading frame to be translated efficiently is the leader peptide itself. The alternative leader peptide and the terminator peptide are both translated at a negligible rate.

Amino Acyl-tRNA Synthetases↗

PubMed1992

An overlap between operons involved in carotenoid and bacteriochlorophyll biosynthesis in Rhodobacter capsulatus.

A new example of superoperonal gene arrangement has been documented in the Rhodobacter capsulatus photosynthetic gene cluster. The promoter for the operon initiated by the bchI gene is embedded within an upstream operon for carotenoid synthesis. The stop codon for the crtA gene, the only gene in the first operon, overlaps the start codon of the downstream bchI gene. As a consequence of this overlap, the promoter(s) for the bch operon must be located within the crtA structural gene. The bchI gene is shown here for the first time to be required for the conversion of protoporphyrin IX to subsequent intermediates in bacteriochlorophyll biosynthesis.

Bacteriochlorophylls↗