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PubMed1996

Bacillus subtilis operon under the dual control of the general stress transcription factor sigma B and the sporulation transcription factor sigma H.

The sigma B transcription factor of Bacillus subtilis is activated in response to a variety of environmental stresses, including those imposed by entry into the stationary-growth phase, and by heat, salt or ethanol challenge to logarithmically growing cells. Although sigma B is thought to control a general stress regulon, the range of cellular functions it directs remains largely unknown. Our approach to understand the physiological role of sigma B is to characterize genes that require this factor for all or part of their expression, i.e. the csb genes. In this study, we report that the transposon insertion csb40::Tn917lac identifies an operon with three open reading frames, the second of which resembles plant proteins induced by desiccation stress. Primer-extension and operon-fusion experiments showed that the csb40 operon has a sigma B-dependent promoter which is strongly induced by the addition of salt to logarithmically growing cells. The csb40 operon also has a second, sigma H-dependent promoter that is unaffected by salt addition. These results provide support for the hypothesis that sigma B controls a general stress regulon, and indicate that the sigma B and sigma H regulons partly overlap. We suggest that in addition to its acknowledged role in the sporulation process, sigma H is also involved in controlling a subclass of genes that are broadly involved in a general stress response.

Amino Acid Sequence↗

PubMed2001

A novel mechanism controls anaerobic and catabolite regulation of the Escherichia coli tdc operon.

The tdc operon is subject to CRP-controlled catabolite repression. Expression of the operon is also induced anaerobically, although this regulation does not rely on direct control by either FNR or ArcA. Recently, the anaerobic expression of the tdc operon was found to be fortuitously induced in the presence of glucose by a heterologous gene isolated from the Gram-positive anaerobe Clostridium butyricum. The gene, termed tcbC, encoded a histone-like protein of 14.5 kDa. Using tdc-lacZ fusions, it was shown that TcbC did not activate tdc expression by functionally replacing any of the operon regulators. In vitro transcription analyses with RNA polymerase and CRP revealed that faithful CRP-dependent transcription initiation occurred only on supercoiled templates. No specific, CRP-dependent transcription initiation was observed on relaxed or linear DNA templates. Surprisingly, purified His-tagged TcbC activated transcription from a relaxed, circular template, but not from supercoiled or linear templates. Examination of the CRP binding site of the tdc promoter revealed that it was located 43.5 bp upstream of the transcription initiation site. Repositioning of the CRP site at -41.5 bp abolished activation by the TcbC protein and allowed CRP-dependent transcription to occur on linear, relaxed and supercoiled templates. TcbC bound DNA non-specifically; however, in topoisomerase I relaxation assays, it was demonstrated that TcbC imposed torsional constraints on negatively supercoiled DNA, which influenced the ability of the enzyme to relax the topoisomers. Taken together, these results strongly suggest that TcbC activates transcription of tdc by altering the local topological status of the tdc promoter and that, in the wild-type tdc promoter, the CRP binding site is misaligned to allow transcription to occur only under optimal conditions. Indeed, in vivo transcription analyses revealed that repositioning of the CRP binding site to -41.5 bp resulted in high-level, CRP-dependent transcription, even under catabolite-repressing conditions, and that transcription was no longer influenced by TcbC. Remarkably, however, anaerobic regulation of the mutant promoter was retained. This indicates that the other tdc regulators, TdcA and TdcR, govern anaerobic transcription activation by CRP.

Anaerobiosis↗

PubMed2004

Integration host factor is involved in transcriptional regulation of the Brucella abortus virB operon.

Type IV secretion systems (T4SSs) are multicomponent machineries that play an essential role in pathogenicity of many facultative intracellular bacteria. The virB operon of Brucella abortus codes for a T4SS essential for virulence and intracellular multiplication. Here, virB expression analyses carried out using lacZ transcriptional fusions showed that virB promoter (PvirB) is temporally activated within J774 cells. Primer extension experiments revealed that virB transcription starts at 27 bp upstream of the first gene of the virB operon. Structural analyses showed that PvirB and regulatory sequences involved in intracellular regulation span 430 bp upstream of the transcription start site. A protein able to bind PvirB was isolated and identified. This protein, homologue to integration host factor (IHF), specifically interacts with PvirB and induces a DNA bending with an angle of 50.36 degrees . DNAse I footprinting experiments showed that IHF protects a 51 bp region that contains two overlapped IHF binding consensus motifs. VirB expression experiments carried out with PvirB-lacZ fusions showed that in B. abortus IHF participates in the regulation of PvirB activity during the intracellular and vegetative growth in different media. A mutant strain with a 20 bp IHF binding site replacement failed to turn on the virB operon during the initial stages of macrophage infection and displayed severe intracellular multiplication defects. These data indicate that IHF plays a key role during intracellular virB operon expression being required for the biogenesis of the endoplasmic reticulum-derived replicative vacuole.

Animals↗

PubMed2005

The six mammalian cell entry proteins (Mce3A-F) encoded by the mce3 operon are expressed during in vitro growth of Mycobacterium tuberculosis.

The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. The mammalian cell entry (mce)3 operon is one of four homologous mce operons that encodes six putative invasin-like exported proteins (Mce3A-F), possibly involved in entry and survival of M. tuberculosis inside macrophages. We have recently shown that Mce3A, Mce3D and Mce3E are expressed and elicit antibody responses in a majority of human subjects during natural infection with M. tuberculosis. In this study, we demonstrate the expression of Mce3A-F proteins and their mRNA during in vitro growth of M. tuberculosis. To demonstrate the expression of mce3A-F proteins, the antibodies were raised in rabbits against three pure proteins (Mce3A, Mce3D and Mce3E), and their specificity was checked by immunoblotting with recombinant Mce1A-F proteins encoded by mce1 operon. The antibodies were also generated against all the six Mce3 proteins, which were expressed and purified as fusion proteins with glutathione S-transferase (GST) as the fusion partner (GST-Mce3A-F). The antibodies reacted, in each case, with a protein of expected molecular mass (Mr) for the corresponding Mce3 protein in the cell wall fraction but not in the soluble fraction of in vitro-grown M. tuberculosis cells. The presence of mRNA for mce3A-F genes was also shown by using mce3A-F gene-specific primers, and total RNA isolated from in vitro-grown M. tuberculosis cells by reverse transcription-polymerase chain reaction (RT-PCR). Pretreatment of the RNA preparation with RNase A abolished amplification in RT-PCR confirming that mce3A-F mRNA rather than genomic DNA was being amplified. The data show that Mce3A-F encoded by the mce3 operon are expressed during in vitro growth of M. tuberculosis.

Animals↗

PubMed1997

Ribosomal RNA (rrn) operons in Streptococcus mutans and nucleotide sequence of tRNA(Pro) gene associated with rrnB.

Using a cloned 0.5-kb probe containing an internal fragment of 23S ribosomal RNA from the rrnB operon of Streptococcus mutans, we analyzed various endonuclease digests of the chromosomal DNA isolated from human-derived strains of mutans streptococci. Thus far, the examined S. mutans strains exhibited five ribosomal operons. Here, we describe a ribotyping technique for S. mutans based on restriction and Southern blot analyses with the biotin-labeled homologous probe and chemiluminescence detection. We cloned and sequenced a unique gene encoding tRNA(Pro) downstream from 23S rRNA gene at the 3' end of the operon. Primers designed to the 3' end of the rrnB operon PCR-amplified a 2.3-kb DNA fragment in all tested strains. Restriction fragment length polymorphism analysis of the amplicon revealed a diversity of the single locus among S. mutans isolates, thus establishing a potential use of the technique for the molecular epidemiology of mutans streptococci.

Base Sequence↗

PubMed1987

Cloning and characterization of the pyrF operon of Salmonella typhimurium.

The pyrF gene of Salmonella typhimurium encoding the sixth enzyme of pyrimidine nucleotide biosynthesis, OMP decarboxylase, was isolated from a pyrF-complementing R' factor. A 2.0-kbp DNA fragment, generated by PvuI cleavage, was subsequently subcloned into the multicopy vector pBR322 and shown to contain the intact pyrF gene. Bacterial strains harbouring the resulting plasmid contain 15-20-fold elevated levels of OMP decarboxylase, and these levels increase 4-5-fold during uracil starvation. Experiments utilizing minicells identified the gene product as a polypeptide with a molecular mass of approximately 27 kDa. Furthermore, it was found that the pyrF gene is expressed as the first gene of a bicistronic operon, wherein the second gene encodes an 11-kDa polypeptide of unknown functions. The complete nucleotide sequence of the pyrF operon was determined. An open reading frame, encoding a polypeptide with a calculated molecular mass of 26213 Da, was deduced to be the coding region for pyrF. Another open reading frame, with a translational start codon which overlaps the translational stop codons of the pyrF gene, encodes a polypeptide of 11513 Da. This open reading frame represents the coding region for the second gene of the operon, orfF. S1-nuclease mapping indicated that pyrF transcription is initiated 54 bases upstream of the translational start. The leader region does not show any features resembling the attenuators found preceding the pyrBI operon and the pyrE gene.

Base Composition↗

PubMed1990

Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides. Characterization of fbc deletion mutants and complementation by a site-specific mutational variant.

The ubiquinol: cytochrome-c oxidoreductase (cytochrome bc1 complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bc1 complex has a dual function. When the cells are grown photosynthetically, the bc1 complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bc1 complex is a necessary component of the cytochrome-c2-dependent respiratory chain. The fact that the bc1 complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site-directed mutagenesis to address questions relating to the structure and function of the bc1 complex. In the current work, the cloning and complete sequence of the fbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe-2S subunit, the cytochrome b subunit, and the cytochrome c1 subunit. Recombination techniques were used to delete the entire fbc operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providing fbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bc1 complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bc1 complex which, as expected, is resistant to the inhibitor. Hence, this system is suitable for the detailed characterization of the bc1 complex, combining site-directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria.

Amino Acid Sequence↗

PubMed1993

Cloning and nucleotide sequence of the gcv operon encoding the Escherichia coli glycine-cleavage system.

P-protein, H-protein and T-protein of the glycine cleavage system have been purified from Escherichia coli. Their N-terminal amino acid sequences were determined, and a set of oligonucleotide probes was designed for gene cloning. The nucleotide sequence of a fragment of DNA around the 62-min region of the E. coli chromosome, containing genes for the components of the glycine-cleavage system has been determined. The sequence includes three structural genes encoding T-protein (363 amino acids, 40013 Da), H-protein (128 amino acids, 13679 Da) and P-protein (956 amino acids, 104240 Da). These genes are named gcvT, gcvH and gcvP, respectively. They are organized in the above-mentioned order on the same strand of DNA with short intercistronic sequences. The presence of a potential promoter preceding gcvT and a typical rho-independent terminator sequence following gcvP indicated that the three genes constitute a single operon. Each component of the E. coli glycine-cleavage system exhibits considerable amino acid sequence similarity with the animal and plant counterparts. When the plasmid containing the gcv operon was transfected in E. coli cells, the gene products of gcvT, gcvH and gcvP were overexpressed under the direction of the promoter of the gcv operon. However, bacteria harboring the plasmid that contained the gcv operon without the promoter region and the 5' terminal portion of gcvT failed to overexpress any of the three components.

Amino Acid Oxidoreductases↗

PubMed2007

Accelerated immunopathological response of mice infected with Mycobacterium tuberculosis disrupted in the mce1 operon negative transcriptional regulator.

Mycobacterium tuberculosis causes a variety of clinical outcomes determined by host as well as bacterial factors. M. tuberculosis disrupted in the mce1 operon causes increased mortality in immunocompetent mice. This operon is negatively regulated by mce1R (Rv0165c). We studied the role of mce1R in infection outcome in mice. At 5 x 10(4) tail vein infectious dose, the median survival time (MST) of mice infected with the mce1R mutant M. tuberculosis H37Rv was 293 days, while mice infected with the wild-type H37Rv survived more than 350 days (P < 0.0001). At a higher dose (5 x 10(6)), the MST of mutant-infected mice was 32 days, compared with 127 days for wild type-infected mice (P < 0.0001). With either tail vein or aerosol infection, mutant-infected mice developed larger granulomatous lesions in their lungs than mice infected with the wild type. Mutant-infected mice were unable to control the bacterial burden in the first 4 weeks of infection, but even after achieving control later, these mice succumbed to granulomatous pneumonia. These observations suggest that the early deregulated expression of the mce1 operon products determines later granulomatous tissue response. mce1 operon may homeostatically regulate the cell wall architecture in vivo that elicits a steady-state granuloma tissue response permitting M. tuberculosis to establish a long-term infection.

Animals↗

PubMed1998

Homologous recombination between rrn operons rearranges the chromosome in host-specialized species of Salmonella.

Partial digestion with I-CeuI, which digests bacterial DNA at the gene coding for the large subunit rRNA, established the rrn genomic skeleton (the distance in kb between rRNA operons) in 56 strains of Salmonella, from Salmonella Reference B (SARB) set. All had seven I-CeuI sites, indicating seven rrn operons. The order of I-CeuI fragments was ABCDEFG in S. typhimurium LT2 and in 31 other species, mostly host-generalists; in S. typhi, S. paratyphi C, S. gallinarum, and S. pullorum (host-specialized species), these fragments are rearranged, due to homologous recombination between the rrn operons. Rearrangements, such as inversions and translocations not involving the rrn operons, are rare. I-CeuI fragments of some species are larger than the norm, suggesting the insertion of unique blocks of DNA by lateral transfer from other species.

Chromosomes, Bacterial↗

PubMed1998

Multiple copies of ammonia monooxygenase (amo) operons have evolved under biased AT/GC mutational pressure in ammonia-oxidizing autotrophic bacteria.

The recent availability of complete sequences of ammonia monooxygenase (16 amoA, 5 amoB and 5 amoC gene sequences) and particulate methane monooxygenase (2 pmoA, pmoB and pmoC gene sequences each) genes allowed for a detailed analysis of their relatedness. Nucleotide sequence analysis was performed in order to identify the origins of the nearly identical operon copies within a given nitrosofier/methanotroph strain. Our data suggest that amo-homologous gene evolution has occurred in individual strains (orthology) under biased AT/GC pressure rather than by horizontal transfer. The multiple operon copies within individual strains are the result of operon duplication (paralogy). While the near identity of the multiple operon copies makes it impossible to determine whether paralogous gene expansion occurred in the last common ancestor of ammonia oxidizers or after speciation took place, we conclude that the duplication events were not recent events. We propose that the elimination of third basepair degeneracy between copies within one organism is implemented by a rectification mechanism resulting in concerted evolution.

Base Composition↗

PubMed1999

The amo operon in marine, ammonia-oxidizing gamma-proteobacteria.

While there is an extensive database of genes encoding ammonia monooxygenase (amo) found in the ammonia-oxidizing beta-proteobacteria, few amo sequences are available representing the gamma-proteobacteria. We sequenced the complete amo operon (amoCAB) for Nitrosococcus oceani (ATCC 19707), a marine, autotrophic, ammonia-oxidizing bacterium belonging to the gamma-subdivision of the proteobacteria. An additional autotrophic, ammonia-oxidizing bacterium isolated from a marine environment (strain C-113) was identified as belonging to the Nitrosococcus genus by 16S rDNA analysis and its amo operon was sequenced. This is the first report of a full-length sequence for the amo operon from a gamma-subdivision autotrophic ammonia-oxidizing bacterium. The N. oceani and C-113 amo genes were 88-90% identical to each other, 49-53% identical to the pmo genes encoding the related particulate methane monooxygenase of Methylococcus capsulatus (Bath), and 39-42% identical to the amo genes of the beta-subdivision autotrophic ammonia-oxidizing bacteria. In both Nitrosococcus strains, the amo operon was found as a single copy and contained three genes, amoC, amoA, amoB, with intergenic spacer regions between amoC and amoA (286 bp) and between amoA and amoB (65 bp). We conclude that the amo genes will allow for a finer scale phylogenetic differentiation than 16S rDNA within the gamma-subdivision AOB.

Amino Acid Sequence↗

PubMed2000

The L-asparagine operon of Rhizobium etli contains a gene encoding an atypical asparaginase.

The L-asparagine operon of Rhizobium etli was cloned and sequenced. Sequence analysis showed four adjacent open reading frames which were designated as ansR, ansP, ansA and ansB. The ansR and ansP genes encoded proteins similar to a transcriptional repressor and an L-asparagine permease, respectively. By Tn5 mutagenesis and complementation analysis we identified the ansA product as a thermolabile asparaginase, and the ansB product as an aspartase. An asparagine-inducible transcript covering ansP, ansA and ansB was detected by reverse transcription (RT)-PCR, indicating that these genes are organized in an operon. Introduction of the R. etli ans operon into Sinorhizobium meliloti induced growth with asparagine as the sole carbon and nitrogen source, suggesting that the ans operon plays the same physiological role in both bacteria. The product of the R. etli ansA gene showed no sequence similarity with previously reported microbial asparaginases, this protein seems to be an atypical asparaginase which evolved apart from bacterial and yeast asparaginases.

Asparaginase↗

PubMed2006

Aerobic activation of transcription of the anaerobically inducible Escherichia coli focA-pfl operon by fumarate nitrate regulator.

Expression of the anaerobically inducible focA-pfl operon in Escherichia coli was activated nearly sevenfold relative to wild-type under aerobic growth conditions by increasing the dosage of the fnr gene on a pBR322-based plasmid (pCH21). No effect on anaerobic expression levels was observed, suggesting that operon expression under these conditions is maximal. Examination of the complex transcript pattern of the focA-pfl operon confirmed that in strains bearing pCH21 all transcripts, with the exception of the promoter 7 transcript, were up-regulated aerobically. Western analysis of strains bearing pCH21 revealed that the fumarate nitrate regulator (FNR) level was increased approximately ninefold relative to the level in strains bearing a single copy of the fnr gene aerobically, but was only overproduced threefold anaerobically. Analysis of an fnr-lacZ fusion indicated that fnr expression was more strongly negatively autoregulated in anaerobic cells compared with aerobic cells when pCH21 was present. Taken together, these findings suggest that high-level overproduction of FNR is prevented anaerobically by active FNR repressing expression of the fnr gene. Furthermore, transcription from promoter 7 of the focA-pfl operon, which depends on both ArcA-P and FNR, cannot be activated aerobically by overproduction of FNR alone, while promoter 6, which is less dependent on ArcA-P, can be activated under these conditions.

Acetyltransferases↗

PubMed2005

Control analysis as a tool to understand the formation of the las operon in Lactococcus lactis.

In Lactococcus lactis the enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are uniquely encoded in the las operon. We used metabolic control analysis to study the role of this organization. Earlier studies have shown that, at wild-type levels, LDH has no control over glycolysis and growth rate, but high negative control over formate production (C(Jformate)LDH=-1.3). We found that PFK and PK exert no control over glycolysis and growth rate at wild-type enzyme levels but both enzymes exert strong positive control on the glycolytic flux at reduced activities. PK exerts high positive control over formate (C(Jformate)PK=0.9-1.1) and acetate production (C(Jacetate)PK=0.8-1.0), whereas PFK exerts no control over these fluxes at increased expression. Decreased expression of the entire las operon resulted in a strong decrease in the growth rate and glycolytic flux; at 53% expression of the las operon glycolytic flux was reduced to 44% and the flux control coefficient increased towards 3. Increased las expression resulted in a slight decrease in the glycolytic flux. At wild-type levels, control was close to zero on both glycolysis and the pyruvate branches. The sum of control coefficients for the three enzymes individually was comparable with the control coefficient found for the entire operon; the strong positive control exerted by PK almost cancels out the negative control exerted by LDH on formate production. Our analysis suggests that coregulation of PFK and PK provides a very efficient way to regulate glycolysis, and coregulating PK and LDH allows cells to maintain homolactic fermentation during glycolysis regulation.

Bacterial Proteins↗

PubMed1996

Differential regulation of the mcb and emr operons of Escherichia coli: role of mcb in multidrug resistance.

The mcb operon (which is responsible for microcin B17 production) and the emr operon (which encodes a multidrug resistance pump) share a common negative regulator, EmrR. Nevertheless, compounds that induce the emr operon repress the mcb operon. The pump dedicated to microcin B17 extrusion can also protect the calls against sparfloxacin and other toxic compounds.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗

PubMed2005

Horizontal transfer of iturin A operon, itu, to Bacillus subtilis 168 and conversion into an iturin A producer.

Iturin A and its derivatives are lipopeptide antibiotics produced by Bacillus subtilis and several closely related bacteria. Three iturin group operons (i.e., iturin A, mycosubtilin, and bacillomycin D) of those antibiotic-producing strains have been cloned and sequenced thus far, strongly implying the horizontal transfer of these operons. To examine the nature of such horizontal transfer in terms of antibiotic production, a 42-kb region of the B. subtilis RB14 genome, which contains a complete 38-kb iturin A operon, was transferred via competent cell transformation to the genome of a non-iturin A producer, B. subtilis 168, using a method based on double-crossover homologous recombination with two short landing pad sequences (LPSs) in the genome. The recombinant was positively selected by confirming the elimination of the cI repressor gene, which was localized between the two LPSs and substituted by the transferred segment. The iturin A operon-transferred strain 168 was then converted into an iturin A producer by the introduction of an sfp gene, which encodes 4'-phosphopantetheinyl transferase and is mutated in strain 168. By inserting the pleiotropic regulator degQ, the productivity of iturin A increased sevenfold and was restored to about half that of the donor strain RB14, without the transfer of additional genes, such as regulatory or self-resistance genes.

Bacillus subtilis↗

PubMed1998

Construction and characterization of a 1,3-propanediol operon.

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose. Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B12, which must be provided in E. coli fermentations. However, the amount of coenzyme B12 needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.

Acetates↗