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PubMed1995

cAMP-cAMP receptor protein complex: five binding sites in the control region of the Escherichia coli mannitol operon.

The control region of the mannitol (mtl) operon of Escherichia coli has been shown to contain five cAMP receptor protein (CRP) binding sequences, the most yet reported for any operon. A DNA fragment encompassing the entire mtl operon regulatory region was generated by PCR, and the binding of the cAMP-CRP complex was studied. Using restrictional analysis to separate, delineate and destroy the various putative CRP binding sites, all five sites were shown to be functional for CRP binding in vitro. Four of these sites bound the cAMP-CRP complex with high affinity while the fifth site (the most distal relative to the transcriptional start site) bound the complex with lower affinity. Simultaneous binding of cAMP-CRP complexes to several of these sites was demonstrated. The results serve to identify and define five dissimilar CRP binding sites in a single operon of E. coli. A model for mtl operon transcriptional initiation and repression complexes is presented.

Base Sequence↗

PubMed1996

Genes responsible for nisin synthesis, regulation and immunity form a regulon of two operons and are induced by nisin in Lactoccocus lactis N8.

Nisin is a small post-translationally modified lanthionine-containing peptide (lantibiotic) produced by certain Lactococcus lactis strains which has a high antimicrobial activity against several pathogenic Gram-positive bacteria. Northern blots and RT/PCR analysis of the nisin-producing strain N8 revealed that the nisZBTCIPRKFEG gene cluster, responsible for nisin biosynthesis, immunity and regulation, consists of two operons, nisZBTCIPRK and nisFEG. The promoter of the nisFEB operon was mapped. The -35 to -1 region upstream of the transcription start of the nisFEG promoter showed 73% identity with the corresponding region upstream of the nisA and nisZ gene. In contrast to earlier reports, nisin was found to be secreted during the early stages of growth was well as later in the growth cycle. The secreted nisin was adsorbed on the surface of the cells and was released to the medium during mid-exponential growth, when the pH in the medium fell below 5.5. In nisZB antisense and nisT deletion mutant strains constructed in this study the transcription of the nisin operons, nisin production and immunity were lost. Provision of external nisin restored the transcription of both operons in the mutant strains, showing that the operons are coordinately regulated by mature nisin. Nisin induction of the mutant strains also resulted in an increased amount of the NisI protein and an increase in the level of immunity. Induction using higher concentrations of nisin yielded a higher level of immunity. These results showed that the nisin promoters are under positive control in an autoregulatory manner and that antimicrobial peptides can also function as signal molecules.

Base Sequence↗

PubMed1999

The genes controlling sucrose utilization in Clostridium beijerinckii NCIMB 8052 constitute an operon.

The sucrose operon of Clostridium beijerinckii NCIMB 8052 comprises four genes, which encode a sucrose-specific enzyme IIBC(Scr) protein of the phosphotransferase system (ScrA), a transcriptional repressor (ScrR), a sucrose hydrolase (ScrB) and an ATP-dependent fructokinase (ScrK). The scrARBK operon was cloned in Escherichia coli in three stages. Initial isolation was achieved by screening a C. beijerinckii genomic library in E. coli for clones able to utilize sucrose, while the remainder of the operon was isolated by inverse PCR and by plasmid rescue of flanking regions from a scrB mutant constructed by targeted gene disruption. Substrate specificity assays confirmed that the sucrose hydrolase was a beta-fructofuranosidase, able to hydrolyse sucrose and raffinose but not inulin or levans, and that the scrK gene encoded an ATP/Mg2+-dependent fructokinase. Both enzyme activities were induced by sucrose in C. beijerinckii. Disruption of the scr operon of C. beijerinckii by targeted plasmid integration into either the scrR or the scrB gene resulted in strains unable to utilize sucrose, indicating that this was the only inducible sucrose catabolic pathway in this organism. RNA analysis confirmed that the genes of the scr operon were co-transcribed on a 5 kb mRNA transcript and that transcription was induced by sucrose, but not by glucose, fructose, maltose or xylose. Primer extension experiments identified the transcriptional start site as lying 44 bp upstream of the scrA ATG start codon, immediately adjacent to the imperfect pelindrome sequence proposed to be a repressor binding site. Disruption of the scrR gene resulted in constitutive transcription of the upstream scrA gene, suggesting that ScrR encodes a transcriptional repressor which acts at the scrA operator sequence. The scrR gene is therefore itself negatively autoregulated as part of the polycistronic scrARBK mRNA

5' Untranslated Regions↗

PubMed2003

The regulatory link between carbon and nitrogen metabolism in Bacillus subtilis: regulation of the gltAB operon by the catabolite control protein CcpA.

Bacillus subtilis assimilates ammonium by the concerted action of glutamine synthetase and glutamate synthase. The expression of the gltAB operon encoding the latter enzyme is impaired in B. subtilis ccpA mutant strains. CcpA is a pleiotropic transcriptional regulator that is the key factor in the regulation of carbon metabolism. However, in addition to their defect in catabolite repression ccpA mutants are unable to grow on minimal media with glucose and ammonium as the single sources of carbon and nitrogen, respectively. In this work, the expression of the gltAB operon was analysed and its role in growth on minimal sugar/ammonium media was studied. Expression of gltAB requires induction by glucose or other glycolytically catabolized carbon sources. In ccpA mutants, gltAB cannot be induced by glucose due to the low activity of the phosphotransferase sugar transport system in these mutants. A mutation that allowed phosphotransferase system activity in a ccpA background simultaneously restored glucose induction of gltAB and growth on glucose/ammonium medium. Moreover, artificial induction of the gltAB operon in the ccpA mutant allowed the mutant strain to grow on minimal medium with glucose and ammonium. It may be concluded that expression of the gltAB operon depends on the accumulation of glycolytic intermediates which cannot occur in the ccpA mutant. The lack of gltAB induction is the bottleneck that prevents growth of the ccpA mutant on glucose/ammonium media. The control of expression of the gltAB operon by CcpA provides a major regulatory link between carbon and amino acid metabolism.

Bacillus subtilis↗

PubMed2003

A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus.

Plasmid analysis of isolates from a small Paracoccus population revealed that all 15 representatives carried at least one endogenous plasmid of 23 or 15 kb in size, in addition to further plasmids of different sizes. It was shown by restriction analysis and hybridization that the 23 and 15 kb plasmids from the different isolates were identical or very similar to each other. By partial sequencing of pOL18/23, one of the 23 kb plasmids, a complete rrn operon with the structural genes for 16S, 23S and 5S rRNA, two genes for tRNA(Ile) and tRNA(Ala) within the spacer between the 16S and 23S rRNA genes, and a final tRNA(fMet) at the end of the operon were discovered. Expression of a green fluorescent protein gene (gfp) after insertion of a DNA fragment from the region upstream of the rRNA genes into a promoter-probe vector demonstrated that the rrn promoter region is functional. The rrn operon encoded by plasmid pOL18/23 is the first complete rrn operon sequenced from a strain of the genus Paracoccus, and only the second example of an rrn operon on a small plasmid.

Base Sequence↗

PubMed2004

Regulation of the expression of the Acidithiobacillus ferrooxidans rus operon encoding two cytochromes c, a cytochrome oxidase and rusticyanin.

The regulation of the expression of the rus operon, proposed to encode an electron transfer chain from the outer to the inner membrane in the obligate acidophilic chemolithoautroph Acidithiobacillus ferrooxidans, has been studied at the RNA and protein levels. As observed by Northern hybridization, real-time PCR and reverse transcription analyses, this operon was more highly expressed in ferrous iron- than in sulfur-grown cells. Furthermore, it was shown by immunodetection that components of this respiratory chain are synthesized in ferrous iron- rather than in sulfur-growth conditions. Nonetheless, weak transcription and translation products of the rus operon were detected in sulfur-grown cells at the early exponential phase. The results strongly support the notion that rus-operon expression is induced by ferrous iron, in agreement with the involvement of the rus-operon-encoded products in the oxidation of ferrous iron, and that ferrous iron is used in preference to sulfur.

Acidithiobacillus↗

PubMed2003

Coexpression of neighboring genes in Caenorhabditis elegans is mostly due to operons and duplicate genes.

In many eukaryotic species, gene order is not random. In humans, flies, and yeast, there is clustering of coexpressed genes that cannot be explained as a trivial consequence of tandem duplication. In the worm genome this is taken a step further with many genes being organized into operons. Here we analyze the relationship between gene location and expression in Caenorhabditis elegans and find evidence for at least three different processes resulting in local expression similarity. Not surprisingly, the strongest effect comes from genes organized in operons. However, coexpression within operons is not perfect, and is influenced by some distance-dependent regulation. Beyond operons, there is a relationship between physical distance, expression similarity, and sequence similarity, acting over several megabases. This is consistent with a model of tandem duplicate genes diverging over time in sequence and expression pattern, while moving apart owing to chromosomal rearrangements. However, at a very local level, nonduplicate genes on opposite strands (hence not in operons) show similar expression patterns. This suggests that such genes may share regulatory elements or be regulated at the level of chromatin structure. The central importance of tandem duplicate genes in these patterns renders the worm genome different from both yeast and human.

Animals↗

PubMed1996

Regulation of the nisin operons in Lactococcus lactis N8.

The antibiotic peptide nisin produced by Lactococcus lactis is used as a food preservative due to its activity against spores and vegetative cells of Gram-positive bacteria. The post-translational maturation of this secreted peptide includes dehydration of serine and threonine residues, lanthionine formation and a proteolytic processing of 23 amino acids from the N-terminus. Mutations in the nisZ, nisB and nisP genes of the biosynthetic nisZBTCIPRK nisin operon were made by gene replacement or integration of a plasmid. The mutations caused a drastic decrease of the transcription from the promoters upstream of the nisZBTCIPRK and nisFEG operons resulting in loss of nisin production and nisin immunity. The transcription of the nisin operons and nisin immunity could be partially restored by adding nisin to the growth medium of the cells. Nisin induction of the mutant strains also increased the level of the putative immunity NisI protein. These results showed that the nisZBTCIPRK operon is positively autoregulated and that the nisFEG operon is in the same regulon.

Cloning, Molecular↗

PubMed1987

Translation of galE and coordination of galactose operon expression in Escherichia coli: effects of insertions and deletions in the non-translated leader sequence.

Using gene-manipulation techniques, we made a set of short insertions and deletions in the Escherichia coli galactose operon between the transcription start site and the Shine-Dalgarno sequence of the first gene of the operon, galE. Translation initiation is severely reduced when the distance between the 5' end of the message and the Shine-Dalgarno sequence drops below 15 bases. Transcription of the gal operon can start at two distinct sites, S1 and S2, separated by 5 bp, situated 16 and 21 bp upstream of the galE Shine-Dalgarno sequence, respectively. When transcription starts at S2, gal operon expression is discoordinate as the galE gene is better translated than promoter-distal genes. Here we report that gal operon expression is discoordinate even when message starting at S2 is shortened. This shows that the better translation of galE from transcripts starting at S2 is not simply due to the fact that they are longer than transcripts starting at S1.

Bacterial Proteins↗

PubMed1992

Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES.

In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene. The complete DNA sequence of this groESL operon was determined. The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85-90% and 59-61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins. Southern blotting analyses with M. leprae groES- and groEL-specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis. This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene. Using five T-cell clones from two leprosy patients as probes, expression of the M. leprae GroES protein in Escherichia coli after heat shock was demonstrated. Four of these clones recognized the same M. leprae-specific GroES-derived peptide in a DR2-restricted fashion. No expression of the groEL gene from this operon was detected in E. coli after heat shock, as tested with a panel of T-cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.

Amino Acid Sequence↗

PubMed1993

Specific transcriptional requirements for positive regulation of the anaerobically inducible pfl operon by ArcA and FNR.

Expression of the pfl operon of Escherichia coli is induced 12- to 15-fold by anaerobiosis and transcription is mediated by seven co-ordinately regulated promoters. The 5' non-translated regulatory region of the operon is approximately 450bp in length and contains two of the seven promoters, termed promoter 6 and promoter 7. Site-directed mutagenesis was used to aid the identification of DNA sequences important in directing transcription from the two promoters and to examine the effects such mutations had on the regulation of anaerobic pfl operon expression. Introduction of chromosomal mutations either in the FNR-binding site or -10 region of promoter 6 blocked transcription from this promoter, as determined by primer extension. Similarly, mutation of the -10 region or the putative FNR half-site located at -50 relative to the transcription start site of promoter 7 severely reduced transcription from that promoter. Prevention of transcription from promoter 6 by the -10 box mutation had no influence on promoter 7 transcription. Surprisingly, however, alteration of the FNR-binding site at promoter 6 did reduce transcription from promoter 7. Thus, a cis mutation located 280 bp downstream on the DNA had a profound effect on promoter 7 transcription. This effect would be commensurate with this mutation disrupting an important interaction between proteins bound at promoter 7 with those bound at promoter 6. Primer extension demonstrated that the promoter 7 mutations had no apparent influence on promoter 6 transcription. By using pfl-lacZ gene fusions it could be shown that the FNR-binding site and -10 region mutations at promoter 6 abolished FNR-dependent anaerobic regulation of pfl operon expression. The equivalent mutations at promoter 7 caused a 25% reduction in anaerobic expression. The residual anaerobic expression in such constructs was FNR-, but no longer ArcA-dependent. A construct in which the -10 region of both promoters 6 and 7 was mutated showed no anaerobic induction of pfl operon expression. This indicates that transcription from both promoters is required for maximal anaerobic regulation by ArcA and FNR.

Acetyltransferases↗

PubMed1993

The arsD gene encodes a second trans-acting regulatory protein of the plasmid-encoded arsenical resistance operon.

The plasmid-encoded arsenical resistance (ars) operon produces resistance to trivalent and pentavalent salts of arsenic and antimony. The first gene in the operon, arsR, was previously shown to encode a repressor protein. A newly identified gene, arsD, is shown here to encode a regulatory protein, the ArsD protein. The gene was identified by construction of an in-frame fusion between the C-terminally truncated arsD gene and the coding region for the mature form of beta-lactamase (blaM). The native arsD gene product was overexpressed and radioactively labelled as a 13 kDa polypeptide. A frameshift mutation within the arsD gene resulted in elevated levels of expression of downstream ars genes. Co-expression of a wild-type arsD gene in trans with the operon containing the mutated arsD gene reduced expression of the downstream genes to wild-type levels. The presence of the arsD gene had no effect on the basal level of operon expression set by the arsR gene product, and the repression produced by the arsD gene product was not affected by inducers of the operon. The results indicate that the ArsD protein is an inducer-independent trans-acting regulatory protein.

Arsenic↗

PubMed1994

Escherichia coli cad operon functions as a supplier of carbon dioxide.

We examined the gene expression of the Escherichia coli cad operon, which consisted of the genes cadB and cadA (lysine decarboxylase), using cells possessing cadB-lacZ fusion gene. The cad operon was expressed when O2 was limited, and the expression was optimal at pH 6.3. The beta-galactosidase activity was lowered by the addition of sodium carbonate to the medium. The expression of the cad operon was reduced in cells containing the plasmid-encoding ornithine decarboxylase, which produced carbon dioxide, indicating that the gene expression of the cad operon was regulated by carbon dioxide (or its derivatives). It is known that the Krebs cycle is a major pathway for producing carbon dioxide, and that its activity is repressed when O2 is limited. Thus, our present results suggested that the physiological role of the cad operon is to supply carbon dioxide when its internal level is lowered under O2-limiting conditions at a low pH.

Amino Acid Transport Systems↗

PubMed1994

'Muprints' of the lac operon demonstrate physiological control over the randomness of in vivo transposition.

A method called Muprinting has been developed that uses PCR to generate a detailed picture of the bacteriophage Mu transposition sites in chosen domains of the bacterial chromosome. Muprinting experiments in Escherichia coli show that the frequency of phage integration changes dramatically near two repressor binding sites in the lac operon. When the lac operon was repressed, hotspots for Mu transposition were found near the O1 and O2 operators that are proposed to make a repression loop. When cells were grown in lactose, Mu transposition near these operators was greatly diminished. Striking changes in transposition frequencies were limited to the control region and were not found in a region of the lacZ gene lying beyond the O2 operator. Muprints of the bgl operon showed a different pattern; hotspots for Mu transposition detected in sequences upstream of the bglC promoter when the operon was silenced changed when the operon became activated by mutation. By targeting transposition to the regulatory regions around non-expressed genes, Mu may demonstrate a self-restraint mechanism that allows the virus to move through its host genome without disrupting the functions that contribute to a healthy cell physiology.

Bacteriophage mu↗

PubMed2005

HxlR, a member of the DUF24 protein family, is a DNA-binding protein that acts as a positive regulator of the formaldehyde-inducible hxlAB operon in Bacillus subtilis.

The HxlR protein from Bacillus subtilis belongs to the DUF24 protein family (InterPro No. IPR002577) of unknown function. The hxlR gene that encodes this protein is located upstream of the hxlAB operon. This operon encodes two key enzymes in the ribulose monophosphate pathway that are involved in formaldehyde fixation, 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. Expression of the hxlAB operon is induced by the presence of formaldehyde. Recombinant HxlR prepared from Escherichia coli showed specific binding to a region of DNA upstream of the hxlAB operon. Using gel-retardation and DNase I footprinting assays, we identified two 25 bp binding regions for HxlR within the upstream DNA. Surface plasmon resonance analyses suggested that two HxlR dimers sequentially bound to the DNA. Finally, we demonstrated that each of the two binding regions for HxlR was necessary for formaldehyde-induced expression of the hxlAB operon in B. subtilis. Thus, we have shown that HxlR is a DNA-binding protein that is necessary for formaldehyde-induced expression of hxlAB in B. subtilis.

Aldehyde-Lyases↗

PubMed2006

Characterization of the putative operon containing arylamine N-acetyltransferase (nat) in Mycobacterium bovis BCG.

Mycobacterium bovis BCG and Mycobacterium tuberculosis possess a single arylamine N-acetyltransferase whose gene is predicted to occur within a six-gene operon. Deletion of the nat gene caused an extended lag phase in M. bovis BCG and a cell morphology associated with an altered pattern of cell wall mycolates. Analysis of cDNA from M. bovis BCG shows that during in vitro growth all the genes in the putative nat operon are expressed and the open reading frames are contiguous, supporting the existence of an operon. Two genes in the operon, Mb3599c and Mb3600c, are predicted to encode homologues of enzymes annotated as a 2,3-dihydroxybiphenyl 1,2-dioxygenase (bphC5) and a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD2), respectively, in Rhodococcus RHA1. As predicted, M. bovis BCG cell lysates metabolized the BphC substrate 2,3-dihydroxybiphenyl (2,3-DHB) to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), a BphD substrate, which was subsequently hydrolysed. Immunoprecipitation of the BphD homologue from these lysates led to an accumulation of HOPDA. M. bovis BCG growth on both solid and liquid media was inhibited with either 2,3-DHB or an inhibitor of BphC, 3-chlorocatechol (3-CC). In addition, incubation with 2,3-DHB affects the lipid composition of the cell wall resulting in a diminished level of mycolates and an altered cell morphology similar to the Deltanat strain. We propose the enzymes encoded by the putative operon have a similar endogenous role to that of the NAT enzyme and are part of a pathway important for cell wall synthesis.

Arylamine N-Acetyltransferase↗

PubMed2006

IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins.

In Escherichia coli, Fe-S clusters are assembled by gene products encoded from the isc and suf operons. Both the iscRSUA and sufABCDSE operons are induced highly by oxidants, reflecting an increased need for providing and maintaining Fe-S clusters under oxidative stress conditions. Three cis-acting oxidant-responsive elements (ORE-I, II, III) in the upstream of the sufA promoter serve as the binding sites for OxyR, IHF and an uncharacterized factor respectively. Using DNA affinity fractionation, we isolated an ORE-III-binding factor that positively regulates the suf operon in response to various oxidants. MALDI-TOF mass analysis identified it with IscR, known to serve as a repressor of the iscRSUA gene expression under anaerobic condition as a [2Fe-2S]-bound form. The iscR null mutation abolished ORE-III-binding activity in cell extracts, and caused a significant decrease in the oxidant induction of sufA in vivo. OxyR and IscR contributed almost equally to activate the sufA operon in response to oxidants. Purified IscR that lacked Fe-S cluster bound to the ORE-III site and activated transcription from the sufA promoter in vitro. Mutations in Fe-S-binding sites of IscR enabled sufA activation in vivo and in vitro. These results support a model that IscR in its demetallated form directly activates sufA transcription, while it de-represses isc operon, under oxidative stress condition.

Base Sequence↗

PubMed1993

The regulation of expression of the Lactococcus lactis lactose operon.

Translational gene fusions between the Escherichia coli beta-galactosidase (lacZ) gene and the Lactococcus lactis lactose operon were constructed such that transcription from the lactose operon promoter could be assessed by measuring beta-galactosidase activity. The level of beta-galactosidase activity was up to 2.5-fold lower when MG5267 cells, which contain a chromosomal copy of the lactose operon, were grown in glucose compared to those grown in lactose. A greater degree of repression was seen in cells containing the multi-copy plasmid-encoded repressor than in those with only the single-copy chromosomal gene, indicating that the repressor protein is at least partly responsible for the reduction in expression when the cells are grown in glucose (i.e. in the absence of inducer). However, the beta-galactosidase activity was found to be 5.5-fold lower in glucose than in lactose in cells which lacked a fully functional lactose operon. The decrease in expression was shown to be due to glucose repression. The levels of expression when the cells were grown in glucose were considerably higher for MG5267 than for MG1363 suggesting perhaps that a product of the chromosomally-encoded operon in MG5267 has a positive effect on transcription.

Cloning, Molecular↗