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PubMed1982

Leftward transcription in the Escherichia coli bio operon does not require products of the rightward transcript.

The amount of leftward transcription in the Escherichia coli bio operon, as measured by hybridization and by beta-galactosidase assays in lac-bio fusion strains, was determined in bacteria lysogenic for lambda bio phage carrying different amounts of DNA corresponding to rightward message, and in bacteria with polar or nonpolar bioB mutations. The positions of the bioB endpoints in relation to the pB promoter were determined by electron microscopy of heteroduplexes. Normal rates of leftward transcription were found in all cases, except that the shortest lambda bio (lambda bio showed a 2- to 3-fold increase in leftward transcription, which was not abolished by the presence of a wild-type bio operon in trans. These results indicate that no product of the rightward transcript is needed to turn on leftward transcription.

Bacteriophage lambda↗

PubMed1984

The selection and characterisation of two novel mutations in the overlapping promoters of the Escherichia coli galactose operon.

Mutations that result in small decreases or increases in expression from the Escherichia coli galactose operon promoter region can be detected by using a plasmid in which the gal promoters were fused to the lac operon. We describe how the level of lac expression was adjusted so that the Lac phenotype of host cells was optimally sensitive to changes in the gal promoter sequence. We have investigated the properties of two new gal promoter mutations both in vivo and in vitro, and have determined their effects on the two overlapping gal promoters, P1 and P2. Although one mutation causes only a small reduction in overall expression in vivo, it completely suppresses transcription initiation at the P1 promoter. However, it also increases expression from the P2 promoter, which compensates for the change at P1. This mutation, a GC to AT transition, falls in a zone just upstream of the P1 Pribnow box, which is essential for P1 activity, whilst improving the homology between the P2 Pribnow box and the consensus sequence. The second mutation causes a small increase in P1 activity. This change, a GC to AT transition at -23, falls in the spacer region between the Pribnow box and the -35 region, a zone containing no known promoter consensus sequences. We suggest that this mutation, which creates a stretch of five AT base pairs, acts by increasing the twist angle of the sequences in the spacer region. We argue that the increase in promoter activity is due to this twist changing the relative orientation of the Pribnow box and -35 regions.

DNA, Bacterial↗

PubMed1987

Improved single and multicopy lac-based cloning vectors for protein and operon fusions.

We describe several new vectors for the construction of operon and protein fusions to the Escherichia coli lacZ gene. In vitro constructions utilize multicopy plasmids containing suitable cloning sites located between upstream transcription terminators and downstream lac operon segments whose lacZ genes retain or lack translational start signals. Single-copy lambda prophage versions of multicopy constructs can be made genetically, without in vitro manipulation. The new vectors, both single and multicopy, are improved in that they have very low levels of background lac gene expression, which makes possible the easy detection and accurate quantitation of very weak transcriptional and translational signals. These vectors were developed for analysis of the expression of IS10's transposase gene, which is transcribed less than, once per generation, and whose transcripts are translated on average less than once each. Both single and multicopy constructs can also be used to select mutations affecting fusion expression, and mutations isolated in single-copy constructs can be crossed genetically back onto multicopy plasmids for further analysis.

Gene Expression Regulation↗

PubMed2005

Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression.

Certain nascent peptide sequences, when within the ribosomal exit tunnel, can inhibit translation termination and/or peptide elongation. The 24 residue leader peptidyl-tRNA of the tna operon of E. coli, TnaC-tRNA(Pro), in the presence of excess tryptophan, resists cleavage at the tnaC stop codon. TnaC residue Trp12 is crucial for this inhibition. The approximate location of Trp12 in the exit tunnel was determined by crosslinking Lys11 of TnaC-tRNA(Pro) to nucleotide A750 of 23S rRNA. Methylation of nucleotide A788 of 23S rRNA was reduced by the presence of Trp12 in TnaC-tRNA(Pro), implying A788 displacement. Inserting an adenylate at position 751, or introducing the change U2609C in 23S rRNA or the change K90H or K90W in ribosomal protein L22, virtually eliminated tryptophan induction. These modified and mutated regions are mostly located near the putative site occupied by Trp12 of TnaC-tRNA(Pro). These findings identify features of the ribosomal exit tunnel essential for tna operon induction.

Biotinylation↗

PubMed1983

Mutations in the promoter regions of the malEFG and malK-lamB operons of Escherichia coli K12.

The malB region of Escherichia coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes. Expression of the malB operons is under the positive control of the malT gene product (MalT) and maltose and of the crp gene product (CRP) and cyclic AMP. Strains in which the lac genes have been fused to malE or malK are unable to use lactose as carbon source if they have been deleted for malT or crp. Mutations in the malB region allowing such fusion strains to grow on lactose have been isolated. These and previously isolated mutations were genetically characterized. As regards the malEp promoter mutations, malEp9, malEp1 and malEp6 create new promoters that are MalT and CRP independent. malEp9 and malEp1 change residues -1 and -2, respectively, of malEp without altering its activity. malEp6 duplicates six base-pairs between residues -22 and -23. malEp3 improves the -10 region hexamer. malEp5 deletes residues -29 to -62. It creates a new promoter that is MalT independent, CRP dependent, likely by fusing together functional regions of malEp that are normally apart. malEp5 also reduces the expression of malK-lamB, suggesting the existence of a link between the malEp and malKp promoters. As regards the malKp mutations, malKp6 changes residue -81 of malKp without altering its activity. It creates a new promoter, which is MalT independent, CRP dependent, likely by using a pre-existing cyclic AMP/CRP binding site. malKp102 changes residue -36, two bases upstream of the -35 region hexamer. It decreases the activity of malKp by at least four orders of magnitude and likely alters the MalT binding site. These results are discussed in terms of regulatory interactions within the malB control region.

Base Sequence↗

PubMed1999

The gene for 2-phosphoglycolate phosphatase (gph) in Escherichia coli is located in the same operon as dam and at least five other diverse genes.

Downstream of the dam gene in the Escherichia coli genome the following three genes are located: first rpe, then a gene encoding a 27 kDa protein and finally trpS. Here we present evidence that the 27 kDa protein has 2-phosphoglycolate phosphatase activity, and we name the gene gph. Phosphoglycolate phosphatase is needed in autotrophic organisms performing the Calvin-Benson-Bassham (CBB) reductive pentose-phosphate cycle. E. coli is not capable of autotrophic growth and probably utilizes Gph activity for other function(s) than in the CBB cycle. We found no physiological effect of deleting gph and its function in E. coli remains unclear. The use of fusion plasmids, where lacZ was inserted into gph and trpS, and deletion derivatives of these fusion plasmids, showed that rpe, gph and trpS are all members of the dam-containing operon. A novel promoter was identified in the distal part of the dam gene. The operon, which contains aroK, aroB, urf74.3, dam, rpe, gph, and trpS, can be termed a superoperon, since it consists of (at least) seven apparently unrelated genes which are under complex regulatory control.

Artificial Gene Fusion↗

PubMed1997

Aerobic regulation of cytochrome d oxidase (cydAB) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation.

The cydAB operon of Escherichia coli encodes the cytochrome d oxidase complex, one of two aerobic terminal oxidases that catalyses the oxidation of ubiquinol-8 and the reduction of oxygen to water. This enzyme has a higher affinity for oxygen than the cytochrome o oxidase complex and accumulates as oxygen becomes limiting. Expression of the cydAB operon is microaerobically controlled by the ArcA/ArcB two-component regulatory system and by Fnr. To understand how ArcA and Fnr contribute to this control, a set of cyd-lacZ reporter fusions were constructed and analysed in vivo. Two cydAB promoters, designated P1 and P2, were identified by primer extension analysis and are located 288 and 173 bp upstream of the start of cydA translation respectively. Transcription from promoter P1 was shown to be regulated by both Fnr and ArcA in response to anaerobiosis. DNasel footprint experiments revealed the locations of two Fnr binding sites at the P1 promoter: one is centred at the start of cyd transcription, while the other is positioned 53.5 bp upstream. A single ArcA-phosphate binding site of 49 bp, centred 93 bp upstream of promoter P1, was identified to be sufficient for the activation of cydAB expression. Based on the results of the in vitro and in vivo studies, a working model for ArcA activation and Fnr repression of cydAB transcription is proposed.

Aerobiosis↗

PubMed1980

Metabolite gene regulation of the L-arabinose operon in Escherichia coli with indoleacetic acid and other indole derivatives.

The ability of indole derivatives to facilitate RNA polymerase transcription of the L-arabinose operon in Escherichia coli was shown to require the catabolite activator protein (CAP) as well as the araC gene product. Adenosine 3',5'-monophosphate (cAMP) was not obligatory for araBAD transcription when the cells were grown in the presence of 1 mM indole-3-acetic acid or in the presence of indole-3-acetamide, indole-3-propionic acid, indole-3-butyric acid, or 5-hydroxyindole-3-acetic acid. However, these indole derivatives were unable to circumvent the cAMP requirement for the induction of the lactose and the maltose operons. Catabolic repression occurred when glucose was added to cells grown in the presence of L-arabinose and 1 mM indoleacetic acid or 1 mM cAMP. This effect was reversed at higher concentrations of indoleacetic acid or cAMP. The induction and the catabolite repression phenomena were quantitated by measuring the differential rate of synthesis of L-arabinose isomerase (the araA gene product). These results indicated that indole metabolites from various living systems may regulate gene expression and may be involved in "metabolite gene regulation."

Arabinose↗

PubMed2003

Genomic rearrangements at rrn operons in Salmonella.

Most Salmonella serovars are general pathogens that infect a variety of hosts. These "generalist" serovars cause disease in many animals from reptiles to mammals. In contrast, a few serovars cause disease only in a specific host. Host-specific serovars can cause a systemic, often fatal disease in one species yet remain avirulent in other species. Host-specific Salmonella frequently have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons while the generalists consistently have a conserved chromosomal arrangement. To determine whether this is the result of an intrinsic difference in recombination frequency or a consequence of lifestyle difference between generalist and host-specific Salmonella, we determined the frequency of rearrangements in vitro. Using lacZ genes as portable regions of homology for inversion analysis, we found that both generalist and host-specific serovars of Salmonella have similar tolerances to chromosomal rearrangements in vitro. Using PCR and genetic selection, we found that generalist and host-specific serovars also undergo rearrangements at rrn operons at similar frequencies in vitro. These observations indicate that the observed difference in genomic stability between generalist and host-specific serovars is a consequence of their distinct lifestyles, not intrinsic differences in recombination frequencies.

Base Sequence↗

PubMed1989

Sigma-G RNA polymerase controls forespore-specific expression of the glucose dehydrogenase operon in Bacillus subtilis.

The gene encoding glucose dehydrogenase (gdh) is part of an operon whose expression is transcriptionally activated specifically in the developing forespore of Bacillus subtilis at stage III of sporulation. The in vivo startpoint of gdh transcription was determined using primer extension analysis. Deletion mapping and site-specific mutagenesis experiments indicated that the region responsible for regulated expression of gdh in vivo was limited to the "-35" and "-10" regions preceding the transcriptional start site. RNA polymerase containing omega G (E omega G) transcribed gdh in vitro with a start site identical to that found in vivo, and transcription of gdh by E omega G in vitro also did not require any specific sequences upstream from "-35" region. These results suggest that the appearance of E omega G in the forespore at stage III of sporulation is sufficient to cause temporal and compartment-specific expression of the gdh operon.

Bacillus subtilis↗

PubMed1998

DNA bending and expression of the divergent nagE-B operons.

Repression of the divergent nagE - B operons requires NagC binding to two operators which overlap the nagE and nagB promoters, resulting in formation of a DNA loop. Binding of the cAMP/CAP activator to its site, adjacent to the nagE operator, stabilizes the DNA loop in vitro. The DNA of the nagE-B intergenic region is intrinsically bent, with the bend centred on the CAP site. We show that displacement of the CAP site by 6 bp results in complete derepression of the two operons. This derepression is observed even in the absence of cAMP/CAP binding and despite the fact that the two NagC operators are still in phase, demonstrating that the inherently bent structure of the DNA loop is important for repression. Since no interaction between NagC and CAP has been detected, we propose that the role of CAP in the repression loop is architectural, stabilizing the intrinsic bend. The cAMP/CAP complex is necessary for activation of the nagE-B promoters. In this case protein-protein contacts between CAP and RNA polymerase are necessary for full activation, but at least a part of the activation is likely due to an effect of CAP binding altering DNA structure.

Acetylglucosamine↗

PubMed1998

Bacillus popilliae cry18Aa operon is transcribed by sigmaE and sigmaK forms of RNA polymerase from a single initiation site.

Bacillus popilliae is an obligate pathogen for larvae of the insect family Scarabaeidae (Coleoptera). It forms parasporal crystals upon sporulation. The gene cry18Aa coding for the parasporal crystal protein and an upstream open reading frame, orf1, were previously isolated from B.popilliae. Here we report an analysis of cry18Aa transcription in Bacillus thuringiensis. The only transcriptional start site of cry18Aa was found 29 bp upstream of the open reading frame orf1, suggesting that orf1 and cry18Aa are transcribed as an operon. lacZ fusion to the cry18Aa promoter was used to follow the time-course of cry18Aa transcription in wild type B.thuringiensis and in various B.thuringiensis sporulation-deficient mutants (spo0A, sigE or sigK). In wild type B.thuringiensis, the cry18Aa promoter was activated 2 h after the end of exponential growth and the expression lasted to the late sporulation phase. The results of promoter activity in Spo+or Spo-backgrounds together with the results of primer extension experiments suggest that the transcription from this promoter can be driven by both sigmaE and sigmaK types of RNA polymerase at a single start site. The promoter region of cry18Aa operon fits the consensus sequences of both sigmaE and sigmaK dependent promoters of Bacillus.

Animals↗

PubMed1983

Regulation of expression of the uxu operon and of the uxuR regulatory gene in Escherichia coli K12.

Gene fusions between the lac structural genes and various genes of the hexuronate system of Escherichia coli K12 were isolated by the technique of Casadaban. Mud(Aprlac) and lambda plac-Mu insertion mutants were constructed in which the lac genes were fused to the regulatory region of the uxu operon. In all the uxu-lac fusion strains, beta-galactosidase expression was shown to be inducible by the natural inducers of the uxu operon (glucuronate and fructuronate) and sensitive to catabolite repression by glucose. In addition we isolated a Mud(Aprlac) fusion where the lac genes were fused to the uxuR regulatory gene. In this fusion the synthesis of beta-galactosidase reflects the regulation of the uxuR gene. In the presence of a wild-type uxuR allele, partial repression of beta-galactosidase expression was found; the repression was removed when inducer was added. This result indicates that while the uxuR gene is subject to autogenous control, the uxuR repressor may have only a low affinity for its own operator.

Coliphages↗

PubMed1998

A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10).

The early secreted antigenic target 6 kDa protein (ESAT-6) is a potent T-cell protein antigen synthesized by Mycobacterium tuberculosis. Its corresponding gene (esat-6) is located in RD1, a 10 kb DNA region deleted in the attenuated tuberculosis vaccine strain Mycobacterium bovis BCG. The promoter region of M. tuberculosis esat-6 was cloned and characterized. A new gene, designated lhp and cotranscribed with esat-6, was identified. Moreover, computer searches in the M. tuberculosis genome identified 13 genes related to the lhp/esat-6 operon, defining a novel gene family. The transcription initiation sites of the lhp/esat-6 operon were mapped using M. tuberculosis RNA. The corresponding promoter signals were not recognized in Mycobacterium smegmatis, in which transcription of lhp/esat-6 is initiated at different locations. The M. tuberculosis lhp gene product was identified as CFP-10, a low-molecular-mass protein found in the short-term culture filtrate. These results show that the genes encoding CFP-10 and ESAT-6 are transcribed together in M. tuberculosis and that both code for small exported proteins.

Amino Acid Sequence↗

PubMed2006

Sequence, organization, transcription and regulation of lactose and galactose operons in Lactobacillus rhamnosus TCELL-1.

AIMS: Understanding the metabolism of lactose and galactose and their regulation in Lactobacillus rhamnosus. METHODS AND RESULTS: A gene cluster containing nine open reading frames (ORFs) involved in the metabolism of lactose and galactose in Lact. rhamnosus TCELL-1 was sequenced and characterized. The order of the ORFs was lacTEGF and galKETRM. Northern blotting experiments revealed that the gene cluster could be transcribed as one lacTEGF-galKETRM mRNA though three major transcripts (lacTEGF, galKETRM and galETRM) were detected for the gene cluster. The transcription of the lac or gal operon was independently induced in the presence of lactose or galactose. Northern blotting and primer extension experiments found the presence of four putative promoters upstream from the ORFs lacT (lacTp), galK (galKp1 and galKp2) and galE (galEp). The measurements of enzymatic activities of GalK, GalE and GalT suggested that the expression of the gal operon was subjected to a galactose activation and glucose repression mechanism. CONCLUSIONS: In Lact. rhamnosus TCELL-1, the galactose moiety of lactose could be metabolized by two alternative pathways (the Leloir and the tagatose 6-phosphate pathways) whereas galactose metabolism could be mediated by the Leloir pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides important information about sugar metabolism in Lact. rhamnosus.

Amino Acid Sequence↗

PubMed1992

The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity.

Bacterial cells possess a subset of genes whose expression correlates with changes in DNA supercoiling brought about by anaerobic growth and by growth at high osmolarity. It has been shown previously that expression of the histidine biosynthetic operon of Salmonella typhimurium is derepressed by relaxation of supercoiled DNA. Here, we confirm that a his::MudJ operon fusion in S. typhimurium can be induced by treatment with the DNA gyrase inhibitor novobiocin in a dose-dependent manner, and show that the level of derepression is higher in stationary phase than in mid-exponential phase cultures. Furthermore, expression of his is repressed by anaerobiosis and by osmolarity, two environmental parameters which increase the negative supercoiling of bacterial DNA. Novobiocin induction of his is also repressed by growing the cells either at high osmolarity or anaerobically. Both environmental repression and novobiocin induction of his require the his attenuator. In addition, derepression of his expression by novobiocin and its repression by anaerobiosis or osmolarity are independent of the stringent response gene, relA.

Anaerobiosis↗

PubMed1998

Histidine utilisation operon (hut) is upregulated at low temperature in the antarctic psychrotrophic bacterium Pseudomonas syringae.

The antarctic psychrotrophic bacterium Pseudomonas syringae was mutagenised using a transposon Tn5-OT182 which facilitates identification of promoter fusions expressing the reporter gene (lacZ) for beta-galactosidase. Most mutants expressed beta-galactosidase both at optimal growth temperature (20-22 degrees C) and at low temperature (4 degrees C). But a small percentage of the mutants (approximately 5%) were unique in that they expressed beta-galactosidase activity predominantly at low temperature. One such mutant was found to have an insertion in the gene for urocanase (hutU) of the histidine utilisation (hut) operon. Direct assay of urocanase and histidase activity in wild-type cells of various antarctic psychrotrophic strains including P. syringae, P. fluorescens and P. putida also suggested that the hut operon is expressed at an elevated level at low temperature.

Amino Acid Sequence↗

PubMed2005

Expression of the psl operon in Pseudomonas aeruginosa PAO1 biofilms: PslA performs an essential function in biofilm formation.

The psl gene cluster, comprising 15 cotranscribed genes from Pseudomonas aeruginosa, was recently identified as being involved in exopolysaccharide biosynthesis and biofilm formation. In this study, we investigated the regulation of the psl gene cluster and the function of the first gene in this cluster, the pslA gene. PslA shows strong similarities to UDP-glucose lipid carriers. An isogenic marker-free pslA deletion mutant of P. aeruginosa PAO1 deficient in attachment and biofilm formation was used for complementation studies. The expression of only the pslA gene, comprising a coding region of 1,437 bp, restored the biofilm-forming phenotype of the wild type, indicating that PslA is required for biofilm formation by nonmucoid P. aeruginosa. The promoter region of the psl gene cluster, which encodes PslA-PslO, was identified by rapid amplification of cDNA 5' ends. Promoter assays using transcriptional fusions to lacZ and gfp indicated a constitutive expression of the psl cluster in planktonic cells and a highly regulated and localized expression in biofilms, respectively. Expression of the psl cluster in biofilms was almost exclusively found in the centers of microcolonies, as revealed by confocal laser scanning microscopy. These data suggest that constitutive expression of the psl operon enables efficient attachment to surfaces and that regulated localized psl operon expression is required for biofilm differentiation.

Bacterial Proteins↗