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PubMed1996

Complete sequence and organization of the Serratia marcescens biotin operon.

The nucleotide sequence of the biotin (bio) operon of wild-type Serratia marcescens Sr41 was determined. Five ORFs were identified to encode BioA (7,8-diaminopelargonic acid aminotransferase), BioB (biotin synthase), BioF (7-keto-8-aminopelargonic acid synthase), BioC (an enzyme catalysing the synthesis of pimeloyl-CoA) and BioD (dethiobiotin synthase), in this order. The operon was deduced to be transcribed divergently to the left into bioA and to the right into the bioBFCD genes. The promoters and a common predicted operator for both bioA and bioBFCD genes were located between the bioA and bioB genes. The predicted amino acid sequences of these enzymes were similar to the sequences of the corresponding enzymes of Escherichia coli. Analysis of expression of the lacZ structural gene fused with the bioA and bioB promoters revealed that the biotin operon was subject to biotin-mediated feedback repression.

Amino Acid Sequence↗

PubMed2000

Isolation and characterization of mutated FhlA proteins which activate transcription of the hyc operon (formate hydrogenlyase) of Escherichia coli in the absence of molybdate(1).

Escherichia coli growing under anaerobic conditions produces H(2) and CO(2) by the enzymatic cleavage of formate catalyzed by formate hydrogenlyase (FHL) consisting of a molybdoenzyme formate dehydrogenase H (fdhF), hydrogenase 3 (hyc), and intermediate electron carriers (hyc). Transcription of both the fdhF and hyc operons requires the activator, FhlA protein, as well as formate and molybdate. Several fhlA mutants with an altered response to the required effector molybdate were isolated and these FhlA mutated proteins activated hyc transcription in the absence of molybdate, but only in the presence of formate. Mutated protein FhlA126 carries a single mutation (R495C) in the conserved central domain of the modular, sigma(54)-dependent, enhancer-binding protein. FhlA57 contains two mutations; one in the unique N-terminal domain (E205K) and a second in the central domain (P442S). Both mutations in FhlA132 are located in the N-terminal domain (A42T and E363K). Both FhlA126 and FhlA132 proteins activated the hyc operon even in the absence of ModE and MoeA, two components of Mo-metabolism which are required for hyc-lac expression in wild-type E. coli. Based on these results, a model is proposed in which the native FhlA protein interacts with a unique form of Mo (MoeA product?) as a second effector for optimum expression of the hyc operon in E. coli.

Bacterial Proteins↗

PubMed1997

Inhibition of the multiple antibiotic resistance (mar) operon in Escherichia coli by antisense DNA analogs.

The multiple antibiotic resistance operon (marORAB) in Escherichia coli controls intrinsic susceptibility and resistance to multiple, structurally different antibiotics and other noxious agents. A plasmid construct with marA cloned in the antisense direction reduced LacZ expression from a constitutively expressed marA::lacZ translational fusion and inhibited the induced expression of LacZ in cells bearing the wild-type repressed fusion. The marA antisense construction also decreased the multiple antibiotic resistance of a Mar mutant. Two antisense phosphorothioate oligonucleotides, one targeted to marO and the other targeted to marA of the mar operon, introduced by heat shock or electroporation reduced LacZ expression in the strain having the marA::lacZ fusion. One antisense oligonucleotide, tested against a Mar mutant of E. coli ML308-225, increased the bactericidal activity of norfloxacin. These studies demonstrate the efficacy of exogenously delivered antisense oligonucleotides targeted to the marRAB operon in inhibiting expression of this chromosomal regulatory locus.

Cloning, Molecular↗

PubMed2004

Enhancement of the mexAB-oprM efflux pump expression by a quorum-sensing autoinducer and its cancellation by a regulator, MexT, of the mexEF-oprN efflux pump operon in Pseudomonas aeruginosa.

nfxC-type cells of Pseudomonas aeruginosa that produce the MexEF-OprN efflux pump exhibit resistance to fluoroquinolones and chloramphenicol and hypersusceptibility to most classical beta-lactam antibiotics. We investigated the molecular mechanism of how the nfxC mutation causes beta-lactam hypersusceptibility. The MexAB-OprM extrusion pump transports and confers resistance to beta-lactam antibiotics. Interestingly, expression of the mexAB-oprM operon reached the highest level during the mid-stationary growth phase in both wild-type and nfxC-type mutant strains, suggesting that expression of the mexAB-oprM operon may be controlled by cell density-dependent regulation such as quorum sensing. This assumption was verified by demonstrating that exogenous addition of the quorum-sensing autoinducer N-butyryl-L-homoserine lactone (C4-HSL) enhanced the expression of MexAB-OprM, whereas N-(3-oxododecanoyl)-L-homoserine lactone had only a slight effect. Furthermore, this C4-HSL-mediated enhancement of mexAB-oprM expression was repressed by MexT, a positive regulator of the mexEF-oprN operon. It was concluded that beta-lactam hypersusceptibility in nfxC-type mutant cells is caused by MexT-mediated cancellation of C4-HSL-mediated enhancement of MexAB-OprM expression.

4-Butyrolactone↗

PubMed1992

Regulation of the sacPA operon of Bacillus subtilis: identification of phosphotransferase system components involved in SacT activity.

The sacT gene which controls the sacPA operon of Bacillus subtilis encodes a polypeptide homologous to the B. subtilis SacY and the Escherichia coli BglG antiterminators. Expression of the sacT gene is shown to be constitutive. The DNA sequence upstream from sacP contains a palindromic sequence which functions as a transcriptional terminator. We have previously proposed that SacT acts as a transcriptional antiterminator, allowing transcription of the sacPA operon. In strains containing mutations inactivating ptsH or ptsI, the expression of sacPA and sacB is constitutive. In this work, we show that this constitutivity is due to a fully active SacY antiterminator. In the wild-type sacT+ strain or in the sacT30 mutant, SacT requires both enzyme I and HPr of the phosphotransferase system (PTS) for antitermination. It appears that the PTS exerts different effects on the sacB gene and the sacPA operon. The general proteins of the PTS are not required for the activity of SacY while they are necessary for SacT activity.

Bacillus subtilis↗

PubMed1995

Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon.

The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence. During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP-activated locus. beta-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ. At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions. However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions. Two different transcripts are produced from the phoPQ operon during exponential growth. These transcripts define two promoters: phoPp1, which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp2, which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins. The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies. In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon. First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP-PhoQ-independent promoter. Then, phospho-PhoP would activate transcription of phoPp1, resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes. A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp1 is inhibited.

Bacterial Proteins↗

PubMed1995

Transcriptional control of the nuo operon which encodes the energy-conserving NADH dehydrogenase of Salmonella typhimurium.

The 14 nuo genes encode the subunits of the type I (energy-conserving) NADH dehydrogenase, a key component of the respiratory chain. Salmonella typhimurium, like Escherichia coli, has two enzymes that can oxidize NADH and transfer electrons to ubiquinone, but only the type I enzyme translocates protons across the membrane to generate a proton motive force. Cells with the type I enzyme are energetically more efficient; the role of the type II enzyme (encoded by ndh) is not established, but it may function like a relief valve to allow more rapid NADH recycling. Here, we have investigated transcription of the nuo gene cluster, primarily in S. typhimurium. Studies with polar insertion mutants demonstrate that these genes are arranged as a single, large operon that is expressed from a complex promoter region upstream of nuoA. The DNA sequence of the promoter region was determined, and primer extension analysis of nuo transcripts was used to map four major RNA 5' ends to this region. A set of lac operon fusions to various DNA segments from the nuo promoter region was also constructed. Analysis of these fusions confirmed the presence of at least two nuo promoters. Mutations in the global regulatory genes arcA, oxrA (fnr), crp, cya, and katF were tested for effects on expression of the nuo operon. However, none of the mutations tested had a large effect on expression of type I NADH dehydrogenase.

Base Sequence↗

PubMed1998

Expression of lacZ from the promoter of the Escherichia coli spc operon cloned into vectors carrying the W205 trp-lac fusion.

The expression of lacZ has been analyzed and compared in a series of promoter cloning vectors by measuring the amount of lacZ mRNA by hybridization and the amount of beta-galactosidase by standard enzymatic assay. Expression was driven by the promoter, Pspc, of the spc ribosomal protein operon. The vectors contained either the standard W205 trp-lac fusion with the trp operon transcription terminator, trpt, located in the lacZ leader sequence, or a deletion derivative that functionally inactivates trpt. In the presence of trpt, lacZ expression was temperature dependent so that increasing the growth temperature reduced the accumulation of lacZ mRNA and beta-galactosidase activity. The frequency of transcript termination at trpt was estimated to be near zero at 20 degreesC and at about 45% at 37 degreesC. The amount of Pspc-derived lacZ mRNA and the amount of beta-galactosidase produced per lacZ mRNA varied, depending on the mRNA 5' leader sequence between Pspc and lacZ. These results demonstrate that the quantitative assessment of promoter activities with promoter cloning vectors requires careful analysis and interpretation. One particular construct without trpt did not seem to contain fortuitous transcription or translation signals generated at the fusion junction. In this strain, lacZ expression from Pspc was compared at the enzyme activity and mRNA levels with a previously constructed strain in which lacZ was linked to the tandem P1 and P2 promoters of the rrnB operon. At any given growth rate, the different activities of beta-galactosidase in these two strains were found to reflect the same differences in their amounts of lacZ mRNA. Assuming that the promoter-lacZ fusions in these strains reflect the properties of the promoters in their normal chromosomal setting, it was possible to estimate the absolute transcription activity of Pspc and the relative translation efficiency of Pspc-lacZ mRNA at different growth rates. Transcription from the spc promoter was found to increase from about 10 transcripts per min at a growth rate of 1.0 doublings/h to a maximum plateau of about 23 transcripts per min at growth rates above 1.5 doublings/h. The translation frequency of lacZ mRNA expressed from Pspc was unaffected by growth rates.

Artificial Gene Fusion↗

PubMed1999

Histidine operon deattenuation in dnaA mutants of Salmonella typhimurium correlates with a decrease in the gene dosage ratio between tRNA(His) and histidine biosynthetic loci.

Expression of the histidine operon of Salmonella typhimurium is increased in dnaA(Ts) mutants at 37 degrees C. This effect requires an intact his attenuator and can be suppressed by increasing the gene copy number of the hisR locus, which encodes the tRNA(His). We present data which suggest that the his deattenuation defect in dnaA(Ts) mutants results from the loss of a gene dosage gradient between the hisR locus, close to oriC, and the his operon, far from oriC. Some of the conclusions drawn here may apply to other operons as well.

Bacterial Proteins↗

PubMed1990

Isolation and characterization of mutants affected in the expression of the nar operon Escherichia coli.

In the nitrate reductase system of Escherichia coli, the maximal expression of the nar operon is obtained under anaerobiosis in the presence of nitrate. Mudl (Ap,lac) insertion mutants, which only grew on lactose anaerobically if supplemented with nitrate were mapped at the chlC locus at min 27 of the map. In these fusion strains which lack benzyl viologen dependent nitrate reductase (NR) activity as well as the formiate-linked NR activity, the synthesis of beta-galactosidase reflects the regulation of the wild type nar operon at the transcriptional level. From these strains, two classes of spontaneous regulatory mutants were isolated: class I mutants which synthesized beta-galactosidase in anaerobiosis in the absence of nitrate and class II mutants in which the synthesis of that enzyme was partially independent of nitrate and it was no longer repressed by oxygen. The class I regulatory mutation was tightly linked to the nar operon as shown by bacteriophage P1 transductions. It probably affects either a closely linked cis-active element or a gene coding for a negative regulatory protein.

Chromosome Mapping↗

PubMed1984

Role of attenuation in growth rate-dependent regulation of the S10 r-protein operon of E. coli.

We have investigated the transcription of the 11 gene S10 ribosomal protein operon of Escherichia coli under various growth conditions. The differential synthesis rate of structural gene message increases 2- to 2.5-fold immediately after a shift-up from glycerol minimal medium to glucose plus amino acids. After the initial increase, the transcription rate goes through several oscillations before reaching the new steady-state rate. By comparing the rates of transcription of leader and structural genes, we conclude that these oscillations are due predominantly to changes in the level of read-through at the S10 attenuator. This regulation of attenuation can account for most of the variations in protein synthesis from the S10 operon after a shift. We also measured the level of read-through in cells growing exponentially in different growth media. Over a 2.5-fold range in growth rates, the read-through changed less than 50%. Thus, regulation of attenuation cannot explain the growth-dependent regulation of ribosomal protein synthesis during steady-state growth. Apparently, additional mechanisms are required to control the expression of the S10 operon in exponentially growing cells.

Escherichia coli↗

PubMed1985

In vivo evidence that the nusA and infB genes of E. coli are part of the same multi-gene operon which encodes at least four proteins.

Previous work has shown that the Escherichia coli nusA gene codes for a protein which regulates transcription termination. The 16.0-kb EcoRI DNA fragment that includes the nusA gene, codes for at least eight bacterial proteins of mol. wts. 48 000 (argG), 21 000 (p21), 64 000 (nusA), 120 000 (IF2 alpha)-(infB), 91 000 (IF2 beta)(infB), 15 000 (p15), 10 000 (rpsO) and 85 000 (pnp). We have constructed several deletion and fusion derivatives from this cloned DNA and examined in vivo the structure and expression of these genes. First, the promoter functional in vivo for the nusA gene was mapped at approximately 800 bp upstream of the nusA structural gene. Second, the synthesis of five proteins, p21, NusA, IF2 alpha, IF2 beta (and p15) proteins, was affected by the deletion of the nusA promoter. Third, these same five proteins were hyperproduced after fusion of the DNA fragment to the lambda pL promoter. In addition, subcloning experiments revealed that the p15 gene is expressed by the read-through transcription from the infB gene. These results lead us to conclude that the genes coding for the p21, NusA, InfB (IF2 alpha and IF2 beta), and p15 proteins form a single-transcriptional unit ('nusA-infB operon') in vivo and that rpsO and pnp genes do not belong to the same operon. The in vivo attenuation site of this operon is described.

Bacterial Proteins↗

PubMed1985

Structure of wild-type and mutant repressors and of the control region of the rbt operon of Klebsiella aerogenes.

Pentitol metabolism in Klebsiella aerogenes is encoded by continuous ribitol (rbt) and D-arabitol (dal) operons transcribed in bipolar fashion and sandwiched between long stretches of homologous DNA. The operons are separated by a central control region (2.2 kb) which encodes both the repressors and all the control sequences. The rbt repressor (270 amino acids) shows homology to the Escherichia coli lac repressor and other DNA-binding proteins. It is transcribed from the strand opposite the rbt operon and the intervening control region (254-bp) contains features which reflect the complex regulation. A rbt-constitutive mutant strain used in previous studies of experimental enzyme evolution encodes a truncated rbt-peptide of 133 residues due to a frameshift mutation.

Amino Acid Sequence↗

PubMed1987

The cox protein of bacteriophage P2 inhibits the formation of the repressor protein and autoregulates the early operon.

The cox gene is the first gene of the early operon of bacteriophage P2. The early promoter Pe and the repressor promoter Pc are located close to each other and in such a way that their transcripts have opposite polarity and show an overlap of about 30 nucleotides. The expression of the early operon and of the C gene was studied in vivo by using fusions to a promoterless cat (chloramphenicol acetyl transferase) gene. The results show that the Cox protein negatively autoregulates the early operon and inhibits the formation of the repressor C. By measuring the efficiency of plating of a series of P2 virulent deletion mutants on bacteria carrying a cloned cox gene, the site of action of the Cox protein was mapped within the Pe-Pc region. The stimulatory effect of the C protein on expression of the Pc promoter was found to be due to inhibition of transcription from Pc; this was demonstrated by mutating Pe which showed that loss of transcription from Pe stimulated transcription from Pc. Hence, this is a case of regulation of gene expression by convergent transcription. By cloning the region C-Pe-Pc-cox such that the cat and kan genes are expressed from Pc and Pe, respectively, it was shown that only one of the resistances (Cm and Km) was expressed. This mimics the choice between lysogeny and lytic growth of the phage. The 'lysogenic' state was very stable whereas the 'lytic' state flipped to the 'lysogenic' at a somewhat higher frequency. The presence of a cloned cox gene drastically stimulated the formation of free phage from a P2-lysogen and dramatically reduced the frequency of lysogenization after P2 infection. We conclude that the pleiotropic effects of the cox (control of excision) gene, namely effects on lysogenization, formation of free phage and site-specific P2 recombination, can be explained by the effect of the Cox protein on the activity of the promoters Pc and Pe.

Amino Acid Sequence↗

PubMed1988

Resistance to macrolides, lincosamides and streptogramin type B antibiotics due to a mutation in an rRNA operon of Streptomyces ambofaciens.

Streptomyces ambofaciens produces spiramycin, a macrolide antibiotic and expresses an inducible resistance to macrolides, lincosamides and streptogramin B antibiotics (MLS). From a mutant of S.ambofaciens exhibiting a constitutive MLS resistance phenotype a resistance determinant was cloned on a low copy number vector (pIJ61) through its expression in Streptomyces lividans. Further characterization has shown that this determinant corresponded to a mutant rRNA operon with a mutation in the 23S rRNA gene. In different organisms, mutations leading to MLS resistance have been located at a position corresponding to the adenine 2058 of Escherichia coli 23S rRNA. In the 23S rRNA from S.ambofaciens a similar position for the mutation has been postulated and DNA sequencing of this region has shown an adenine to guanine transition at a position corresponding to 2058. S.ambofaciens possesses four rRNA operons which we have cloned. In Streptomyces, contrary to other bacteria, a mutation in one among several rRNA operons confers a selectable MLS resistance phenotype. Possible reasons for this difference are discussed.

Anti-Bacterial Agents↗

PubMed1989

Autoregulation of RNase III operon by mRNA processing.

RNase III has been implicated in the control of gene expression by the processing of mRNA. We have found that the rnc operon is autoregulated; rnc- mutant strains oversynthesize the operon's mRNA and protein products. A site in the 5'-noncoding region of the operon's message is cleaved by RNase III. This site-specific cleavage appears to be the initial step in the functional inactivation of the message, since the half-life of the cut message is dramatically shorter than that of the uncut message.

Base Sequence↗

PubMed1991

Analysis of the Rhodobacter capsulatus puc operon: the pucC gene plays a central role in the regulation of LHII (B800-850 complex) expression.

Formation of the light harvesting complex B800-850 (LHII) of Rhodobacter capsulatus requires the expression of more than the three known genes specific for that complex (pucA, pucB and pucE) encoding the alpha, beta and gamma subunits of LHII, respectively. In this work evidence is presented that the product of the gene pucC, which is located downstream from pucA, is essential for high-level transcription of the pucBACDE operon and formation of LHII. Plasmids were constructed containing deletions in one or several puc genes and transferred to a pucC::Tn5 mutant in which the puc operon is not expressed. It was found that the LHII- phenotype of the mutant was due to the missing PucC protein and that all known puc genes are located in one operon. To dissect the pucC, pucD and pucE genes from pucB and pucA and independently regulate them, they were placed under control of the nifHDK promoter. Only under nitrogen-fixing growth conditions was the LHII- pucC::Tn5 mutant complemented by this construction. It is concluded that expression of pucC is essential for formation of the LHII complex in R.capsulatus. Analysis of the pucD and pucE genes led to the conclusion that the products of these genes stabilize the B800-850 complex.

Blotting, Northern↗

PubMed1991

Regulation of the acetate operon in Escherichia coli: purification and functional characterization of the IclR repressor.

Growth of Escherichia coli on acetate requires operation of the anaplerotic sequence known as the glyoxylate bypass. In this pathway three different enzymes are activated: malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase which are encoded by genes aceB, aceA and aceK, respectively. These three genes are clustered, in that order, in the same acetate (ace) operon whose expression is under the transcriptional control of the iclR gene located downstream from aceK. We have cloned the iclR gene in the pKK233-2 vector which allows optimization of both transcription and translation initiation. The IclR repressor has been overproduced, then purified to homogeneity in a one-step procedure by cation exchange chromatography after ammonium sulfate fractionation. Its specific interaction with the operator/promoter region of the ace operon has been analyzed by gel retardation and DNase I footprinting experiments. The IclR repressor has been shown to recognize a 35 bp palindromic sequence which largely overlaps the -35 recognition site of RNA polymerase. Moreover, the formation of the complex between IclR and the operator/promoter region has been found to be impaired by phosphoenol pyruvate but insensitive to acetate, acetyl-CoA, pyruvate, and oxaloacetate. These results are discussed in terms of primary regulation of the expression of the ace operon.

Acetates↗