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PubMed1995

Different structure and expression of the operons encoding the membrane-bound hydrogenases from Methanosarcina mazei Gö1.

The expression of the vho and vht operons from Methanosarcina mazei Gö1, which each encode a membrane-bound hydrogenase and a cytochrome b, was analyzed under various growth conditions. Synthesis of both hydrogenases was induced at the level of transcription during methanogenesis from H2/CO2 or methanol. Transcripts of the vho operon were also detected when Ms. mazei Gö1 was grown on acetate, indicating that this operon is constitutively expressed. In contrast, mRNA from the vht operon was not found in acetate-grown cells. Downstream of the structural genes vhtG and vhtA and the cytochrome-b-encoding gene vhtC, an additional open reading frame (vhtD; 486 bp) was identified. vhtD is followed by six tandem repeats of an 11-bp sequence, which is probably a termination site of transcription. Northern blots revealed that vhtD is part of the vht operon. In the vho operon, a vhtD-like gene and a terminator composed of tandem repeats could not be identified. The physiological function of two genetically distinct, membrane-bound hydrogenases from Ms. mazei Gö1 is discussed.

Amino Acid Sequence↗

PubMed2005

Regulation of the nap operon encoding the periplasmic nitrate reductase of Paracoccus pantotrophus: delineation of DNA sequences required for redox control.

Expression of the nap operon, encoding the periplasmic nitrate reductase in Paracoccus pantotrophus, is maximal when cells are grown aerobically, but not anaerobically, with butyrate. Two promoters, termed P1 and P2, control operon expression and the operon-proximal P2 promoter is primarily responsible for increased nap expression in the presence of butyrate. A near-perfect palindromic sequence is centred at +7, relative to the P2 transcription start site. Mutation of this palindrome demonstrated that it is important for regulation of nap operon expression in response to both the redox and the oxidation state of the carbon substrate. A 5' deletion analysis of the nap promoter fused to lacZ revealed that full redox control of expression was retained when the DNA sequence up to position -49 bp, relative to the operon-distal P1 transcription start site, was removed. Encroaching beyond this position resulted in an approximately 4-fold reduction in expression when cells were grown aerobically with butyrate. Additionally, point mutations at position -38 and -45 relative to P1 also resulted in a reduction in expression during aerobic growth with butyrate. A GC-rich region of nap promoter DNA, centred on position -41 relative to the P1 transcription start site is thus proposed as a second DNA motif that is important for efficient expression of the nap operon.

Bacterial Proteins↗

PubMed1996

Cloning and sequence analysis of the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus.

Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin-like pigment. An operon, designated mel, containing a gene that encodes tyrosinase, which is involved in the synthesis of melanin pigment, was cloned from the chromosomal DNA of the microorganism into the high-copy plasmid pAK114 and expressed in S. lividans. The tyrosinase activity of the transformed cells was at approximately a 110-fold higher level than that of the same host carrying the plasmid pIJ702, which has the same replication origin as pAK114 and carries the mel operon from S. antibioticus. The sequence analysis of the S. castaneoglobisporus mel operon revealed that an open-reading frame consisting of 378 base pairs(bp), designated ORF378, was found upstream of the tyrosinase gene (TYRC) consisting of 819 bp. In the present study, we constructed a chimeric mel operon consisting of ORF378 from S. castaneoglobisporus and the tyrosinase gene (TYRA) from S. antibioticus. The chimeric mel operon or the S. antibioticus mel operon, which consists of ORF438 and TYRA, expressed the tyrosinase activity in Escherichia coli intracellularly when located under the control of lacZ promoter, and the tyrosinase activity from the former was at a 30-fold higher level than that from the latter. This suggests that the gene contributing to the high expression of the tyrosinase activity in S. castaneoglobisporus is ORF378, rather than TYRC.

Amino Acid Sequence↗

PubMed1985

Control of phenylalanyl-tRNA synthetase genetic expression. Site-directed mutagenesis of the pheS, T operon regulatory region in vitro.

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant M13 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a lambda phage carrying a pheS, T-lacZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 base-pairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in the translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.

Amino Acyl-tRNA Synthetases↗

PubMed1988

Positive and negative regulators for glucitol (gut) operon expression in Escherichia coli.

Expression of the glucitol (gut) operon in Escherichia coli is regulated by an unusual, complex system which consists of an activator (encoded by the gutM gene) and a repressor (encoded by the gutR gene) in addition to the cAMP-CRP complex (CRP, cAMP receptor protein). The activator and repressor are predicted to possess 119 (Mr = 12,955) and 257 (Mr = 28,240) aminoacyl residues, respectively, as deduced from the nucleotide sequences of their structural genes. Both of the genes encoding the two regulators are located downstream from the other known gut structural genes. Reverse transcriptase mapping revealed that the gutM gene is a promoter-distal constituent of the gut operon. The gutR gene has its own promoter, but expression of this gene is primarily due to readthrough from the gut operon operator-promoter. Thus, the gut operon consists of at least five structural genes and has the following gene order: gutOPABDMR. Interestingly, synthesis of the mRNA, which initiates at the promoter specific to the gutR gene, occurs within the gutM gene. Expressional control of the gut operon appears to occur as a consequence of the antagonistic action of the products of the autogenously regulated gutM and gutR genes. An additional cistron of the gut operon, of unknown function, may follow the gutR gene.

Base Sequence↗

PubMed1989

Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species.

The trmD operon is a four-cistron operon in which the first and fourth genes encode ribosomal proteins S16 (rpsP) and L19 (rplS), respectively. The second gene encodes a 21,000 Mr polypeptide of unknown function and the third gene (trmD) encodes the enzyme tRNA(m1G37)methyltransferase, which catalyzes the formation of 1-methylguanosine (m1G) next to the 3' end of the anticodon (position 37) of some tRNAs in Escherichia coli. Here we show under all regulatory conditions studied, transcription initiates at one unique site, and the entire operon is transcribed into one polycistronic mRNA. Between the promoter and the first gene, rpsP, an attenuator-like structure is found (delta G = -18 kcal; 1 cal = 4.184 J), followed by four uridine residues. This structure is functional in vitro, and terminates more than two-thirds of the transcripts. The different parts of the trmD operon mRNA decay at a uniform rate. The stability of the trmD mRNA is not reduced with decreasing growth rate, which is in contrast to what has been found for other ribosomal protein mRNAs. Furthermore, earlier experiments have shown the existence of differential expression as well as non-co-ordinate regulation within the operon. Our results are consistent with the regulation of the trmD operon being due to some mechanism(s) operating at the post-transcriptional level, and do not involve differential degradation of different mRNA segments, internal promoters or internal terminators.

Bacterial Proteins↗

PubMed1989

Bacteriophage P1 tail-fibre and dar operons are expressed from homologous phage-specific late promoter sequences.

Two plasmid systems, containing the easily assayable galK and lacZ functions, were employed to study the regulation of the bacteriophage P1 tail-fibre and dar operons. Various P1 DNA fragments carrying either the 5' end of lydA (the 1st gene in the dar operon) or the tail-fibre gene 19 precede the promoterless coding region of galK or were fused, in-frame, to the lacZ gene. In the presence of an induced P1 prophage, GalK and LacZ activities were both detected after a 20 to 30 minute lag period, indicating that the dar and tail-fibre operons are expressed from positively regulated, late promoters. The corresponding DNA operons are expressed from positively regulated, late promoters. The corresponding DNA region of the closely related p15B plasmid exhibits comparable promoter properties. Deletion analysis mapped the promoter of a gene 19-lacZ fusion to a DNA region upstream from gene R, an open reading frame that precedes the coding frame of gene 19. The tail-fibre gene thus forms the second gene in a three gene operon (genes R, 19 (S) and U). Sequence comparison between this promoter region, upstream sequences of the lydA gene and the corresponding portions of the p15B genome allowed the identification of a highly conserved 38 base-pair sequence, which most likely represents a P1-specific late promoter. This was confirmed by 5' mapping of P1 mRNA. Transcription of both the tail-fibre and dar operons is initiated at sites five and six base-pairs, respectively, downstream from the first conserved nucleotide of this sequence. The conserved motif consists of a standard Escherichia coli -10 region followed by a nine base-pair palindromic sequence located centrally about position -22.

Base Sequence↗

PubMed1991

Characterization and sequence of the Escherichia coli stress-induced psp operon.

We describe a new Escherichia coli operon, the phage shock protein (psp) operon, which is induced in response to heat, ethanol, osmotic shock and infection by filamentous bacteriophages. The operon includes at least four genes: pspA, B, C and E. PspA associates with the inner membrane and has the heptad repeats characteristic of proteins that can form coiled coils. The operon encodes a factor that activates psp expression, and deletion analyses indicate that this protein is PspC; PspC is predicted to possess a leucine zipper, a motif present in many eukaryotic transcription factors. The pspE gene is expressed in response to stress as part of the operon, but is also transcribed from its own promoter under normal conditions. In vitro studies suggest that PspA and C are modified in vivo. Expression of the psp genes does not require the heat shock sigma factor, sigma32. The increased duration of psp induction in a sigma32 mutant suggests that a product (or products) of the heat shock response down-regulates expression of the operon.

Amino Acid Sequence↗

PubMed1992

Mutagenesis of the Bacillus subtilis "-12, -24" promoter of the levanase operon and evidence for the existence of an upstream activating sequence.

The levanase operon of Bacillus subtilis is controlled by RNA polymerase associated with sigma 54 factor and by the LevR activator that is homologous to the NifA/NtrC family of regulators. A "-12, -24" promoter is present at the appropriate distance from the transcription start site. The drastic down effect of base substitutions in the TGGCAC, TTGCA consensus sequence on the expression of the levanase operon confirmed the involvement of the "-12, -24" region in promoter function. Deletion derivatives of the upstream sequence of the operon promoter were constructed using translational levD'-'lacZ fusions and were integrated as single copies at the amyE locus of the B. subtilis chromosome. A cis-acting DNA sequence that is required for activation of the operon promoter by LevR was identified. This regulatory sequence is about 50 base-pairs long and is centered 125 base-pairs upstream from the transcription start site in a region containing a 16 base-pair palindromic structure. This region of dyad symmetry functions as a regulatory element when placed up to at least 600 base-pairs upstream from the "-12, -24" promoter, although the efficacy of activation is lowered. Thus, in common with most sigma 54-dependent promoters, an upstream activating sequence (UAS) is involved in the control of expression of the levanase operon. The isolation and characterization of eight mutations in the UAS region confirmed the importance of the palindromic structure in promoter activation. Moreover, the expression of the levanase operon was inhibited by placing the UAS in trans on a multicopy plasmid, probably through titration of the LevR polypeptide. In conclusion, the levanase promoter region can be divided into two regulatory sequences: the "-12, -24" promoter recognized by the sigma 54 RNA polymerase holoenzyme and the UAS, an inverted repeat sequence that is probably the LevR binding site.

Amino Acid Sequence↗

PubMed1983

The operon that encodes the sigma subunit of RNA polymerase also encodes ribosomal protein S21 and DNA primase in E. coli K12.

The sigma subunit of E. coli RNA polymerase is encoded by the rpoD gene. Within the sequence upstream from rpoD, we have identified the structural genes rpsU and dnaG, which encode the 30S ribosomal protein S21 and DNA primase, respectively. The three genes are in the order rpsU, dnaG rpoD, and are all encoded by the same DNA strand. Analysis of in vivo transcripts from this region shows that these genes are all within the same operon. By correlating the 5' and 3' ends of in vivo transcripts with our DNA sequence, we have identified several regulatory features of the operon. These features include tandem promoters upstream from rpsU, a terminator between rpsU and dnaG, an RNA processing site separating dnaG and rpoD, and the operon terminator just downstream from rpoD. Immediately upstream of the operon promoters is an active promoter for an unidentified gene. We discuss the regulatory significance of the operon features and the biological significance of an operon encoding proteins essential for translation, replication and transcription.

Bacterial Proteins↗

PubMed1984

Regulation of E.coli phenylalanyl-tRNA synthetase operon in vivo.

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNAPhe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.

Amino Acyl-tRNA Synthetases↗

PubMed1980

Cloning of an EcoRI-generated fragment of the leucine operon of Salmonella typhimurium.

Recombinant plasmids carrying part of the leucine operon of Salmonella typhimurium were isolated following transformation of an Escherichia coli leucine auxotroph to prototrophy with a ligated mixture of EcoRI-treated Salmonella DNA and plasmid pSC101 DNA. Plasmids pCV11 and pCV13, containing a 3.4-10(6) dalton DNA fragment ligated to the vector, had the leu operon oriented in opposite directions. The orientation of the leu operon relative to plasmid genes was determined. The 3.4-10(6) dalton fragment was ligated in to the EcoRI site of plasmid pMB9 yielding plasmids pCV12 (orientation as in pCV11) and pCV14 (orientation as in pCV13). The results of enzyme assays and complementation tests indicated that these plasmids carry functional leuA, leuB, and leuC genes but not a functional leuD gene. Furthermore, the following results indicated that they have a functional leu control region and promoter. Expression of plasmid leu genes was markedly enhanced under conditions of leucine limitation whereas introduction of a leu promoter mutation into the operon oriented in either direction with respect to plasmid genes had a strong negative effect upon leu operon expression. Transcriptional readthrough from plasmid promoters, if it occurs at all, must be small in comparison with transcription initiated at the leu promoter. RNA was isolated from leucine auxotrophs grown under conditions of repression and derepression and from prototrophic strains derepressed for the leucine operon as a result of mutations in leuO, leuS, and flrB. The rate of synthesis of leu mRNA, measured by hybridization to plasmid pCV12 DNA, was proportional in each case to leu enzyme levels.

Cloning, Molecular↗

PubMed1996

A mgl-like operon in Treponema pallidum, the syphilis spirochete.

A 38-kDa lipoprotein of Treponema pallidum subsp. pallidum (T. pallidum), the syphilis spirochete, previously was identified as a putative homolog of E. coli MglB [Becker et al. (1994) Infect. Immun. 62, 1381-1391]. In the present study, genome walking in regions adjacent to the T. pallidum 38-kDa lipoprotein gene has identified three contiguous genes (tp-mglB [formerly tpp38], tp-mglA, and tp-mglC) which appear to comprise a mgl-like operon in T. pallidum. A prominent transcript corresponding to tp-mglB, the first gene of the operon which encodes the carbohydrate receptor, is synthesized by T. pallidum along with lesser abundant transcript(s) corresponding to the entire T. pallidum mgl operon. An active promoter 135 bp upstream of tp-mglB is believed to direct mRNA synthesis for the operon. This is the first membrane protein-encoding operon of T. pallidum for which a putative function (glucose import) has been assigned. Furthermore, by analogy with E. coli MglB which interacts with the sensory transducer Trg to induce a chemotactic response, it is possible that T. pallidum also contains a homolog of E. coli Trg or other methyl-accepting chemotaxis proteins. The existence of a mgl operon in T. pallidum thus may have important implications with respect to T. pallidum survival, tissue dissemination, and sensory transduction during virulence expression.

Amino Acid Sequence↗

PubMed2004

Organization and transcriptional regulation of myo-inositol operon in Clostridium perfringens.

myo-Inositol operon of Clostridium perfringens strain 13 consists of 13 genes with an upstream divergent regulator, iolR. Transcriptional analysis showed three separate transcripts for the operon of 15.6, 4.6 and 2.0 kb in length. iolR mutation studies showed that IolR is a negative regulator of the operon at transcriptional level. All the transcripts were induced by myo-Inositol in dose- and time-dependent manner. Glucose repressed the expression of all the transcripts of myo-Inositol operon. We also found that the operon was positively regulated by the two-component VirR/VirS system both in the presence and absence of myo-Inositol. This study shows that the global regulatory VirR/VirS system controls the expression of genes related to energy production (e.g. myo-Inositol operon) in addition to the virulence genes of C. perfringens strain 13.

Bacterial Proteins↗

PubMed2003

Separate and coordinate transcriptional control mechanisms link expression of the potentially lethal KilB spread locus to the upstream transmission operon on Streptomyces plasmid pIJ101.

Efficient conjugation of the high copy plasmid pIJ101 among members of the bacterial genus Streptomyces depends on a single plasmid gene (tra) for initial inter-mycelial transfer, and involves three additional pIJ101 functions (spdA, spdB, and kilB), which may promote intra-mycelial spread of the plasmid upon its entrance into the recipient. The genes tra, spdA, and spdB are co-transcribed as part of an operon, whose expression is negatively controlled by the pIJ101 repressor KorA. Downstream of this transmission operon and in the same orientation, the kilB spread gene possesses its own promoter, which is recognized by the pIJ101 KorB repressor protein; binding of KorB appears to prevent the lethal overexpression of the KilB protein, which otherwise shows a temporally increasing pattern of production or accumulation during the streptomycete life cycle. To define better the mechanism(s) controlling the concentration of the potentially toxic KilB protein in cells, a variety of transcriptional analyses involving the kilB promoter and kilB-specific mRNA were performed. These studies demonstrated that transcription originating from the kilB promoter on pIJ101 is dramatically reduced by KorB binding under non-mating conditions; more significantly, however, as judged by evidence of readthrough transcription across the kilB promoter region and polarity effects of upstream insertion and deletion mutations, kilB was found to be expressed also as part of the transmission operon with optimal KilB production being necessarily tied to such co-transcription. Our data indicate that the genes tra, spdA, spdB, and kilB comprise an unusual operon in which separate tight control of the distal gene (kilB) by the KorB repressor is superimposed on coordinate regulation of full operon transcription by KorA. Moreover, our results imply that potential interactions between elongating RNA polymerase molecules synthesizing transmission operon transcripts and KorB repressor bound to the intercistronic kilB promoter region are important for modulating kilB expression.

Bacterial Proteins↗

PubMed2004

Evidence against the selfish operon theory.

According to the selfish operon hypothesis, the clustering of genes and their subsequent organization into operons is beneficial for the constituent genes because it enables the horizontal gene transfer of weakly selected, functionally coupled genes. The majority of these are expected to be non-essential genes. From our analysis of the Escherichia coli genome, we conclude that the selfish operon hypothesis is unlikely to provide a general explanation for clustering nor can it account for the gene composition of operons. Contrary to expectations, essential genes with related functions have an especially strong tendency to cluster, even if they are not in operons. Moreover, essential genes are particularly abundant in operons.

Animals↗

PubMed2005

New insights into regulation of the tryptophan biosynthetic operon in Gram-positive bacteria.

The tryptophan operon of Bacillus subtilis serves as an excellent model for investigating transcription regulation in Gram-positive bacteria. In this article, we extend this knowledge by analyzing the predicted regulatory regions in the trp operons of other fully sequenced Gram-positive bacteria. Interestingly, it appears that in eight of the organisms examined, transcription of the trp operon appears to be regulated by tandem T-box elements. These regulatory elements have recently been described in the trp operons of two bacterial species. Single T-box elements are commonly found in Gram-positive bacteria in operons encoding aminoacyl tRNA synthetases and proteins performing other functions. Different regulatory mechanisms appear to be associated with variations of trp gene organization within the trp operon.

Bacillus↗

PubMed1990

Upstream regulatory sequence for transcriptional activator XylR in the first operon of xylene metabolism on the TOL plasmid.

Transcription of the first operon coding for m-xylene-degrading enzymes on the TOL plasmid of Pseudomonas putida is activated by the xylR gene product in the presence of m-xylene. The operon has the consensus sequence of the ntr/nif promoters at -24 and -12 regions, and the transcription is dependent on an RNA polymerase containing a sigma factor NtrA (RpoN or sigma 54). Deletion derivatives of the upstream sequence of the operon promoter were made in vitro and connected with the xylE gene on a plasmid. Their promoter activities were analyzed in Escherichia coli by monitoring catechol 2,3-dioxygenase activity, the xylE gene product. A cis-acting DNA element was identified, which is required for activation of the operon promoter by XylR protein in the presence of the inducer. This regulatory sequence of about 40 base-pairs in length was located 150 base-pairs upstream from the transcription start site. Analysis of the mutants containing insertions between the upstream regulatory sequence and the promoter sequence demonstrated strong dependence of the activation upon helical periodicity of DNA. The regulatory sequence functioned in the inverse orientation or at a distance of more than 1 x 10(3) base-pairs upstream from the promoter though less efficient. These results indicated that this upstream regulatory sequence might be the binding site for XylR protein. DNA-loop formation through protein-protein interaction between XylR protein attached to the upstream sequence and the NtrA-containing RNA polymerase bound by the promoter sequence was suggested for activation of the operon transcription. A sequence similar to the regulatory sequence of the first operon of xylene metabolism was found in the upstream region of the xylS gene, which is also activated by XylR protein in the presence of m-xylene.

Base Sequence↗