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PubMed1978

Promoter- and attenuator-related metabolic regulation of the Salmonella typhimurium histidine operon.

Expression of the histidine (his) operon in Salmonella typhimurium was found to be positively correlated with the intracellular level of guanosine tetraphosphate (ppGpp). Limitation for amino acids other than histidine elicited a histidine-independent metabolic regulation of the operon. In bacteria grown at decreased growth rates, his operon expression was metabolically regulated up to a point, after which further decreases in growth rate no longer resulted in further enhancement of operon expression. Studies using strains carrying various regulatory and deletion mutations indicated that metabolic regulation is achieved predominantly by increased RNA chain initiations at the primary (P1) and internal (P2) promoters. Metabolic regulation ordinarly did not involve changes in RNA chain terminations at the attenuator site of the his operon. A model is proposed that involves ppGpp-induced changes in RNA polymerase initiation specificity at particular promoters. A second, special form of metabolic regulation may operate which also is histidine independent, but does involve relief of attenuation.

Alanine↗

PubMed1981

An amber mutation in a ribosomal protein gene: ineffective suppression stimulates operon-specific transcription.

The effects of inefficient suppression and the accompanying polarity of an amber mutation in the ribosomal protein gene rplC (L3) on the transcriptional activities of several ribosomal protein operons were investigated. The L3 gene is proximally located in the S10 operon. Inefficient suppression of the mutation specifically stimulated transcription of regions both proximal and distal to the site of mutation by about twofold. In contrast, no effect on the transcriptional activity of regions within the alpha and spc ribosomal protein operons was observed. Thus, transcription of the S10 operon could be actively and specifically regulated in response to a deficiency in one or more of the protein products from the operon. We presume that this transcriptional control acts at the level of transcription initiation, but we cannot exclude the possibility of some other unknown transcriptional regulatory mechanism.

Bacterial Proteins↗

PubMed1981

Regulation of fatty acid degradation in Escherichia coli: analysis by operon fusion.

Fusion of the lacZ gene coding for beta-galactosidase to the fadA,B and fadE operons was accomplished by using the phage Mu d (Apr lac). In such fusion strains, beta-galactosidase was induced by long-chain fatty acids and repressed by glucose, as is the normal pattern of control for the enzymes of the fad regulon. The level of induction seen was approximately 10-fold for both the fadA and fadE operons. These results demonstrate that the previously observed regulation of both the fadA and fadE operons is at the transcriptional level. When an insertion mutation in the fadR (repressor) gene was introduced into the fusion strains, beta-galactosidase was produced constitutively. A series of fatty acids of different chain lengths were tested as inducers. Acids of chain lengths of 10 carbon atoms or less failed to induce, those of 12 carbon atoms induced partly, and those of 14 or more carbon atoms induced fully. Imidazole was found to counteract the glucose repression of the fadA operon as recently demonstrated for the ara operon.

Acyl-CoA Dehydrogenase↗

PubMed1982

Evolution of the D-ribose operon on Escherichia coli B/r.

The D-ribose operon (rbs) of Escherichia coli K-12 maps at 83 min and is inducible. The rbs operon of E. coli B/r maps at 2 min and is constitutive. Evidence is presented showing that a second inducible copy of the rbs operons is present in E. coli B/r mapping at 83 min. The data indicated that the duplication of the rbs operon represented a transposition of the 83-min region to 2 min. The identification of a second copy of the rbs operon in B/r and the determination of its inducibility were based on the reactivation, through mutagenesis, of inducible rbs expression, mapping by P1 transduction of the mutation site to 83 min, and merodiploid complementation analysis of the D-ribokinase expression in E. coli B/r. We also show that the rbs transposition to 2-min continued to generate transposable elements coding for the 1- to 2-min region of the chromosome and transposing onto extrachromosomal DNA target molecules such as pBR322.

Chromosome Mapping↗

PubMed1983

Evidence that repression mechanisms can exert control over the thr, leu, and ilv operons of Escherichia coli K-12.

Mutants of Escherichia coli K-12 resistant to either the threonine analog DL-alpha-amino-beta-hydroxyvaleric acid or the leucine analog 5',5',5'-trifluoro-DL-leucine were isolated. One DL-alpha-amino-beta-hydroxyvaleric acid-resistant mutant strain, designated SP572, constitutively expressed the thr and ilv operons. The mutant allele, avr-16, was localized between trpR and the thr operon at min 0. The wildtype allele of avr-16, designated ileR, is trans dominant. One 5',5',5'-trifluoro-DL-leucine-resistant mutant strain, designated FLR9, expressed the leu and ilv operons constitutively. The mutant allele, flr-9, is linked to entA at min 13. The constitutive expression of the thr, leu, and ilv operons in mutants avr-16 and flr-9 was partly reversed in cells harboring a plasmid, which leads to elevated levels of the trpR gene product, the Trp aporepressor protein. Operator-like sequences situated upstream from the transcription startpoints of the thr, leu, and ilv operons are plausible candidates for targets of systems of repressor-operator control functioning in parallel with attenuation.

Chromosome Mapping↗

PubMed1985

Evidence for an internal promoter in the Escherichia coli threonine operon.

We constructed plasmids carrying the two first genes of the threonine operon from which the major promoter was deleted in vitro by digestion with BAL 31 nuclease. These plasmids continued to express the second gene (thrB) of the operon as judged by their ability to complement a threonine auxotroph. These data indicate that, in addition to the major promoter thrP, there was an internal promoter, thrBp, which could be used for the transcription of the thrB, the second gene of the operon. Additional evidence was given by subcloning a 230-base-pair segment of the operon in a plasmid suitable for detection of translation initiation signals and promoters. The thrBp promoter was thus shown to lie within a 61-base-pair fragment at the 3' end of the first gene, thrA, of the threonine operon.

Cloning, Molecular↗

PubMed1985

Role of translation and attenuation in the control of pyrBI operon expression in Escherichia coli K-12.

Expression of the pyrBI operon of Escherichia coli K-12, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated by the intracellular levels of UTP. Previous experiments suggested a unique model for regulation of operon expression in which low UTP levels cause close coupling of transcription and translation of the pyrBI leader region. This close coupling suppresses transcriptional termination at an attenuator preceding the structural genes. In this study, we examined the regulatory role of translation and attenuation in operon expression. To determine whether the leader region is translated, we constructed a plasmid, designated pBHM17, in which the pyrBI promoter(s) and the first 11 codons for a putative 44-amino acid leader polypeptide are fused to codon 9 of lacZ. A transformant carrying this plasmid synthesized a beta-galactosidase fusion protein with the amino-terminal sequence of the leader polypeptide, demonstrating that the signals required for leader polypeptide synthesis function in vivo. Synthesis of the fusion protein was nearly insensitive to pyrimidine availability. In uracil-grown cells, the level of fusion protein synthesis encoded by plasmid pBHM17 was much greater than that encoded by a similar plasmid containing a pyrB::lacZ gene fusion, in which the pyrBI promoter-regulatory region is intact. These results indicate that the downstream leader sequence which includes the attenuator is required for regulation and functions as a transcriptional barrier. Oligonucleotide-directed mutagenesis was used to change the ATG leader polypeptide initiation codon of the intact pyrBI operon to ACG, which was shown to strongly inhibit translational initiation. This mutation greatly reduced operon expression and regulation as predicted by the attenuation control model.

Amino Acid Sequence↗

PubMed1987

Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals.

The nar operon, which encodes the three subunits of nitrate reductase in Escherichia coli, is fully induced under anaerobic conditions with nitrate. Two distinct regulatory domains have been delineated in the 5' region of the operon which respond respectively to positive induction by the fnr gene product under anaerobic conditions and to positive induction by the narL gene product in the presence of nitrate (S.F. Li, T. Rabi, and J.A. DeMoss, J. Bacteriol. 164:25-32). To characterize these two regulatory regions, we determined the DNA sequence for a 500-base-pair (bp) region extending upstream from the first structural gene of the nar operon. Analysis of subsequent subclones of the operon established that the 5' limit of the nar operon lies between 215 and 260 bp upstream from the translational start site of the first structural gene. The region required for induction by the fnr gene product is located within 160 bp from the translation start site, while the region responding to induction by nitrate extends an additional 100 bp upstream. Protein fusions of lacZ with the N-terminal sequence of the narG gene were constructed so that beta-galactosidase formation was under the control of the nar promoter and one or both regulatory domains. Analysis of strains bearing these fusion plasmids indicated that the expression of the hybrid proteins paralleled that of nitrate reductase by the parent plasmids, demonstrating that the regulatory signals did not extend significantly into the first structural gene. The transcriptional start site and the level of the transcription were determined by the S1 mapping procedure. One major transcript was identified which initiated -50 bp from the translational start site of the first structural gene. The synthesis of the transcript was repressed aerobically, was fully induced by nitrate anaerobically, and was greatly reduced in an Fnr- mutant. Possible regulatory sequences were identified in the 200-bp regulatory region extending upstream from the transcription start site.

Aerobiosis↗

PubMed1987

Structural analysis of the Escherichia coli K-12 hisT operon by using a kanamycin resistance cassette.

We constructed a series of recombinant plasmids containing a kanamycin resistance (Kmr) cassette upstream from, within, and downstream from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I. These Kmr insertions were then crossed directly into the bacterial chromosome. We determined growth characteristics, assayed in vivo hisT expression, and mapped in vivo hisT operon transcripts for the Kmr insertion mutants. We also analyzed polypeptides synthesized in minicells from plasmids containing Kmr cassettes. The combined results from these experiments demonstrate new features concerning the structure and expression of the complex operon that contains hisT. We show that the minimum size of the operon is approximately 3,500 base pairs and that it contains at least four genes, which are arranged in the order usg-2 (pdxB), usg-1, hisT, and dsg-1 and encode polypeptides with apparent molecular masses of 42,000, 45,000, 31,000, and 17,000 daltons, respectively. Of these genes, only the functions of usg-2 (pdxB) and hisT are known, and genetic evidence suggests that these two genes do not require usg-1 or dsg-1 for function, usg-2 (pdxB) is required for growth of bacteria on minimal medium at 37 degrees C. In contrast, the three genes at the end of the hisT operon are dispensable and form a transcription unit that is expressed from a relatively strong internal promoter. The phenotypes of the Kmr insertion mutants and results from gene expression experiments further confirm the position of the internal promoter and locate additional genetic signals in the DNA sequence around hisT. The experiments reported here also indicate several interesting properties of the Kmr cassette as a tool for probing complex operons.

Base Sequence↗

PubMed1987

Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes.

Wild-type Escherichia coli cells are unable to grow on beta-glucosides. Spontaneous mutants arise, however, which are able to utilize certain aromatic beta-glucosides such as salicin or arbutin as carbon sources, revealing the presence of a cryptic operon called bgl. Mutations activating the operon map within (or close to) the promoter region of the operon and are due to the transposition of an IS1 or IS5 insertion element into this region. This operon was reported to consist of three genes coding for a phospho-beta-glucosidase, a specific transport protein (enzyme IIBgl), and a positively regulating protein. We have defined the extent and location of three structural genes, bglC, bglS, and bglB, and have determined their DNA sequence. The amino acid sequences deduced from the open reading frames together with deletion and subcloning analyses suggest that the first gene, bglC, codes for the regulatory protein, the second, bglS, codes for the transport protein, and the third, bglB, for phospho-beta-glucosidase. A fourth gene may exist which codes for a product of unknown function. We discuss structural features of the DNA sequence which may bear on the regulation of the operon. Homologies to sequences preceding the gene for an excreted levansucrase of Bacillus subtilis, which are known to be involved in the regulation of this gene, and to sequences preceding the gene for an excreted beta-endoglucanase of B. subtilis, for which data pertaining to regulation are not yet available, suggest a close evolutionary relationship among the regulatory components of all three systems.

Amino Acid Sequence↗

PubMed1987

Promoter of the pertussis toxin operon and production of pertussis toxin.

Pertussis toxin (PT), the major virulence factor of Bordetella pertussis, is composed of five different subunits whose genes are organized as an operon. We report the mapping of the promoter region of the PT operon and show that this promoter is only weakly active in Escherichia coli. Bordetella parapertussis and Bordetella bronchiseptica, which do not produce any PT, are shown to have a weaker promoter sequence for this operon and not to produce any detectable PT mRNA. We show that transcription of the PT operon in B. pertussis was constant throughout until the late stationary phase, when transcription significantly decreased. Analysis of the transposon Tn5 mutant BP347 showed that the product of the vir locus was required for transcription of the PT operon. Characterization of the Tn5 mutant BP356 showed that subunit S3 was required for the release of PT into the extracellular medium.

Biological Transport↗

PubMed1987

Identification of the promoter of the Bacillus subtilis sdh operon.

The Bacillus subtilis sdhCAB operon contains the structural genes for the three subunits of the membrane bound succinate dehydrogenase complex. An sdh-specific transcript of about 3,450 nucleotides was detected in vegetative bacteria. S1 nuclease mapping experiments showed that the sdh operon is transcribed from a sigma-43 promoter; the transcript starts at a guanosine residue 90 base pairs upstream from the first gene of the operon, sdhC. No sdh transcript was found in B. subtilis carrying the sdh-115 mutation, which decreases expression of the sdh operon by more than 99%. The sdh-115 mutation is a G-to-A transition in the -35 region of the sigma-43 promoter. The sdh operon is sensitive to glucose repression. When the sdh promoter region was used to drive transcription of the cat-86 gene this gene also became glucose repressed.

Bacillus subtilis↗

PubMed1987

Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product.

The fumarate reductase enzyme complex, encoded by the frdABCD operon, allows Escherichia coli to utilize fumarate as a terminal electron acceptor for anaerobic oxidative phosphorylation. To analyze the expression of fumarate reductase, protein and operon fusions were constructed between the frdA and the lacZ genes and introduced onto the E. coli chromosome at the lambda attachment site. Expression of beta-galactosidase from either fusion was increased 10-fold during anaerobic versus aerobic cell growth, increased an additional 1.5-fold by the presence of fumarate, the substrate, and decreased 23-fold by nitrate, a preferred electron acceptor. The addition of trimethylamine-N-oxide as an electron acceptor did not significantly alter frdA'-'lacZ expression. Control of frd operon expression is therefore exerted at the transcriptional level in response to the availability of the electron acceptors oxygen, fumarate, and nitrate. Anaerobic induction of frdA'-'lacZ expression was impaired in an fnr mutant and was restored when the fnr+ gene was provided in trans, thus establishing that the fnr gene product, Fnr, is responsible for the anaerobic activation of frd operon expression. Nitrate repression of frdA'-'lacZ expression was observed under either aerobic or anaerobic cell growth conditions in both wild-type and fnr mutant strains, demonstrating that the mechanism for nitrate repression is independent of nitrate respiration and oxygen control imparted by Fnr. Studies performed with a fnr'-'lacZ protein fusion confirmed that the fnr gene is expressed both aerobically and anaerobically. A model is proposed for the regulation of frdABCD operon expression in response to the availability of the alternate terminal electron acceptors oxygen, nitrate, and fumarate.

Aerobiosis↗

PubMed1988

Feedback regulation of the spc operon in Escherichia coli: translational coupling and mRNA processing.

The spc operon of Escherichia coli encodes 10 ribosomal proteins in the order L14, L24, L5, S14, S8, L6, L18, S5, L30, and L15. This operon is feedback regulated by S8, which binds near the translation start site of L5 and inhibits translation of L5 directly and that of the distal genes indirectly. We constructed plasmids carrying a major portion of the spc operon genes under lac transcriptional control. The plasmids carried a point mutation in the S8 target site which abolished regulation and resulted in overproduction of plasmid-encoded ribosomal proteins upon induction. We showed that alteration of the AUG start codon of L5 to UAG decreased the synthesis rates of plasmid-encoded distal proteins, as well as L5, by approximately 20-fold, with a much smaller (if any) effect on mRNA synthesis rates, indicating coupling of the distal cistrons' translation with the translation of L5. This conclusion was also supported by experiments in which S8 was overproduced in trans. In this case, there was a threefold reduction in the synthesis rates of chromosome-encoded L5 and the distal spc operon proteins, but no decrease in the mRNA synthesis rate. These observations also suggest that transcription from ribosomal protein promoters may be special, perhaps able to overcome transcription termination signals. We also analyzed the state of ribosomal protein mRNA after overproduction of S8 in these experiments and found that repression of ribosomal protein synthesis was accompanied by stimulation of processing (and degradation) of spc operon mRNA. The possible role of mRNA degradation in tightening the regulation is discussed.

Blotting, Northern↗

PubMed1988

In vivo analysis of puf operon expression in Rhodobacter sphaeroides after deletion of a putative intercistronic transcription terminator.

The intercistronic region of the mRNA derived from the puf operon of Rhodobacter sphaeroides is capable of forming two stable stem-loop structures, the first of which resembles a factor-independent transcription terminator. A puf operon construction lacking the putative transcription terminator was made in vitro and crossed into the chromosome of R. sphaeroides PUFB1 to yield a single chromosomal copy in the terminator-deleted strain. The mutant strain, designated PUF delta 348-420 which was otherwise isogenic with the wild-type strain 2.4.1, showed a normal growth rate at high light intensity compared with the wild type, with the levels of the B875 and reaction center spectral complexes being approximately 7% and 25%, respectively, of those found in the wild type. The deletion mutation correlated with a reduction in the size of the fixed photosynthetic unit from 15:1 in the wild type to 4:1 in the mutant. The level of the B800-850 complex was increased approximately twofold in the mutant strain. However, substantial amounts of the B875 and reaction center polypeptides were not incorporated into spectrally active complexes, suggesting the importance of other factors in the assembly of these complexes. Removal of the intercistronic stem-loops resulted in increased readthrough of the puf operon terminator to regions downstream, as well as altering the stability of the resulting puf operon-specific transcripts. A model is proposed which links ribosome stalling within the open reading frame K leader region of the puf operon transcript with chain termination.

Bacteria↗

PubMed1988

narI region of the Escherichia coli nitrate reductase (nar) operon contains two genes.

In previous studies it has been established that in Escherichia coli the three known subunits of anaerobic nitrate reductase are encoded by the narGHI operon. From the nucleotide sequence of the narI region of the operon we conclude that, in addition to the narG and narH genes, the nar operon contains two other open reading frames (ORFs), ORF1 and ORF2, that encode proteins of 26.5 and 25.5 kilodaltons, respectively. Protein fusions to each of the genes in the operon showed that expression of all four genes was similarly regulated. The reading frames of ORF1 and ORF2 were verified, and the N-terminal sequence for the ORF1 fusion protein was determined. The nar operon therefore contains four genes designated and ordered as narGHJI.

Anaerobiosis↗

PubMed1989

Nucleotide sequence of the transcriptional control region of the osmotically regulated proU operon of Salmonella typhimurium and identification of the 5' endpoint of the proU mRNA.

Southern blot analysis of 15 proU transposon insertions in Salmonella typhimurium indicated that this operon is at least 3 kilobase pairs in length. The nucleotide sequence of 1.5-kilobase-pair fragment that contains the transcriptional control region of the proU operon and the coding sequences specifying 290 amino acids of the first structural gene of the operon was determined. The predicted amino acid sequence of the product of this gene shows extensive similarity to the HisP, MalK, and other proteins that are inner membrane-associated components of binding protein-dependent transport systems. S1 mapping and primer extension analysis of the proU mRNAs revealed several species with different 5' ends. Two of these endpoints are sufficiently close to sequences that have weak similarities to the consensus -35 and -10 promoter sequences that they are likely to define two transcription start sites. However, we cannot rule out the possibility that some or all of the 5' endpoints detected arose as a result of the degradation of a longer mRNA. The expression of proU-lacZ operon fusions located on plasmids was normal in S. typhimurium regardless of the plasmid copy number. The sequences mediating normal, osmoregulated expression of the proU operon were shown by subcloning to be contained on an 815-base-pair fragment. A 350-base-pair subclone of this fragment placed onto a lacZ expression vector directed a high-level constitutive expression of beta-galactosidase, suggesting that there is a site for negative regulation in the proU transcriptional control region which has been deleted in the construction of this plasmid.

Amino Acid Sequence↗

PubMed1989

Overlap between pdxA and ksgA in the complex pdxA-ksgA-apaG-apaH operon of Escherichia coli K-12.

We report that pdxA, which is required for de novo biosynthesis of pyridoxine (vitamin B6) and pyridoxal phosphate, belongs to an unusual, multifunctional operon. The pdxA gene was cloned in the same 3.5-kilobase BamHI-EcoRI restriction fragment that contains ksgA, which encodes the 16S rRNA modification enzyme m6(2)A methyltransferase, and apaH, which encodes diadenosine tetraphosphatase (ApppA hydrolase). Previously, Blanchin-Roland et al. showed that ksgA and apaH form a complex operon (Mol. Gen. Genet. 205:515-522, 1986). The pdxA gene was located on recombinant plasmids by subcloning, complementation, and insertion mutagenesis, and chromosomal insertions at five positions upstream from ksgA inactivated pdxA function. DNA sequence analysis and minicell translation experiments demonstrated that pdxA encoded a 35.1-kilodalton polypeptide and that the stop codon of pdxA overlapped the start codon of ksgA by 2 nucleotides. The translational start codon of pdxA was tentatively assigned based on polypeptide size and on the presence of a unique sequence that was also found near the translational start of PdxB. This conserved sequence may play a role in translational control of certain pyridoxine biosynthetic genes. RNase T2 mapping of chromosomal transcripts confirmed that pdxA and ksgA were members of the same complex operon, yet about half of ksgA transcripts arose in vivo under some culture conditions from an internal promoter mapped near the end of pdxA. Transcript analysis further suggested that pdxA is not the first gene in the operon. These structural features support the idea that pyridoxine-biosynthetic genes are members of complex operons, perhaps to interweave coenzyme biosynthesis genetically with other metabolic processes. The results are also considered in terms of ksgA expression.

Amino Acid Sequence↗