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PubMedDate not supplied

The regulation of microcin B, C and J operons.

Microcins are ribosomally encoded small peptide antibiotics produced by Gram(-) enterobacteria. Microcin production-biosynthesis, maturation and secretion to the medium-is encoded by gene clusters organized in operons. Production of the best known plasmid-encoded microcins (MccB, MccC and MccJ) switches on when cells reach the stationary growth phase. This production is doubly regulated at transcriptional level by (a). the growth phase: microcin operons silent/repressed during exponential growth are induced/derepressed when cells sense nutrient starvation and stop exponential growth, and (b). global bacterial regulators acting as inducers or repressors of operon expression. The role played by these regulators (CRP, EmrR, IHF, H-NS, LRP, OmpR, Sigma-38 and SpoT) in the expression of specific microcin operons is reviewed.

Bacteriocins↗

PubMed1999

Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon.

The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions.

Journal Article↗

PubMed1981

Cryptic operon for beta-glucoside metabolism in Escherichia coli K12: genetic evidence for a regulatory protein.

Escherichia coli K12 does not metabolize beta-glucosides such as arbutin and salicin because of lack of expression of the bglBSRC operon, which contains structural genes for transport (bglC) and hydrolysis (bglB) of phospho-beta-glucosides. Mutants carrying lesions in the cis-acting regulatory site bglR metabolize beta-glucosides as a consequence of expression of this cryptic operon (Prasad and Schaefler 1974). We isolated mutations promoting beta-glucoside metabolism that were unlinked to bglR; some of these mutations were shown to be amber. All of them were mapped at 27 min on the E. coli K12 linkage map and appeared to define a single gene, for which we propose the designation bglY. Utilization of beta-glucosides in bglY mutants appeared to be a consequence of expression of the bglBSRC operon, since bglB bglR and bglB bglY double mutants had the same phenotype. All bglY mutations analyzed were recessive to the wild-type bglY+ allele. Phospho-beta-glucosidase B and beta-glucoside transport activities are inducible in bglY mutants, as they are in bglR mutants. Metabolism of beta-glucosides in both bglR and bglY mutants required cyclic AMP. We propose that bglY encodes a protein acting as a repressor of the bglBSRC operon, active in both the presence and absence of beta-glucosides, whose recognition site would be within the bglR locus.

Carrier Proteins↗

PubMed1986

Identification and nucleotide sequence of the promoter region of the Bacillus subtilis gluconate operon.

The nucleotide sequence (742 bp) of the promoter region of the Bacillus subtilis gluconate (gnt) operon is presented. Nuclease Sl mapping revealed the start point of the transcription and suggested that the expression of this operon is probably regulated at the transcriptional level. The sequences of the -35 and -10 regions suggested that RNA polymerase possessing sigma-43 may recognize this structure. The 223 bp fragment containing 100 bp upstream from the transcription start site actually exhibited a promoter activity when cloned in a promoter probe vector of pPL603B. This promoter activity was highly derepressed and although still under catabolite repression. The fragment on a high copy plasmid could titrate a regulator of the gnt operon so that the expression of the operon on the host chromosome also became derepressed.

Amino Acid Sequence↗

PubMed1992

Regulation of the Escherichia coli cad operon: location of a site required for acid induction.

The cad operon encodes lysine decarboxylase and a protein homologous to amino acid antiporters. These two genes are induced under conditions of low pH, anaerobiosis, and excess lysine. The upstream regulatory region of the cad operon has been cloned into lacZ expression vectors for analysis of the sequences involved in these responses. Deletion analysis of the upstream region and cloning of various fragments to make cadA::lacZ or cadB::lacZ protein fusions or operon fusions showed that cadA was translated more efficiently than cadB and localized the pH-responsive site to a region near an upstream EcoRV site. Construction of defined end points by polymerase chain reaction further localized the left end of the regulatory site. The presence of short fragments bearing the regulatory region on high-copy-number plasmids greatly reduced expression from the chromosomal cad operon, suggesting that titration of an essential activator protein was occurring. With nonoptimal polymerase chain reaction conditions, a set of single point mutants were made in the upstream regulatory region. Certain of these altered regulatory regions were unable to compete for the regulatory factor in vivo. The locations of these essential bases indicate that a sequence near the EcoRV site is very important for the activator-DNA interaction. In vivo methylation experiments were conducted with cells grown at pH 5.5 or at pH 8, and a difference in protection was observed at specific G residues in and around the region defined as important in pH regulation by the mutation studies. This work defines essential sequences for acid induction of this system involved in neutralization of extracellular acid.

Base Sequence↗

PubMed2002

Cloning, molecular characterization, and transcriptional analysis of dnaK operon in a methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726.

Three structural genes that consist of a dnaK operon in a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 were cloned and characterized. The genes were clustered in the transcription order grpE-dnaK-dnaJ. The cloned grpE, dnaK, and dnaJ genes had open-reading frames of 474, 1,926, and 1,116 nucleotides, coding for proteins with calculated molecular masses of 17,390, 69,761, and 41,050, respectively. The overall identities in the deduced amino acid sequences of GrpE, DnaK, and DnaJ with those of the Escherichia coli homologs were 45.2, 74.5, and 61.2%, respectively. Northern blot analyses with grpE-, dnaK-, and dnaJ-specific probes revealed that the three genes are co-transcribed as a 4.0-kb mRNA. A primer extension analysis revealed that the transcription of the dnaK operon started at the nucleotide A that is located 28 bp upstream of the grpE start codon. The transcription start site was preceded by a putative promoter region 15'-CCCCGCTTGAA(13-bp)CCCCAATTT-3'], which is highly homologous to the consensus sequences of the E. coli sigma32-type heat shock promoter. The putative promoter worked under both normal and heat shock conditions in E. coli. The nature of the nucleotide sequence in the second half of the -35 region played a critical role during transcription. The heat shock mRNA was maximally produced at about 10 min after transfer of the Methylovorus sp. strain SS1 from 30 to 42 degrees C. The dnaK operon was also induced by ethanol, hydrogen peroxide, and NaCl shocks. The cloned dnaK operon complemented the E. coli dnaK mutant.

Bacterial Proteins↗

PubMed1989

Identification of an operator sequence for the Bacillus subtilis gnt operon.

Expression of the Bacillus subtilis gluconate (gnt) operon is negatively regulated by the gnt repressor which is antagonized by D-gluconate or D-glucono-delta-lactone. The repressor specifically binds to a gnt operator near the gnt promoter. From the results of gel retardation assaying of the gnt repressor and various restriction enzyme digests of a fragment carrying the gnt promoter and the gntR gene, the gnt operator was found to be located within a 141-base pair region containing the gnt promoter. Deoxyribonuclease I protection experiments revealed that the gnt repressor protected a gnt promoter region, between -10 and +15, which contains a region showing dyad symmetry with a sequence showing half-symmetry, ATACTTGTA. A 35-base pair synthetic duplex DNA containing this region showing dyad symmetry specifically bound to the gnt repressor. Through cleavage at two AccI sites in the protected region an 8-base pair deletion was introduced into the region showing dyad symmetry, which made expression of the gnt promoter constitutive. A DNA fragment carrying this deletion did not bind to the repressor. These results clearly indicated that the gnt operator sequence is a sequence containing the region showing dyad symmetry located at the transcription initiation site of the gnt operon. It was also suggested that the gnt operon contains only one operator. The similarity in the recognition between the repressor-operator interaction of the gnt operon and those of Escherichia coli TrpR and its operators was discussed.

Amino Acid Sequence↗

PubMed1984

Physical structure, genetic content and expression of the alkBAC operon.

We cloned sequences of the alk (alkane utilization) operon of Pseudomonas and characterized them physically and genetically. These sequences were used to construct a DNA restriction map of the alkBAC region. We physically mapped alk::Tn7 insertions and delta alkBA deletions, and we were able to show complementation or marker rescue of alk point mutations by cloned DNA sequences. Our results confirmed the existence of an operon containing structural loci encoding activities for membrane alkane hydroxylase component (alkB), soluble alkane hydroxylase component (alkA) and membrane alcohol dehydrogenase (alkC). Physical mapping of alkC::Tn7 insertions and complementation of alkC point mutations by cloned sequences from the alkBA region showed that we were previously mistaken in inferring the existence of a separate unlinked alkC cluster. Studies with an alkB-lacZ transcription fusion construct established that the operon is transcribed in the order alkBAC and is under positive regulation by alkR regulatory functions.

Alkanes↗

PubMed1982

Involvement of a gene of the chl E locus in the regulation of the nitrate reductase operon.

A strain of E. coli carrying a Mudl insertion leading to chlorate resistance was found to lack nitrate reductase and formate dehydrogenase activities, but to synthesize b-type cytochrome constitutively. Introduction of this insertion mutation into a strain bearing a fusion between the nitrate reductase operon (chl C, chl I) and the lac structural genes resulted in the constitutive expression of the lac genes of this last fusion. Identical results were found when the Mudl was eliminated promoting a deletion in the original insertion site. This mutation was located midway between gal and aro A, at the Chl E locus. Study of a chl E strain already described revealed similar behaviour. Absence of nitrate reductase activity in these strains which constitutively express the structural genes of the nitrate reductase operon was tentatively attributed to the simultaneous lack of a cofactor of the nitrate reductase terminal enzyme, possibly cofactor Mo-X, and of a repressor of the operon.

Apoproteins↗

PubMed1982

Regulation of the nitrate reductase operon: effect of mutations in chlA, B, D and E genes.

Introduction of chlA, B or E mutant alleles into strains carrying fusions between the lac structural genes and the promoter of the nitrate reductase operon led to the partial or total constitutive expression of the fusion. Presence of chlD mutated alleles in the same strains did not result in constitutive expression of the fusion and allowed full induction by nitrate only in the presence of molybdenum. It is proposed that the molybdenum cofactor, Mo-X, of the nitrate reductase is also corepressor of the operon. The chlA, B and E genes would be involved in the biosynthesis of the X-moity. Mutations in these genes would give an altered X-moity which still binds to molybdenum but leads to a less efficient repressor complex; chlD gene would code for an enzyme inserting molybdenum in the X-moity of the cofactor. Mutations in chlD give an empty cofactor leading to a complex which permanently represses the operon unless molybdenum is added.

Escherichia coli↗

PubMed1999

A novel mutation, of the Bacillus subtilis hut operon that relieves both catabolite repression and amino acid repression.

A mutation, designated hutCR11, which resulted in high expression of the hut operon and release of the catabolite repression and amino-acid repression of hut expression, was isolated and determined to be a T-to-G transversion at position +30 (+1 indicates the transcription-initiation site). In the hutCR11 mutant, levels of hutP mRNA were 5-fold higher than those in wild-type cells under conditions of non-induction and induction and 11-fold higher under conditions of catabolite repression and amino-acid repression. Mutation analysis showed that two types of base change (T-->A and T-->C) at position +30 did not cause high expression of the hut operon, indicating that this was specifically caused by the single base substitution (T-->G) at position +30. The base substitution of A for T at position +30 also led to partial relief of both catabolite repression and amino-acid repression. These results indicate that the nucleotide sequence at +30 is important for regulation of both catabolite repression and amino-acid repression of the hut operon.

Bacillus subtilis↗

PubMed2001

Effects of transcription induction homogeneity and transcript stability on expression of two genes in a constructed operon.

A synthetic operon was constructed using the reporter genes gfp and lacZ and the arabinose-inducible araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 3' and 5' ends of the genes and a putative RNase E site was placed between the genes. These mRNA control elements have been shown to affect transcript processing and decay, resulting in altered protein levels. These constructs were transformed into cells harboring the native arabinose-inducible araE gene encoding the arabinose transport protein and engineered cells harboring a constitutively expressed araE. In the strains with arabinose-dependent transport the linear response in the production of both reporter proteins to inducer concentration occurred over a narrow range of arabinose concentrations. In the strains with constitutive transport the linear range of gene expression occurred over a much larger arabinose concentration range than in strains with the arabinose-inducible transport. Strains with the arabinose-inducible transport harboring different operon constructs produced the two reporter proteins at very different levels at low arabinose concentrations; as inducer concentrations increased, differences in relative expression levels decreased. In contrast, strains with constitutive transport harboring different operon constructs produced the reporter proteins at very different levels across the entire range of inducer concentrations, pointing to the importance of optimizing gene expression control at various levels to control the production of heterologous proteins.

Arabinose↗

PubMed2026

A complete hlyCABD-like RTX operon marks a virulence-associated subset of trh-positive Vibrio parahaemolyticus from Hangzhou Bay, China.

Vibrio parahaemolyticus remains a major cause of seafood-associated gastroenteritis, yet routine surveillance still relies largely on the canonical hemolysin markers thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh). To determine whether this framework overlooks accessory virulence determinants in trh-positive lineages, we analyzed 193 V. parahaemolyticus isolates collected between 2022 and 2025 from clinical, environmental, and seafood-associated sources in the Hangzhou Bay region of China. Serotyping identified 45 serotypes, with O10:K4 predominating among clinical isolates. Both clinical and non-clinical populations showed open pan-genomes, although the non-clinical group carried a larger accessory gene pool. We identified a complete hlyCABD-like RTX operon in 10 trh-positive isolates with T3SS2-associated virulence backgrounds. These RTX-positive isolates were distributed across seven sequence types and three of five phylogenetic groups. This distribution was lineage-restricted but non-clonal. In the representative hybrid-assembled genome, the operon occurred within a mosaic genomic region containing additional virulence- and mobility-associated genes, indicating a composite pathogenicity island-like element. In the tested subset, RTX-positive isolates showed significantly greater hemolytic activity than RTX-negative trh-positive isolates. This significant difference was consistently observed in both plate-based and liquid assays, and within the RTX-positive subset, hlyA expression correlated with hemolytic activity, whereas the trh gene and the tlh (thermolabile hemolysin) gene did not. A complete hlyCABD-like RTX operon therefore identifies a hemolysis-associated subset of trh-positive V. parahaemolyticus and supports its further evaluation as an additional target for food safety surveillance.

Vibrio parahaemolyticus↗

PubMed2003

NaCl-sensitive mutant of Staphylococcus aureus has a Tn917-lacZ insertion in its ars operon.

Staphylococcus aureus is a Gram-positive bacterium that is extremely halotolerant. To investigate the molecular mechanisms by which S. aureus can cope with osmotic stress, Tn917-lacZ-induced NaCl-sensitive mutants were isolated. An NaCl-sensitive mutant showed a longer lag period, slower growth rate, and lower final culture turbidity than the parent strain in liquid medium containing 1.5 M NaCl. Electron microscopic observation of the NaCl-sensitive mutant under NaCl stress conditions revealed large, pseudo-multicellular cells. Addition of exogenous osmoprotectants, such as glycine betaine, choline, L-proline, and proline betaine, did not relieve the NaCl sensitivity of the mutant. The region flanking the transposon insertion site in the NaCl-sensitive S. aureus chromosome was sequenced. The mutated gene was 99% identical to arsR, the arsenic operon regulatory protein present on the pI258 plasmid of S. aureus. The ars operon from pI258 was subcloned into the shuttle vector pLI50 and transferred into the NaCl-sensitive mutant. The ars operon in trans restored NaCl tolerance in the mutant, suggesting that NaCl sensitivity is due to the mutation in arsR.

Bacterial Proteins↗

PubMed1990

Secretion of the Bordetella pertussis adenylate cyclase from Escherichia coli containing the hemolysin operon.

The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1983

Use of uxu-lac fusion strains to study the regulation of the uxuAB operon in Escherichia coli K12.

In the hexuronate system of Escherichia coli, the uxuAB operon is negatively controlled by the UxuR and ExuR repressors. A Mudlac phage was used, by the method of Casadaban, to construct strains where a truncated lacZ was fused to uxuB. From these fusion strains, deletions of various lengths extending into the uxu region were created in vivo by selecting temperature-insensitive mutants. Operator-constitutive mutations were also selected for in such strains and their preliminary analysis is presented. Large amounts of either ExuR or UxuR repressor caused a strong decrease of the constitutive expression of the uxuAB operon in the operator mutants. The implications of this repression for the presence of one or two operator sites in the uxuAB operon are discussed.

Chromosome Deletion↗

PubMed1993

Rhodobacter capsulatus puc operon: promoter location, transcript sizes and effects of deletions on photosynthetic growth.

Gene deletions of the puc operon of Rhodobacter capsulatus showed that the pucC and pucE genes, but not pucD, were required for formation of wild-type levels of the LHII complex. Deletion of pucC or pucE also impaired photosynthetic growth. The effects of pucC deletion were suppressed by secondary mutations that mapped outside the puc operon. Fusion of a lac'Z gene to pucE' showed that most of pucE transcription originated from upstream of pucB. RNA blot analysis revealed a 2.4 kb transcript that hybridized to probes specific for the pucBA, pucC and pucDE regions, indicating that some puc operon messages extend from just before the pucB gene to just after the pucE gene.

Bacterial Proteins↗

PubMed2001

N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli.

The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

ATP-Binding Cassette Transporters↗