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At least 91 records · Page 5Linked to original sources
PubMed2005

Gene structure and transcriptional organization of the dnaK operon of Bifidobacterium breve UCC 2003 and application of the operon in bifidobacterial tracing.

The incorporation and delivery of bifidobacterial strains as probiotic components in many food preparations expose these microorganisms to a multitude of environmental insults, including heat and osmotic stresses. We characterized the dnaK gene region of Bifidobacterium breve UCC 2003. Sequence analysis of the dnaK locus revealed four genes with the organization dnaK-grpE-dnaJ-ORF1, whose deduced protein products display significant similarity to corresponding chaperones found in other bacteria. Northern hybridization and real-time LightCycler PCR analysis revealed that the transcription of the dnaK operon was strongly induced by osmotic shock but was not induced significantly by heat stress. A 4.4-kb polycistronic mRNA, which represented the transcript of the complete dnaK gene region, was detected. Many other small transcripts, which were assumed to have resulted from intensive processing or degradation of this polycistronic mRNA, were identified. The transcription start site of the dnaK operon was determined by primer extension. Phylogenetic analysis of the available bifidobacterial grpE and dnaK genes suggested that the evolutionary development of these genes has been similar. The phylogeny derived from the various bifidobacterial grpE and dnaK sequences is consistent with that derived from 16S rRNA. The use of these genes in bifidobacterial species as an alternative or complement to the 16S rRNA gene marker provides sequence signatures that allow a high level of discrimination between closely related species of this genus.

Adenosine Triphosphatases↗
PubMed2006

Identification of a nisI promoter within the nisABCTIP operon that may enable establishment of nisin immunity prior to induction of the operon via signal transduction.

Certain strains of Lactococcus lactis produce the broad-spectrum bacteriocin nisin, which belongs to the lantibiotic class of antimicrobial peptides. The genes encoding nisin are organized in three contiguous operons: nisABTCIP, encoding production and immunity (nisI); nisRK, encoding regulation; and nisFEG, also involved in immunity. Transcription of nisABTCIP and nisFEG requires autoinduction by external nisin via signal transducing by NisRK. This organization poses the intriguing question of how sufficient immunity (NisI) can be expressed when the nisin cluster enters a new cell, before it encounters external nisin. In this study, Northern analysis in both Lactococcus and Enterococcus backgrounds revealed that nisI mRNA was present under conditions when no nisA transcription was occurring, suggesting an internal promoter within the operon. The nisA transcript was significantly more stable than nisI, further substantiating this. Reverse transcriptase PCR analysis revealed that the transcription initiated just upstream from nisI. Fusing this region to a lacZ gene in a promoter probe vector demonstrated that a promoter was present. The transcription start site (TSS) of the nisI promoter was mapped at bp 123 upstream of the nisI translation start codon. Ordered 5' deletions revealed that transcription activation depended on sequences located up to bp -234 from the TSS. The presence of poly(A) tracts and computerized predictions for this region suggested that a high degree of curvature may be required for transcription initiation. The existence of this nisI promoter is likely an evolutionary adaptation of the nisin gene cluster to enable its successful establishment in other cells following horizontal transfer.

Bacterial Proteins↗
PubMed1993

Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein?

The regulon controlled by the leucine-responsive regulatory protein (Lrp) of Escherichia coli consists of over 40 genes and proteins whose expression is regulated, either positively or negatively, by Lrp. The gltBDF operon, encoding glutamate synthase, was originally identified as a member of the Lrp regulon through a two-dimensional electrophoretic analysis of polypeptides from isogenic strains containing or lacking a functional Lrp protein. We have now demonstrated that Lrp regulates the transcription of gltBDF::lacZ operon fusions. Relative to expression in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium, gltBDF::lacZ expression in an lrp+ strain is repressed 2.2-fold in the presence of 10 mM exogenous leucine and 16-fold in Luria broth. Repression of gltBDF::lacZ expression by leucine or Luria broth is not seen for an isogenic strain containing a Tn10 insertion in lrp, and expression of gltBDF::lacZ is 44-fold lower than in the lrp+ strain when both are grown in glucose minimal MOPS medium. Lrp binds specifically to DNA fragments containing the gltBDF promoter region. Saturating levels of leucine do not abolish binding of Lrp upstream of gltBDF but merely increase its apparent dissociation constant from 2.0 to 6.9 nM. Electrophoretic analysis of the Lrp regulon established that target proteins differ greatly in the degree to which the effect of Lrp on their expression is antagonized by leucine. On the basis of our present results, we present a model for positive regulation of target genes by Lrp. Insensitivity to leucine would be expected when the effective intracellular concentration of Lrp is high relative to the affinity of Lrp binding sites required for transcription of the target gene. At lower concentrations of Lrp, transcription of the target gene should be sensitive to leucine. This model suggests that regulation of the concentration of active Lrp is critical to control of the Lrp regulon.

Bacterial Proteins↗
PubMed1985

Characterization of the ileS-lsp operon in Escherichia coli. Identification of an open reading frame upstream of the ileS gene and potential promoter(s) for the ileS-lsp operon.

The preceding paper presents evidence for the co-transcriptional expression of the ileS and lsp genes in Escherichia coli. To identify the promoter for the ileS-lsp operon, we have determined the nucleotide sequence of an 1.8-kilobase DNA fragment between the rpsT and IleS genes. The sequence data have revealed an open reading frame, designated gene X, which encodes a polypeptide with 312 amino acid residues. Both in vivo and in vitro expressions of the x gene result in the synthesis of a soluble protein with an apparent Mr of 35,000. The x gene is transcribed in the same direction as that of the ileS-lsp operon and opposite to that of the upstream adjacent rpsT gene. No transcription termination sequence can be discerned in the intercistronic region between the x and ileS genes. DNase I footprinting experiment revealed a RNA polymerase binding site at 170-151 base pairs upstream of the x gene.

Bacterial Proteins↗
PubMed1981

Operon fusions in the nitrate reductase operon and study of the control gene nir R in Escherichia coli.

Strains carrying operon fusions between the promotor of the chl I gene and the lac structural genes were constructed. From these strains in which the expression of the lac genes is under the control of both nitrate and oxygen, spontaneous regulatory mutants were selected: (i) mutants which synthesize beta-galactosidase constitutively in anaerobiosis; (ii) mutants in which beta-galactosidase synthesis is no longer repressed by oxygen. Introduction of the nir R mutated allele into strains carrying these fusions resulted in the total loss of beta-galactosidase synthesis, confirming that nir R is a regulatory gene controlling the expression of the biosynthesis of the nitrate reductase.

Anaerobiosis↗
PubMed1978

Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system.

The synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes. A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate. Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants. Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.

Aldehyde-Lyases↗
PubMed1986

The serC-aro A operon of Escherichia coli. A mixed function operon encoding enzymes from two different amino acid biosynthetic pathways.

Sub-cloning experiments aimed at precisely locating the E. coli aroA gene, which encodes the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase, showed that in certain constructions, which remain capable of complementing an auxotrophic aroA mutation, expression of aroA is reduced. DNA sequence analysis revealed that a sequence approx. 1200 base pairs (bp) upstream of aroA is necessary for its expression. An open reading frame was identified in this region which encodes a protein of 362 amino acids with a calculated Mr of 39,834 and which ends 70 bp before the start of the aroA coding sequence. This gene has been identified as serC, the structural gene for 3-phosphoserine aminotransferase, an enzyme of the serine biosynthetic pathway. Both genes are expressed as a polycistronic message which is transcribed from a promotor located 58 bp upstream of serC. Evidence is presented which confirms that the aroA and serC genes constitute an operon which has the novel feature of encoding enzymes from two different amino acid biosynthetic pathways.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1984

Mercuric ion-resistance operons of plasmid R100 and transposon Tn501: the beginning of the operon including the regulatory region and the first two structural genes.

The mercuric ion-resistance operons of plasmid R100 (originally from Shigella) and transposon Tn501 (originally from a plasmid isolated in Pseudomonas) have been compared by DNA sequence analysis. The sequences for the first 1340 base pairs of Tn501 are given with the best alignment with the comparable 1319 base pairs of R100. The homology between the two sequences starts at base 58 after the end of the insertion sequence IS-1 of R100. The sequences include the transcriptional regulatory region, and the homology is particularly strong in regions just upstream from potential transcriptional initiation sites. The trans-acting regulatory gene merR consists of 180 base pairs in both cases and codes for a highly basic polypeptide of 60 amino acids, which is also rich in serine. The Tn501 and R100 merR genes differ in 25 of the 180 base positions, and the resulting polypeptides differ in seven amino acids. The regulatory region before the major transcription initiation site contains potential -35 and -10 sequences and dyad symmetrical sequences, which may be the merR binding sites for transcriptional regulation. The first structural gene, merT, encodes a highly hydrophobic polypeptide of 116 amino acids. The R100 and Tn501 merT genes differ in 17% of their positions, leading to 14 (12%) amino acid changes. This region had previously been shown to encode a protein governing membrane transport of mercuric ions. The second structural gene, merC, would give a 91 amino acid polypeptide with a hydrophobic amino-terminal segment. The Tn501 and R100 merC genes differ at 37 base positions, leading to 10 amino acid changes.

Amino Acid Sequence↗
PubMed1986

Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIIC in spo mutants of Bacillus subtilis: a branched pathway of expression of sporulation operons.

The sporulation gene spoIIIC from Bacillus subtilis was fused to the lacZ gene from Escherichia coli, so that the transcription of the lacZ gene was under the control of the spoIIIC promoter. Production of beta-galactosidase, under conditions of sporulation, was then used as an indicator to study the expression of spoIIIC in relation to other sporulation loci. Expression of spoIIIC, which occurred only in the mother cell compartment, was prevented by mutations in all of the stage 0 and stage II loci, and also by spoIIID, spoIVA and spoIVB mutations. By contrast, the last three operons are not needed for expression of spoVA, which has previously been shown to be spore-specific. In consequence, a branched pathway of gene expression is proposed. One branch leads to expression of spoVA within the spore compartment, the other to expression of spoIIIC in the mother cell.

Bacillus subtilis↗
PubMed1995

Molecular analysis of the cso operon of enterotoxigenic Escherichia coli reveals that CsoA is the adhesin of CS1 fimbriae and that the accessory genes are interchangeable with those of the cfa operon.

A deletion mutation in csoA, the gene encoding the structural subunit protein of CS1 fimbriae of enterotoxigenic Escherichia coli of serotype O6:K15:H16 or H-, was constructed in the subcloned CS1 genetic determinant. The mutation resulted in the abolition of CS1 fimbrial adhesiveness. Complementation, in trans, involving the determinant with the csoA deletion mutation and the gene encoding the structural subunit protein, CsoA, expressed from compatible plasmids, restored the expression and adhesive ability of CS1 fimbriae. In addition, trans-complementation was achieved between the cso determinant with the aforementioned deletion mutation and the cfaB gene encoding the structural subunit protein (CfaB) of CFA/I fimbriae, resulting in the expression of CFA/I fimbriae. The observation that heterologous assembly was possible between these two fimbrial systems, together with the knowledge that the adhesin of CFA/I fimbriae is the structural subunit, was exploited to investigate whether CsoA had adhering properties. A deletion mutation in cfaB was created in the CFA/I fimbrial determinant. Complementation of this mutation with csoA in trans resulted in expression of the CsoA antigen on the bacterial cell surface and restoration of bacterial adherence. As no minor subunits act as the adhesin in CFA/I fimbriae, adhesion was mediated by CsoA. Nucleotide sequencing of the DNA region downstream from csoA confirmed the absence of genes encoding minor subunits which might act as the adhesin. Two open reading frames were revealed which encoded proteins sharing considerable homology with proteins encoded by corresponding ORFs in the CFA/I fimbrial operon. These proteins underlie the functional similarities between the CS1 and CFA/I fimbrial systems, allowing heterologous expression of their respective subunits.

Adhesins, Bacterial↗
PubMed1990

The Chlamydia trachomatis hyp operon is homologous to the groE stress response operon of Escherichia coli.

The Chlamydia trachomatis serovar A hyp operon was cloned, sequenced, and expressed in Escherichia coli. Two cotranscribed open reading frames, hypA and hypB, encoded polypeptides of 17 and 57 kilodaltons, respectively. The deduced amino acid sequences of serovar A HypA and HypB proteins were (respectively) 85 and 94% identical with HypA and HypB proteins of Chlamydia psittaci GPIC, and HypB was greater than 50% identical to 60-kilodalton stress response proteins from other procaryotes and eucaryotes. The sequence should be useful in defining the antigenic structure of the Chlamydia trachomatis HypB protein, a necessary step toward understanding the relationship between the immune response to this protein and the pathogenesis of human chlamydial diseases.

Amino Acid Sequence↗
PubMed2006

Transcription of the Bacillus subtilis gerK operon, which encodes a spore germinant receptor, and comparison with that of operons encoding other germinant receptors.

The gerA, gerB, and gerK operons, which encode germinant receptors in spores of Bacillus subtilis, were transcribed only in sporulation, and their mRNA levels peaked initially approximately 3 h before the initiation of accumulation of the spore's dipicolinic acid. After a rapid fall, levels of these mRNAs peaked again approximately 5 h later. In one wild-type strain (PS832), gerA mRNA was the most abundant, with levels of gerB and gerK mRNAs approximately 50% of that of gerA mRNA, whereas gerB mRNA was the most abundant in another wild-type strain (PY79). The synthesis of gerK mRNA in sporulation was abolished by loss of the forespore-specific RNA polymerase sigma factor, sigma(G), and induction of sigma(G) synthesis in vegetative cells led to synthesis of gerK mRNA. SpoVT, a regulator of sigma(G)-dependent gene expression, repressed gerK expression. The gerK promoter showed sequence similarities to sigma(G)-dependent promoters, and deletion of elements of this putative promoter abolished gerK expression in sporulation.

Bacillus subtilis↗
PubMed1993

Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I.

The nucleotide sequence of the Serratia marcescens threonine operon (thrA1A2BC) was determined. Three long open reading frames were identified; these open reading frames code for aspartokinase I (AKI)-homoserine dehydrogenase I (HDI), homoserine kinase, and threonine synthase, in that order. The predicted amino acid sequences of these enzymes were similar to the amino acid sequences of the corresponding enzymes in Escherichia coli. The AKI-HDI protein is apparently a tetramer composed of monomer polypeptides that are 819 amino acids long. A deletion analysis revealed that the central and C-terminal region was responsible for threonine-resistant HDI activity, a monomeric fragment extending from the N terminus to residue 306 was responsible for threonine-resistant AKI activity, and an N-terminal portion containing 468 residues was responsible for threonine-sensitive AKI activity. The thrA(1)1A(2)1 and thrA(1)5A(2)5 mutations of threonine-excreting strains HNr21 and TLr156, which result in the loss of threonine-mediated feedback inhibition of both AKI activity and HDI activity, cause single amino acid substitutions (Gly to Asp at position 330 and Ser to Phe at position 352, respectively) in the central region of the AKI-HDI protein. The thrA1+A(2)2 mutation of strain HNr59, which results in a threonine-sensitive AKI and a threonine-resistant HDI, also causes a single amino acid substitution (Ala to Thr at position 479).

Allosteric Regulation↗
PubMed1978

[Regulation of the activity of Escherichia coli deo-operon structural genes: the mutation mapped within the operon boundaries and affecting drm and pup gene activity].

The mutant AIR38 is isolated from Escherichia coli K-12 strain deficient in thymidilate synthetase and deoxyriboaldolase (HfrH, thy, dra)--by selection for low thymine requirement on the medium containing inosine as the carbon source. Under the conditions mentioned the mutant AIR38 (thy, dra) grows at low thymine concentration (2 mkg/ml), and is uncapable to grow in the presence of thymidine (40 mkg/ml). Dra+ derivatives of the AIR38 do no catabolize inozine in the presence of thymidine as well. The mutation AIR38 is mapped within the deo-operon between drm and pup mutation markers. The levels of phosphodeoxyribomutase and purine nucleoside phosphorylase in cell extracts of AIR38 are 2.5-6-fold decreased. In transductional experiments with phage P1 and the mutant AIR38 as recipient the delayed haploidization of merozygotes dra+, AIR+/dra, AIR38, thy and the dominant expression of the sensitivity to thymidine in the presence of inosine as the carbon source are observed. It is supposed that the mutation AIR38 affects the structural gene of purine nucleoside phosphorilase by altering the mode of interaction of this enzyme with the membrane under the conditions of thymine starvation.

Chromosome Mapping↗
PubMed1991

PCR based gene engineering of the Vibrio harveyi lux operon and the Escherichia coli trp operon provides for biochemically functional native and fused gene products.

The polymerase chain reaction (PCR) was applied to clone the luxA and luxB genes from Vibrio harveyi, and the trp poL (promoter operator leader) region and the trpB and trpA genes from Escherichia coli. PCR-derived luxA/B and trpB/A genes were shown to express bacterial luciferase and tryptophan synthase respectively, when introduced into E. coli on a plasmid cloning vehicle. The trp poL was used successfully to control the expression of lac alpha, luxAB, trpB and trpA. PCR was also used to construct a functional luxAB translational fusion protein. Primers for this were designed to facilitate precise gene fusion and to provide a silent mutation within an EcoRI site in the luxB gene. Production of functional genes was verified in vitro and in vivo using polyacrylamide gel electrophoresis (PAGE) analysis of transcription-translation products and crude cell extracts, and by monitoring enzyme activity.

Base Sequence↗