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At least 55 records · Page 3Linked to original sources
PubMed1996

Organisation of the tmb catabolic operons of Pseudomonas putida TMB and evolutionary relationship with the xyl operons of the TOL plasmid pWW0.

In Pseudomonas putida (Pp) TMB the genes involved in the catabolism of methyl-substituted aromatic hydrocarbons 1,2,4-trimethylbenzene, m- and p-xylene (tmb operon), are functionally and genetically homologous to the xyl genes of the plasmid pWW0, but are chromosomally encoded. We have analysed by cloning. Southern blotting and sequencing of selected regions the organisation of the tmb cluster. This analysis shows that the structural and regulatory genes of the tmb and xyl systems exhibit a high degree of homology and are similarly organised in operons. However the operons are differently arranged on the Pp TMB chromosome and on the pWW0 plasmid. Comparison of the two systems suggests that the operon arrangement found in pWW0 may have originated from that found in Pp TMB via cointegration mediated by replicative transposition or by intermolecular recombination between two copies of the insertion element IS1246.

Base Sequence↗
PubMed1974

Multiple regulation of the galactose operon-genetic evidence for a distinct site in the galactose operon that responds to capR gene regulation in Escherichia coli K-12.

Previous results demonstrated that the capR (lon) locus, which is not linked to the gal operon, independently controls the synthesis of the gal operon enzymes and gal mRNA, i.e., galO(+)capR9 strains are derepressed 4- to 6-fold as compared to galO(+)capR(+) strains. A mutation has been isolated and localized in the galactose operator region that defines a new and distinct site of control. Mutation in this site, designated galO(capR+), causes a 4-fold increase in the galactose enzymes, galactokinase (EC 2.7.1.6) and UDP-galactose-4-epimerase (EC 5.1.3.2), in a capR(+) background. These mutants exhibit a reduced response to regulation by the unlinked regulator gene capR (lon). However, the galO(capR+) mutants are still subject to control by the galR repressor, since they can be further derepressed by growth in the presence of D-fucose. They also synthesize more galactokinase when grown in glycerol as compared to glucose. Thus there are now at least three, and probably four, sites for control of mRNA synthesis in the operator-promoter regions of the gal operon, making it one of the most complex control systems to date for a single operon in bacteria. The complexity is sufficient to accommodate models for differentiation in higher organisms that require more than one "switch" to control a single group of genes.

Alleles↗
PubMed1990

The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis.

We have cloned and characterized the mtr operon of Bacillus subtilis. This operon encodes a presumed RNA-binding regulatory protein that is required for attenuation control of the trp operon. We have shown that the mtr operon consists of two structural genes, mtrA and mtrB, predicted to encode 22-kDa and 8-kDa polypeptides, respectively. MtrB shows homology with RegA, an RNA-binding regulatory protein of bacteriophage T4. The lesions in several mtr mutants were localized to mtrB or the putative mtr promoter. Several mtrB alleles were dominant to mtr+, suggesting that the regulatory factor is a multimeric protein. The in vivo action of the mtrA and mtrB gene products was analyzed in an E. coli strain containing a trpE-lacZ gene fusion under control of the B. subtilis trp promoter/attenuator region. Both MtrA and MtrB were necessary for regulation of beta-galactosidase production.

Amino Acid Sequence↗
PubMed1999

The Na(+)-F(1)F(0)-ATPase operon from Acetobacterium woodii. Operon structure and presence of multiple copies of atpE which encode proteolipids of 8- and 18-kda.

Eight genes (atpI, atpB, atpE(1), atpE(2), atpE(3), atpF, atpH, and atpA) upstream of and contiguous with the previously described genes atpG, atpD, and atpC were cloned from chromosomal DNA of Acetobacterium woodii. Northern blot analysis revealed that the eleven atp genes are transcribed as a polycistronic message. The atp operon encodes the Na(+)-F(1)F(0)-ATPase of A. woodii, as evident from a comparison of the biochemically derived N termini of the subunits with the amino acid sequences deduced from the DNA sequences. The molecular analysis revealed that all of the F(1)F(0)-encoding genes from Escherichia coli have homologs in the Na(+)-F(1)F(0)-ATPase operon from A. woodii, despite the fact that only six subunits were found in previous preparations of the enzyme from A. woodii. These results unequivocally prove that the Na(+)-ATPase from A. woodii is an enzyme of the F(1)F(0) class. Most interestingly, the gene encoding the proteolipid underwent quadruplication. Two gene copies (atpE(2) and atpE(3)) encode identical 8-kDa proteolipids. Two additional gene copies were fused to form the atpE(1) gene. Heterologous expression experiments as well as immunolabeling studies with native membranes revealed that atpE(1) encodes a duplicated 18-kDa proteolipid. This is the first demonstration of multiplication and fusion of proteolipid-encoding genes in F(1)F(0)-ATPase operons. Furthermore, AtpE(1) is the first duplicated proteolipid ever found to be encoded by an F(1)F(0)-ATPase operon.

Amino Acid Sequence↗
PubMed2004

Interchangeability and distinct properties of bacterial Fe-S cluster assembly systems: functional replacement of the isc and suf operons in Escherichia coli with the nifSU-like operon from Helicobacter pylori.

The assembly of iron-sulfur (Fe-S) clusters, a key step in the post-translational maturation of Fe-S proteins, is mediated by a complex apparatus. In E. coli, this process involves two independent systems called ISC and SUF encoded by the iscSUA-hscBA-fdx gene cluster and sufABCDSE operon, respectively. Another system, termed NIF (nifSU), is required for the maturation of nitrogenase in nitrogen-fixing bacteria. We have developed a novel genetic system to gain further insight into these multi-component systems, and to determine how ISC, SUF and NIF might differ in their roles in Fe-S assembly. We have constructed an E. coli mutant lacking both the isc and suf operons, and this strain can only survive in the presence of a complementing plasmid. Using the plasmid replacement technique, we examined the isc and suf operons, and identified the genes essential for the function. Additionally, we have found that nifSU-like genes cloned from Helicobacter pylori are functionally exchangeable with the isc and suf operons. Thus, the NIF-like system participates in the maturation of a wide variety of Fe-S proteins. An increased ability of NIF to complement isc and suf loss was seen under anaerobic conditions. This may explain why the NIF system is only found in a limited number of bacterial species, and most other organisms prefer the ISC and/or SUF systems. While the differences between ISC and SUF were small with respect to the complementing activity, the SUF system appears to be more advantageous for bacterial growth in the presence of hydrogen peroxide.

Bacterial Proteins↗
PubMed1987

Analysis of the terminator region after the deoCABD operon of Escherichia coli K-12 using a new class of single copy number operon-fusion vectors.

We describe the construction of low copy number operon-fusion vectors, and use one of these vectors for the cloning and transcriptional analysis of the terminator region after the deo operon of Escherichia coli K-12. The new vectors are miniderivatives of plasmid R1 containing the parB stability locus of this plasmid and the lac genes as a selectable marker. Since the copy number of the vectors is only one per genome-equivalent at temperatures below 37 degrees C this system is ideally suited for isolation and characterization of transcriptional and translational signals from E. coli. Our results show that a very strong terminator (deot), which resembles Rho-independent terminators, is located 60 bp downstream from the fourth structural gene of the deo operon. This confirms that deoD is the last gene in the operon. In addition, we have identified a new promoter just after the deot terminator and a short DNA sequence that is able to reduce lacZ expression by 85% when inserted between the deoP2 promoter and the lac genes.

Base Sequence↗
PubMed2001

Pseudomonas putida CE2010 can degrade biphenyl by a mosaic pathway encoded by the tod operon and cmtE, which are identical to those of P. putida F1 except for a single base difference in the operator-promoter region of the cmt operon.

Psudomonas putida CE2010 can assimilate biphenyl despite its high similarity to P. putida F1. Biphenyl degradation in strain CE2010 was achieved using a mosaic of pathways consisting of the cmt and tod operons. CmtE hydrolysed 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage product of 2,3-dihydroxybiphenyl. This enzyme was expressed differently in strains CE2010 and F1. A cmtE disruption mutant, a tod operon disruption mutant and a cmt operon disruption mutant were unable to utilize biphenyl. The introduction of the cmtE gene enabled the cmt operon disruption mutant to grow on biphenyl. A single base difference was found in the cmt promoter-operator region in strain CE2010, compared with that of strain F1. CymR protein was purified from Escherichia coli and binding assays were performed, the results of which suggested that the protein bound less strongly to the CE2010 operator sequence than to the F1 operator sequence. Exchanging the F1 promoter-operator fragment into strain CE2010 resulted in a loss of biphenyl degradation capacity. These results indicate that cmtE is not effectively repressed by CymR in strain CE2010, leading to low constitutive expression and, therefore, low growth on biphenyl.

Bacterial Proteins↗
PubMed2001

The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons.

Transcriptional activities of closely spaced divergent promoters are affected by the accumulation of local negative superhelicity in the region between transcribing RNA polymerase molecules (transcriptional coupling). The effect of this transcription-induced DNA supercoiling on these promoters depends on their intrinsic properties. As the global superhelical density of the chromosome is controlled by the energy charge of the cell, which is affected by environmental stresses and transitions from one growth state to another, the transcriptional coupling that occurs between divergently transcribed promoters is likely to serve a physiological purpose. Here, we suggest that transcriptional coupling between the divergent promoters of the ilvYC operon of Escherichia coli serves to co-ordinate the expression of this operon with other operons of the ilv regulon during metabolic adjustments associated with growth state transitions. As DNA supercoiling-dependent transcriptional coupling between the promoters of other divergently transcribed operons is investigated, additional global gene regulatory mechanisms and physiological roles are sure to emerge.

Bacterial Proteins↗
PubMed1988

Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon.

An 8-kilobase HindIII fragment carrying the histidase gene (hutH) and its regulatory region (hutP), from the Bacillus subtilis histidine utilization (hut) operon, was cloned in the temperate bacteriophage phi 105. Histidine utilization was restored in a hutH1 mutant by the specialized transducing phage (phi 105hutH11). The histidase gene in phi 105hutH11 was inducible and was shown to be under catabolite repression. The nucleotide sequence of 3,932 base pairs including the hutH and hutP loci revealed three open reading frames (ORFs). The molecular weights of ORF1 and ORF2 proteins were calculated to be 16,576 (151 amino acid residues) and 55,675 (508 amino acid residues), respectively. Reverse transcriptase mapping experiments showed that the putative promoter for the hut operon could be recognized by RNA polymerase sigma 43. The transcript starts at an adenosine residue 32 base pairs upstream from the initiation codon of ORF1. hutH+-transforming activity was found in ORF2, indicating that ORF2 encoded the histidase. A hutP1 mutation was determined as a substitution of an amino acid in ORF1. By using a specialized transducing phage containing the wild-type ORF1 gene, it was demonstrated that the presence of ORF1 protein in trans was absolutely required for the induction of the hut operon in a hutP1 mutant. These data strongly suggested that ORF1 encodes a positive regulator of the hut operon.

Amino Acid Sequence↗
PubMed1993

Identification and transcriptional analysis of the Escherichia coli htrE operon which is homologous to pap and related pilin operons.

We have characterized a new Escherichia coli operon consisting of two genes, ecpD and htrE. The ecpD gene encodes a 27-kDa protein which is 40% identical at the amino acid level to the pilin chaperone PapD family of proteins. Immediately downstream of the ecpD gene is the htrE gene. The htrE gene encodes a polypeptide of 95 kDa which is processed to a 92-kDa mature species. The HtrE protein is 38% identical to the type II pilin porin protein PapC. The ecpD htrE operon is located at 3.3 min on the genetic map, corresponding to the region from kbp 153 to 157 of the E. coli physical map. The htrE gene was identified on the basis of a Tn5 insertion mutation which resulted in a temperature-sensitive growth phenotype above 43.5 degrees C. The transcription of this operon is induced with a temperature shift from 22 to 37 or 42 degrees C but not to higher temperatures, e.g., 50 degrees C. Consistent with this result, the temperature-induced transcription was shown to be independent of the rpoH gene product (sigma 32). The transcription of this operon was further shown to require functional integration host factor protein, since himA or himD mutant bacteria possessed lower levels of ecpD htrE transcripts. Among the three transcriptional start sites discovered, one, defined by the P2 promoter, was found to be under the positive regulation of the katF (rpoS) gene, which encodes a putative sigma factor required for the transcription of many growth phase-regulated genes.

Adhesins, Escherichia coli↗
PubMed1994

Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization.

The synthesis of an extracellular ribbon of cellulose in the bacterium Acetobacter xylinum takes place from linearly arranged, membrane-localized, cellulose-synthesizing and extrusion complexes that direct the coupled steps of polymerization and crystallization. To identify the different components involved in this process, we isolated an Acetobacter cellulose-synthesizing (acs) operon from this bacterium. Analysis of DNA sequence shows the presence of three genes in the acs operon, in which the first gene (acsAB) codes for a polypeptide with a molecular mass of 168 kDa, which was identified as the cellulose synthase. A single base change in the previously reported DNA sequence of this gene, resulting in a frameshift and synthesis of a larger protein, is described in the present paper, along with the sequences of the other two genes (acsC and acsD). The requirement of the acs operon genes for cellulose production was determined using site-determined TnphoA/Kanr GenBlock insertion mutants. Mutant analysis showed that while the acsAB and acsC genes were essential for cellulose production in vivo, the acsD mutant produced reduced amounts of two cellulose allomorphs (cellulose I and cellulose II), suggesting that the acsD gene is involved in cellulose crystallization. The role of the acs operon genes in determining the linear array of intramembranous particles, which are believed to be sites of cellulose synthesis, was investigated for the different mutants; however, this arrangement was observed only in cells that actively produced cellulose microfibrils, suggesting that it may be influenced by the crystallization of the nascent glucan chains.

Amino Acid Sequence↗
PubMed2006

Cloning and comparison of third beta-glucoside utilization (bglEFIA) operon with two operons of Pectobacterium carotovorum subsp. carotovorum LY34.

A third bgl operon containing bglE, bglF, bglI, and bglA was isolated from Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34). The sequences of BglE, BglF, and Bgll were similar to those of the phosphotransferase system (PTS) components IIB, IIC, and IIA respectively. BglF contains important residues for the phosphotransferase system. The amino acid sequence of BglA showed high similarity to various 6-phospho-beta-glucosidases and to a member of glycosyl hydrolase family 1. Sequence and structural analysis also revealed that these four genes were organized in a putative operon that differed from two operons previously isolated from Pcc LY34, bglTPB (accession no. AY542524) and ascGFB (accession no. AY622309). The transcription regulator for this operon was not found, and the EII complexes for PTS were encoded separately by three genes (bglE, bglF, and bglI). The BglA enzyme had a molecular weight estimated to be 57,350 Da by SDS-PAGE. The purified beta-glucosidase hydrolyzed salicin, arbutin, rhoNPG, rhoNPbetaG6P, and MUG, exhibited maximal activity at pH 7.0 and 40 degrees C, and displayed enhanced activity in the presence of Mg2+ and Ca2+. Two glutamate residues (Glu178 and Glu378) were found to be essential for enzyme activity.

Amino Acid Sequence↗
PubMed1984

Control of expression of the Tn10-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the tet operon regulatory region.

The promoter and operator sequences of the Tn10-encoded tetracycline resistance operon are determined in vitro by transcription studies of purified DNA restriction fragments, protection of guanosine from methylation by dimethylsulphate, and DNase I footprinting employing the purified TET repressor protein. In vitro transcription reveals three promoters with overlapping consensus sequences. Two of them, designated PR1 and PR2, are directed towards the tet repressor gene and the third, called PA, initiates transcription of the tet resistance gene. All three promoters are regulated simultaneously by the TET repressor protein, as demonstrated by in vitro transcription. Tetracycline functions as an inducer in these experiments. Two palindromic operator sequences in the tet operon control region, called O1 and O2, are occupied simultaneously by the TET repressor. Four guanosine residues in symmetric positions close to the centre of the palindromic operator sequences are protected from methylation in the repressor-operator complex. However, only one guanosine residue exhibits an enhanced reaction with dimethylsulphate under these conditions. Footprinting experiments reveal protection of phosphodiester bonds against DNase I slightly further than the palindromic sequence arrangement. Several phosphodiester bonds between the two operators are accessible for cleavage by DNase I in the repressor-operator complex. Two phosphodiester bonds within each operator sequence are cleaved by DNase I. This feature shows a clear assymmetry with the two inside cleavage positions of O1 and O2 being much less accessible for DNase I as compared to the two outside positions. A molecular mechanism of regulation of the Tn10-encoded tetracycline resistance operon is presented based on these and previous results.

Base Sequence↗
PubMed1998

Sequence of the putative alanine racemase operon in Staphylococcus aureus: insertional interruption of this operon reduces D-alanine substitution of lipoteichoic acid and autolysis.

A gene cluster comprising the alanine racemase gene alr was identified 5' to the sigB operon in Staphylococcus aureus. It is flanked upstream by four ORFs of which one shows similarity to the dpj gene of Escherichia coli, and downstream by two ORFs of which the last shows similarity to the E. coli pemK gene. Preliminary data suggest that the seven ORFs orf1-orf2-orf3-dpj-alr-orf6-pemK may form an operon. Disruption of the proposed operon by insertional mutagenesis leads to a drastic loss in the d-alanine (d-Ala) substitution of lipoteichoic acid and to delayed autolysis, without affecting the d-Ala substitution of the wall teichoic acid.

Alanine↗
PubMed1990

Post-transcriptional control in the polycistronic operon environment: studies of the atp operon of Escherichia coli.

Post-transcriptional control mechanisms assume special significance in polycistronic operons. Differential gene expression in the atp operon of Escherichia coli is primarily attributable to translational control and, to a lesser extent, to control of mRNA stability. At the same time, the polycistronic environment influences, to varying degrees, the relative importance of the different types of post-transcriptional control. The present article briefly reviews more recent results obtained through studies of the atp operon. Investigations of the pathway and kinetics of mRNA decay have yielded new information about the role of degradative mechanisms in the overall scheme of control. Moreover, translational coupling has been shown to feature as a major form of interaction between the atp genes. The relevance of these and other data is discussed in the wider context of the post-transcriptional control mechanisms generally available to E. coli.

Escherichia coli↗
PubMed1996

The molybdenum formylmethanofuran dehydrogenase operon and the tungsten formylmethanofuran dehydrogenase operon from Methanobacterium thermoautotrophicum. Structures and transcriptional regulation.

Methanobacterium thermoautotrophicum contains a tungsten formylmethanofuran dehydrogenase (FwdABCD) and a molybdenum formylmethanofuran dehydrogenase (FmdABC). The fwdHFGDACB operon encoding the tungsten enzyme has recently been characterized. We report here on the structure and expression of the gene cluster encoding the molybdenum enzyme. This gene cluster is composed of three open reading frames (fmdECB). The fmdB gene was found to encode the molybdopterin-dinucleotide-binding subunit harboring the enzyme's active site; FmdB is thus functionally equivalent to FwdB. fmdC encodes a protein with sequence similarity to FwdC in its N-terminal part and with sequence similarity to FwdD in its C-terminal part; FmdC is thus functionally equivalent to FwdC and FwdD. Interestingly, the fmd operon lacks a gene fmdA encoding the subunit FmdA of the molybdenum enzyme. FmdA has the same apparent molecular mass and the same N-terminal amino acid sequence as FwdA and only one DNA sequence encoding for this N-terminal amino acid sequence was found in the M. thermoautotrophicum genome. It is therefore proposed that FmdA and FwdA are encoded by the same gene namely fwdA in the fwd operon. In agreement with this proposal is the finding that fwdA is expressed constitutively: northern-blot analysis of RNA from tungstate- and molybdate-grown cells of M. thermo-autotrophicum revealed that the fwdHFGDACB gene cluster is transcribed in the presence of either molybdate or tungstate in the growth medium whereas the fmdECB gene cluster was only transcribed when molybdate was present.

Aldehyde Oxidoreductases↗
PubMed1996

Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression.

The region upstream of the multiple antibiotic resistance efflux operon mexA-mexB-oprM in Pseudomonas aeruginosa was sequenced, and a gene, mexR, was identified. The predicted MexR product contains 147 amino acids with a molecular mass of 16,964 Da, which is consistent with the observed size of the overexpressed mexR gene product. MexR was homologous to MarR, the repressor of MarA-dependent multidrug resistance in Escherichia coli, and other repressors of the MarR family. A mexR knockout mutant showed a twofold increase in expression of both plasmid-borne and chromosomal mexA-reporter gene fusions compared with the MexR+ parent strain, indicating that the mexR gene product negatively regulates expression of the mexA-mexB-oprM operon. Furthermore, the cloned mexR gene product reduced expression of a plasmid-borne mexA-lacZ fusion in E. coli, indicating that MexR represses mexA-mexB-oprM expression directly. Consistent with the increased expression of the efflux operon in the mexR mutant, the mutant showed an increase (relative to its MexR+ parent) in resistance to several antimicrobial agents. Expression of a mexR-lacZ fusion increased threefold in a mexR knockout mutant, indicating that mexR is negatively autoregulated. OCR1, a nalB multidrug-resistant mutant which overproduces OprM, exhibited a greater than sevenfold increase in expression of a chromosomal mexA-phoA fusion compared with its parent. Introduction of a mexR knockout mutation in strain OCR1 eliminated this increase in efflux gene expression and, as expected, increased the susceptibility of the strain to a variety of antibiotics. The nucleotide sequences of the mexR genes of OCR1 and its parental strain revealed a single base substitution in the former which would cause a predicted substitution of Trp for Arg at position 69 of its mexR product. These data suggest that MexR possesses both repressor and activator function in vivo, the activator form being favored in nalB multidrug-resistant strains.

Alkaline Phosphatase↗
PubMed1994

Genetic map of the Actinobacillus pleuropneumoniae RTX-toxin (Apx) operons: characterization of the ApxIII operons.

Actinobacillus pleuropneumoniae RTX-toxin III (ApxIII) is implicated as an important virulence factor of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia. Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized. The toxin appeared to be a member of the RTX-toxin family, as are the other two secreted toxins of A. pleuropneumoniae, i.e., ApxI and ApxII. In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8. This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively. Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB. We examined the presence of apxIIICABD sequences in the 12 serotypes of A. pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII. Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences. Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes. The differences were located between the glycine-rich repeats and the secretion signal. The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A. pleuropneumoniae. We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the toxins in pathogenesis.

Actinobacillus pleuropneumoniae↗