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PubMed1996

A nuclear mutant of Arabidopsis with impaired stability on distinct transcripts of the plastid psbB, psbD/C, ndhH, and ndhC operons.

The high-chlorophyll fluorescence photosynthesis mutant hcf109 of Arabidopsis was characterized in detail to gain insights into the regulatory mechanism of RNA processing in higher plants. By using electron transport, chlorophyll fluorescence, and immunoblot studies, we assigned the mutational lesion to photosystems I and II and the plastid NAD(P)H dehydrogenase complex. The functional pleiotropy was reflected in RNA deficiencies. Although all nuclear-encoded photosynthetic RNAs analyzed revealed no difference in size or steady state level between mutant and wild type, the RNA patterns of the plastome-encoded psbB-psbT-psbH-petB-petD, psbD-psbC-ycf9, ndhC-ndhK-ndhJ, and ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndh D transcription units were severely disturbed. These operons encode subunits of photosystems I (psa) and II (psb), the cytochrome bGf complex (pet), the plastid NAD(P)H dehydrogenase (ndh), and the unidentified open reading frame ycf9. With the exception of the ndhC operon, the RNA deficiencies observed were specific and restricted to particular segments of the psbB, psbD/C, and ndhH operons, that is, the psbB-psbT, ycf9, and psaC regions. Run-on transcription studies with isolated chloroplasts showed that the failure of these transcripts to accumulate was due to RNA stability and not transcription. Other polycistronic transcription units analyzed were not affected by the mutation. This result indicates that the trans-regulatory factor encoded by the hcf109 gene is not a general RNA stability factor but that it specifically controls the stability of only these distinct transcripts. Because the hcf109 locus was mapped at a distance < 0.1 centimorgans from the phytochrome C gene, its molecular characterization by positional cloning is possible.

Arabidopsis↗

PubMed1999

Chromosome-determined zinc-responsible operon czr in Staphylococcus aureus strain 912.

A novel operon, czrAB (zinc-responsible genes), was identified in the chromosome of Staphylococcus aureus. The operon consists of two genes, czrA and czrB. The czrA gene, coding for an 11.5 kDa protein, was homologous to cadC, arsR of S. aureus plasmid pI258 and smtB of Synechococcus PCC7942. The czrB, coding for a 36 kDa membrane spanning protein, was homologous to the czcD gene, cobalt, zinc and the cadmium-resistant factor of Bacillus subtilis and Alcaligenes eutrophus. In the presence of zinc (0.1-10 mM), the transcription of czrAB was enhanced in a concentration-dependent manner. Other heavy metals, such as cobalt, copper, manganese and nickel showed no effect on czrAB expression. The disruptant of the czrB gene became sensitive to zinc ion (MIC, 2 mM; MBC, 10 mM), and the complementation with the plasmid recovered the resistance to zinc at the same concentration as a parental strain (MIC, 5 mM; MBC, 20 mM). The disruptant accumulated intracellular zinc up to 0.4 mg per g dry weight of the organism, while that of the parental strain was 0.25 mg per g dry weight. The findings indicated that the novel operon czrAB should play a role in the transportation of zinc across the cell membrane to maintain the proper intracellular concentration.

Amino Acid Sequence↗

PubMed2000

Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens.

The 5'-flanking region of the metB-cysK-ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C. perfringens, was analyzed. The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa-toxin (collagenase), respectively. Northern analysis revealed that the ycgJ gene was transcribed as a 4.9-kb operon together with the metB-cysK-ygaG genes and that this operon was negatively regulated by the VirR/VirS system. It is indicated that the pfoA (theta-toxin or perfringolysin O), colA, and ycgJ-metB-cysK-ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5-kb region of the chromosome, but do not form a pathogenic island.

Bacterial Proteins↗

PubMed1988

Role of the intercistronic region in post-transcriptional control of gene expression in the histidine transport operon of Salmonella typhimurium: involvement of REP sequences.

The high-affinity histidine permease of Salmonella typhimurium is encoded by a four-gene operon containing a large intercistronic region located between the first gene (hisJ) and the three distal genes (hisQ, hisM, hisP). The level of expression of hisJ is 30-fold greater than that of hisP. In order to investigate the role of the intercistronic region in intra-operonic control of gene expression, we have isolated MudII-mediated lacZ gene fusions to hisQ, hisM and hisP. We have used these fusions to isolate and analyse mutants that have altered levels of expression of the hisQ gene, the first gene downstream from the intercistronic region. The results indicate that intra-operonic regulation is due to a combination of factors including efficiency of translational initiation, mRNA degradation, and retroregulation of hisJ expression. They also suggest that the REP (Repetitive Extragenic Palindromic) sequences, which are located in the hisJ-hisQ intercistronic region, may interfere with translation of the hisQ gene and affect upstream messenger RNA stability by protecting it from 3' to 5' nuclease degradation (in agreement with data presented by Newbury et al., 1987).

Base Sequence↗

PubMed1988

Nucleotide sequence of the mannitol (mtl) operon in Escherichia coli.

The nucleotide sequence of the known portions of the mannitol operon in Escherichia coli (mtlOPAD) has been determined. Both the operator-promoter region and the intercistronic region between the mtlA and mtlD genes (encoding the mannitol-specific Enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase, respectively) show parallels with corresponding regions of the glucitol (gut) operon, but neither the mtlA nor the mtlD gene products show obvious homology with the corresponding gene products of the glucitol operon. Five potential cyclic AMP receptor protein binding sites were identified in the mtlOP region, all showing near identity with the consensus sequence. Four regions of dyad symmetry (four to seven bases in length), serving as potential repressor binding sites, overlap with the potential cyclic AMP receptor protein binding sites. Repetitive extragenic palindromic (REP) sequences, forming stem-loop structures in the intercistronic region between mtlA and mtlD and following the mtlD gene were identified. Probable terminator sequences were not found in any of these three regulatory regions. Mannitol-1-phosphate dehydrogenase exhibits two overlapping, potential NAD+ binding sites near the N-terminus of the protein. Computer techniques were used to analyse the mtlD gene and its product.

Amino Acid Sequence↗

PubMed1990

Mapping of the msDNA operon in the chromosome of Escherichia coli B.

An msDNA operon, consisting of genes for msDNA and a reverse transcriptase, is present in Escherichia coliB and absent from E. coliK12. We have found that the msDNA operon is located on a DNA fragment, longer than 15kb, that is absent from E. coliK12. Using conjugation, P1 transduction, and nucleic acid hybridization between E. coliB and E. coliK12 strains, we have located the position of the msDNA operon on the E. coliB chromosome at a site that corresponds to minute 19 on the genetic map and to position 900 on the physical map of the E. coliK12 chromosome.

Blotting, Southern↗

PubMed1990

Characterization of the virB operon of an Agrobacterium tumefaciens Ti plasmid: nucleotide sequence and protein analysis.

The virulence regulon of the Agrobacterium tumefaciens TiC58 plasmid is composed of six operons, virA, virB, virG, virC, virD and virE, which direct the transfer of T-DNA into plant cells. The 9.5 kbp virB operon is the largest of these operons and its entire nucleotide sequence was determined and found to contain eleven open reading frames (ORFs). Gene fusions of each VirB ORF to T7 phi 10 were made and overexpressed in Escherichia coli to confirm that they encode proteins of predicted size. Hydrophobic analysis of these peptide sequences revealed nine proteins that contain hydrophobic spanning regions including signal-peptide-like sequences. These data suggest that the majority of VirB proteins may associate with bacterial cell membranes, while the two additional proteins possess a potential ATP-binding site. Strong homologies in amino acid sequences were observed between nopaline- and octopine-type plasmids. Specific differences in amino acid sequence encoded by VirB ORFs of nopaline and octopine Ti plasmid and a functional role of the gene products are discussed.

Adenosine Triphosphate↗

PubMed1991

Analysis of the gluconate (gnt) operon of Bacillus subtilis.

The gluconate (gnt) operon of Bacillus subtilis includes the gntR, gntK, gntP, and gntZ genes, respectively encoding the transcriptional repressor of the operon, gluconate kinase, the gluconate permease, and an unidentified open reading frame (Fujita and Fujita, 1987). We have compared the proteins encoded by the gnt operon of B.subtilis with published sequences and showed that (i) the gluconate repressor is homologous to several putative regulatory proteins in Escherichia coli, (ii) the gluconate kinase of B. subtilis is homologous to xylulose kinase, glycerol kinase and fucose kinase in E. coli (20-26% identity; 12-59 S.D.), (iii) the gluconate permease exhibits a C-terminal domain which is homologous to a hydrophobic protein encoded by an unidentified open reading frame (dsdAp) which precedes the dsdA gene of E. coli (39% identity; 19 S.D.), and (iv) the gntZ gene product is homologous to 6-phosphogluconate dehydrogenases of other bacteria and of animals (48-56%; 82-178 S.D.), thereby suggesting that the B. subtilis gntZ encodes 6-phosphogluconate dehydrogenase. Several conserved regions of the sequenced 6-phosphogluconate dehydrogenases can serve as signature patterns of this protein. Computer analyses have indicated that the previously reported sequences of the porcine and ovine 6-phosphogluconate dehydrogenases, as well as the hypothetical DsdAp protein, are probably erroneous. The probable reasons for the errors are reported along with the proposed revised sequences.

Amino Acid Sequence↗

PubMed1991

Differential gene expression from the Escherichia coli atp operon mediated by segmental differences in mRNA stability.

The atp operon of Escherichia coli directs synthesis rates of protein subunits that are well matched to the requirements of assembly of the membrane-bound H(+)-ATPase (alpha 3 beta 3 gamma 1 delta 1 epsilon 1a1b2c10-15). Segmental differences in mRNA stability are shown to contribute to the differential control of atp gene expression. The first two genes of the operon, atpl and atpB, are rapidly inactivated at the mRNA level. The remaining seven genes are more stable. It has previously been established that the translational efficiencies of the atp genes vary greatly. Thus differential expression from this operon is achieved via post-transcriptional control exerted at two levels. Neither enhancement of translational efficiency nor insertion of repetitive extragenic palindromic (REP) sequences into the atplB intercistronic region stabilized atpl. We discuss the implications of these results in terms of the pathway of mRNA degradation and of the role of mRNA stability in the control of gene expression.

Base Sequence↗

PubMed1992

gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression.

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.

Amino Acid Sequence↗

PubMed1993

Conservation and evolution of the rpsU-dnaG-rpoD macromolecular synthesis operon in bacteria.

The macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD) whose products (S21, primase, sigma-70) are necessary for the initiation of protein, DNA, and RNA synthesis respectively. PCR amplifications with primers complementary to conserved regions within these three genes, and subsequent DNA sequencing of rpsU-dnaG PCR products, demonstrate that the three genes appear to be contiguous in 11 different Gram-negative species. Within the Gram-negative enteric bacterial lineage, the S21 amino acid sequence is absolutely conserved in 10 species examined. The putative nuteq antiterminator sequence in rpsU consists of two motifs, boxA and boxB, conserved in primary sequence and secondary structure. The terminator sequence, T1, located between rpsU and dnaG is conserved at 31 positions in nine enterobacterial species, suggesting the importance of primary sequence in addition to secondary structure for transcription termination. The intergenic region between rpsU and dnaG varies in size owing to the presence or absence of the Enterobacterial Repetitive Intergenic Consensus (ERIC) DNA element. The rpoD gene contains rearrangements involving a divergent sequence, although two carboxy-terminal regions which encode functional domains are conserved in primary sequence and spacing. Our data suggest that primary sequence divergence and DNA rearrangements in both coding and non-coding sequences account for the interspecies variation in operon structure. However, MMS operon gene organization and cis-acting regulatory sequences appear to be conserved in diverse bacteria.

Bacteria↗

PubMed1993

Molecular analysis of the hsp (groE) operon of Leptospira interrogans serovar copenhageni.

A chromosomal gene library of Leptospira interrogans serovar copenhageni strain Wijnberg was constructed in phage lambda gt11. Plaque immunoassay with R alpha P64 antiserum identified one clone expressing a putative groEL homologue. DNA sequence analysis of the 2.4 kb EcoRI-Bam HI cloned fragment from strain Wijnberg revealed two open reading frames encoding polypeptides of 10.5 kDa (Hsp10) and 58 kDa (Hsp58). Sequence comparison of the deduced amino acid sequences of these ORFs confirmed the operon as the groE equivalent of Leptospira. Transcriptional analysis suggested that this operon is primarily under the control of an E sigma 70 promoter element. However, both Hsp10 and Hsp58 were overexpressed under heat-shock conditions as determined by [35S]-methionine pulse labelling experiments. As no functional heat-shock promoter could be identified, a 9bp inverted repeat, located between the transcription and translation start sites, may play a role in the upregulation of this operon under heat-shock conditions, similar to mechanisms described for several Gram-positive organisms.

Amino Acid Sequence↗

PubMed1994

Vitamin B12 repression of the cob operon in Salmonella typhimurium: translational control of the cbiA gene.

Expression of the cob operon is repressed by B12 via a post-transcriptional control mechanism which requires sequence elements within the leader region of the mRNA and the first gene of the operon, the cbiA gene. Here we show that B12 repression of cbiA gene expression occurs at the level of translation initiation through sequestration of the ribosomal binding site (rbs) in an RNA hairpin. Analysis of mutations that destabilize or restabilize the secondary structure demonstrates that folding of the hairpin is essential for repression. The existence of the hairpin was confirmed by a secondary structure analysis of RNA from the wild type and three mutants. Deletions that remove the upstream part of the leader confer a drastic reduction in translation efficiency. This low-level translation is caused by the hairpin, as indicated by the finding that suppressor mutations that destabilize the hairpin restore efficient translation. Thus, the native upstream RNA functions as a translation enhancer and acts to relieve the hairpin's inhibitory effect on translation initiation. The inhibitory effect of the hairpin was confirmed by a ribosomal toeprinting analysis. We propose that the translational control of the cbiA gene mediates repression of the entire cob operon.

Base Sequence↗

PubMed1994

Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis.

Conjugative transfer of DNA that occurs between bacteria also operates between bacteria and higher organisms. The transfer of DNA between Gram-negative bacteria requires initial contact by a sex pilus followed by DNA traversing four membranes (donor plus recipient) using a transmembrane pore. Accumulating evidence suggests that transfer of the T-DNA from Agrobacterium tumefaciens to plants may also occur via a conjugative mechanism. The virB operon of the Ti plasmid exhibits close homologies to genes that are known to encode the pilin subunits and pilin assembly proteins. The proteins encoded by the PilW operon of IncW plasmid R388 share strong similarities (average similarity = 50.8%) with VirB proteins. Similarly, the TraA, TraL and TraC proteins of IncF plasmid F have similarities to VirB2, VirB3 and VirB4 respectively (average similarity = 45.3%). VirB2 protein (12.3 kDa) contains a signal peptidase-I cleavage sequence that generates a polypeptide of 7.2 kDa. Likewise, the 12.8 kDa propilin protein TraA of plasmid F also possesses a peptidase-I cleavage site that generates the 7.2 kDa pilin structural protein. Similar amino acid sequences of the conjugative transfer genes of F, R388 as well as plasmid RP4 and the genes of the ptI operon of Bortedella pertussis suggest the existence of a superfamily of transmembrane proteins adapted to the promiscuous transfer of DNA-protein complexes.

Agrobacterium tumefaciens↗

PubMed1994

A seven-gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria.

The DNA sequence of the regulatory region and the structural gene, nrfA, for cytochrome c552 of Escherichia coli K-12 have been reported. We have now established that nrfA is the first gene in a seven-gene operon, designated the nrf operon, at least five of which are essential for formate-dependent nitrite reduction to ammonia. This operon terminates just upstream of the previously sequenced gltP gene encoding a sodium-independent, glutamate and aspartate transporter. Expression of lac fused to nrfA, nrfE or nrfG is regulated by oxygen repression, FNR-dependent anaerobic induction, nitrite induction and nitrate repression during anaerobic growth, exactly as previously reported for the nrfA promoter. In contrast, expression of the gltP-lac fusion was FNR-independent. The open reading frame immediately downstream of nrfA encodes NrfB, a hydrophilic, penta-haem cytochrome c with an M(r) of 20,714. The structure of the N-terminal region is typical of a signal peptide for a periplasmic protein: cleavage at the putative signal peptide cleavage site, Ala-26, would result in a periplasmic cytochrome with a molecular mass of 18 kDa. The NrfC polypeptide, M(r) 24,567, contains 16 cysteine residues arranged in four clusters typical of the CooF super-family of non-haem iron-sulphur proteins. The NrfD sequence predicts a 318-residue hydrophobic protein with a distribution of acidic and basic amino acids which suggests that NrfD is an integral transmembrane protein with loops in both the periplasm and the cytoplasm. Proteins most similar to NrfD include the PsrC subunit of polysulphide reductase from Wolinella, but, as seven of the 10 most similar proteins are NADH-ubiquinone oxidoreductases, we propose that NrfD participates in the transfer of electrons from the quinone pool into the terminal components of the Nrf pathway. NrfE, M(r) 60,851, is predicted to be another hydrophobic, integral membrane protein homologous to the CdI1 protein of Rhodobacter capsulatus, which has been implicated in the assembly of periplasmic c-type cytochromes. The sequence of the 127 residue NrfF polypeptide, M(r) 14,522, is strikingly similar to the CcI2 protein of R. capsulatus, especially in the putative haem-binding motif, RCPQCQNQN.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Substrate induction and catabolite repression of the Streptomyces coelicolor glycerol operon are mediated through the GylR protein.

The pathway for glycerol catabolism in Streptomyces coelicolor is determined by the gylCABX operon, which is transcribed from two closely spaced glycerol-inducible, glucose-repressible promoters. Glucose (or catabolite) repression of gyl is known to be exerted by a general catabolite repression system in which the soluble glucose kinase plays a central role. The gylR gene is contained in a separate glycerol-inducible, weakly glucose-repressible transcription unit immediately upstream from the gyl operon. The role of gylR in the regulation of gyl transcription was assessed by introducing specific null mutations into the chromosomal gylR gene. Direct quantification of gyl transcripts from the gylR null mutants grown on different carbon sources demonstrated that GylR is the repressor of the gylCABX operon and also revealed that GylR functions as a negative autoregulator. Moreover, the transcriptional analysis revealed that the gylR null mutants were relieved of glucose repression of both gylCABX and gylR. We conclude that both substrate induction and catabolite repression of gyl are mediated through the GylR protein. This is the first direct evidence that catabolite repression in Streptomyces is not exerted at the transcriptional level by a general 'catabolite repressor protein'. Models for catabolite repression are discussed.

Bacterial Proteins↗

PubMed1995

In Rhizobium meliloti, the operon associated with the nod box n5 comprises nodL, noeA and noeB, three host-range genes specifically required for the nodulation of particular Medicago species.

In Rhizobium meliloti, the genes required for nodulation of legume hosts are under the control of DNA regulatory sequences called nod boxes. In this paper, we have characterized three host-specific nodulation genes, which form a flavonoid-inducible operon down-stream of the nod box n5. The first gene of this operon is identical to the nodL gene identified by Baev and Kondorosi (1992) in R. meliloti strain AK631. The product of the second gene, NoeA, presents some homology with a methyltransferase. nodL mutants synthesize Nod factors lacking the O-acetate substituent. In contrast, in strains carrying a mutation in either noeA or noeB, no modification in Nod-factor structure or production could be detected. On particular hosts, such as Medicago littoralis, mutants of the n5 operon showed a very weak nodule-forming ability, associated with a drastic decrease in the number of infection threads, while nodulation of Medicago truncatula or Melilotus alba was not affected. Thus, nodL noeA and noeB are host-specific nodulation genes. By using a gain-of-function approach, we showed that the presence of nodL, and hence of O-acetylated Nod factors, is a major prerequisite for confering the ability to nodulate alfalfa upon the heterologous bacterium Rhizobium tropici.

Acetylation↗

PubMed1995

Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12.

Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin- and Congo red-binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non-polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase-specific sigma factor sigma s in wild-type cells, but not in mutants lacking the nucleoid associated protein H-NS. Insertions in csgD abolish completely trancription from the csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.

Bacterial Proteins↗