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PubMed1994

Genes coding for the selenocysteine-inserting tRNA species from Desulfomicrobium baculatum and Clostridium thermoaceticum: structural and evolutionary implications.

The genes (selC) coding for the selenocysteine-inserting tRNA species (tRNA(Sec)) from Clostridium thermoaceticum and Desulfomicrobium baculatum were cloned and sequenced. Although they differ in numerous positions from the sequence of the Escherichia coli selC gene, they were able to complement the selC lesion of an E. coli mutant and to promote selenoprotein formation in the heterologous host. The tRNA(Sec) species from both organisms possess all of the unique primary, secondary, and tertiary structural features exhibited by E. coli tRNA(Sec) (C. Baron, E. Westhof, A. Böck, and R. Giegé, J. Mol. Biol. 231:274-292, 1993). The structural and functional properties of the tRNA(Sec) species from prokaryotes analyzed thus far support the notion that tRNA(Sec) may be an evolutionarily conserved structure whose function in the primordial genetic code was to decode UGA with selenocysteine.

Base Sequence↗
PubMed2003

A transcriptional pause synchronizes translation with transcription in the tryptophanase operon leader region.

Regulation of transcription of the tryptophanase operon requires that translation of its leader peptide coding region, tnaC, be coupled with its transcription. We show in vitro that a transcription pause site exists at the end of the tnaC coding region and that translation of tnaC releases the paused transcription complex, coupling transcription with translation.

Amino Acid Sequence↗
PubMed1991

Amplification by the polymerase chain reaction of a specific target sequence in the gene coding for Escherichia coli verotoxin (VTe variant).

Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to target a specific sequence in the gene coding for the A subunit of Escherichia coli verotoxin (VTe-variant, VTev). This PCR protocol permits the VTe-variant target sequence to be distinguished from closely related sequences in the same coding regions for type 1, type 2, and type 2 variant E. coli verotoxins. This procedure will be a valuable adjunct to other DNA amplification techniques currently being used for molecular epidemiological studies of verotoxigenic E. coli.

Bacterial Toxins↗
PubMed1994

Functional analysis of the Escherichia coli K-12 cyn operon transcriptional regulation.

The cynTSX operon enables Escherichia coli K-12 to degrade and use cyanate as a sole nitrogen source. The promoter of this operon is positively regulated by cyanate and the CynR protein. CynR, a member of the LysR family of regulatory proteins, binds specifically to a 136-bp DNA fragment containing both the cynR and the cynTSX promoters. In this study, we report the results of DNase I digestion studies showing that CynR protects a 60-bp region on the cynR coding strand and a 56-bp sequence on the cynTSX coding strand. CynR binding was not affected by cyanate or its structural homolog azide, a gratuitous inducer of the operon. However, CynR-induced bending of two different DNA fragments was detected. The amount of bending was decreased by cyanate.

Azides↗
PubMed1991

Variations and coding features of the sequence spanning the replication terminus of Bacillus subtilis 168 and W23 chromosomes.

In a comparative study of the sequences of the 3-kb regions of DNA spanning the replication terminus, terC, of Bacillus subtilis strains 168 and W23, it was found that the latter contained an insertion of a large open reading frame (ORF405) whose translated protein product is a member of the cytochrome P-450 family. The insertion was about 34 nucleotides upstream from a putative promoter for the rtp gene. The sequenced regions contained a number of other ORFs. The translation product of one (ORF238) is a member of a previously identified oxidoreductase superfamily. The translation product of another (ORF257) is significantly similar to the proC product of Escherichia coli, but this ORF does not code for a functional proC product of B. subtilis.

Amino Acid Sequence↗
PubMed1993

Mechanism of expression of DNA repair gene vsr, an Escherichia coli gene that overlaps the DNA cytosine methylase gene, dcm.

The DNA cytosine methylase gene of Escherichia coli, dcm, overlaps an open reading frame (ORF) that continues in +1 register past the end of dcm. This ORF codes for a gene, vsr, that is required for a T:G to C:G base mismatch correction process. In this study, mutants that affect the level of expression of the two genes were constructed and characterized. Further, a previously isolated mutant, dcm-6, was cloned and mutations within it were identified. Northern blots were used to identify dcm-specific RNA species in wild type and dcm-6 cells. Based on these studies we conclude that there is a six-codon overlap between vsr and dcm. The two proteins appear to be made from a single RNA transcript and translation of dcm is required for the efficient synthesis of Vsr. Further, Vsr is active by itself and may not be produced as a fusion with Dcm. This is the first example of chromosomal genes that overlap in their coding regions and produce proteins with distinct functions.

Alleles↗
PubMed1990

Complementation of Escherichia coli sigma 54 (NtrA)-dependent formate hydrogenlyase activity by a cloned Thiobacillus ferrooxidans ntrA gene.

The ntrA gene of Thiobacillus ferrooxidans was cloned by complementation of an Escherichia coli ntrA mutant that was unable to produce gas via the sigma 54 (NtrA)-dependent formate hydrogenlyase pathway. Analysis of the DNA sequence showed that the T. ferrooxidans ntrA gene coded for a protein of 475 amino acids (calculated Mr, 52,972). The T. ferrooxidans NtrA protein had 49, 44, 33, and 18% amino acid similarity with the NtrA proteins of Klebsiella pneumoniae, Azotobacter vinelandii, Rhizobium meliloti, and Rhodobacter capsulatus, respectively. The ability of the T. ferrooxidans NtrA protein to direct transcription from sigma 54-dependent promoters was demonstrated in E. coli by using fdhF-lacZ and nifH-lacZ fusions. An open reading frame coding for a protein of 241 amino acids (calculated Mr, 27,023) was situated 12 base pairs upstream of the T. ferrooxidans ntrA gene. Comparison of this protein with the product of the open reading frame ORF1, located upstream of the R. meliloti ntrA gene, showed that the two proteins had 55% amino acid similarity. The cloned T. ferrooxidans ntrA gene was expressed in E. coli from a promoter located within the ORF1 coding region.

Acidithiobacillus thiooxidans↗
PubMed1992

Cloning and nucleotide sequence of a negative regulator gene for Klebsiella aerogenes arylsulfatase synthesis and identification of the gene as folA.

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment. Transfer of the plasmid, which contains the cloned fragment, into constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsR; the synthesis of arylsulfatase was repressed in the presence of inorganic sulfate or cysteine, and this repression was relieved, in each case, by the addition of tyramine. The nucleotide sequence of the 1.6-kb fragment was determined. From the amino acid sequence deduced from the DNA sequence, we found two open reading frames. One of them lacked the N-terminal region but was highly homologous to the gene which codes for diadenosine tetraphosphatase (apaH) in Escherichia coli. The other open reading frame was located counterclockwise to the apaH-like gene. This gene was highly homologous to the gene which codes for dihydrofolate reductase (folA) in E. coli. We detected 30 times more activity of dihydrofolate reductase in the K. aerogenes strains carrying the plasmid, which contains the arylsulfatase regulator gene, than in the strains without plasmid. Further deletion analysis showed that the K. aerogenes folA gene is consistent with the essential region required for the repression of arylsulfatase synthesis. Transfer of a plasmid containing the E. coli folA gene into atsR mutant cells of K. aerogenes resulted in repression of the arylsulfatase synthesis. Thus, we conclude that the folA gene codes a negative regulator for the ats operon.

Amino Acid Sequence↗
PubMed1996

Differential mRNA decay within the transfer operon of plasmid R1: identification and analysis of an intracistronic mRNA stabilizer.

Processing of the transfer operon mRNA of the conjugative resistance plasmid R1-19 results in the accumulation of stable traA mRNAs. The stable traA transcripts found in vivo have identical 3' ends within downstream traL sequences, but vary at their 5' ends. The 3' ends determined coincide with the 3' base of a predicted large clover-leaf-like RNA secondary structure. Here we demonstrate that this putative RNA structure, although part of a coding sequences, stabilizes the upstream traA mRNA very efficiently. We also show that the 3' ends of the stable mRNAs are formed posttranscriptionally and not by transcription termination. Half-life determinations reveal the same half-lives of 13 +/- 2 min for the traA mRNAs transcribed from hybrid lac-traAL-cat test plasmids, the R1-19 plasmid, and the F plasmid. Protein expression experiments demonstrate that the processed stable traA mRNA is translationally active. Partial deletions of sequences corresponding to the predicted secondary structure within the traL coding region drastically reduce the chemical and functional half-life of the traA mRNA. The results presented here unambiguously demonstrate that the proposed secondary structure acts as an efficient intracistronic mRNA stabilizer.

Bacterial Outer Membrane Proteins↗
PubMed2003

An appraisal of the potential for illegitimate recombination in bacterial genomes and its consequences: from duplications to genome reduction.

An exhaustive search for shortly spaced repeats in 74 bacterial chromosomes reveals that they are much more numerous than is usually acknowledged. These repeats were divided into five classes: close repeats (CRs), tandem repeats (TRs), simple sequence repeats (SSRs), spaced interspersed direct repeats, and "others." CRs are widespread and constitute the most abundant class, particularly in coding sequences. The other classes are less frequent, but each individual element shows a higher potential for recombination, when the number of repeats and their distances are taken into account. SSRs and TRs are more frequent in pathogens, as expected given their role in contingency loci, but are also widespread in the other bacteria. The analysis of CRs shows that they have an important role in the evolution of genomes, namely by generating duplications and deletions. Several cases compatible with a significant role of small CRs in the formation of large repeats were detected. Also, gene deletion in Buchnera correlates with repeat density, suggesting that CRs may lead to sequence deletion in general and genome reductive evolution of obligatory intracellular bacteria in particular. The assembly of these results indicates that shortly spaced repeats are key players in the dynamics of genome evolution.

Chromosome Mapping↗
PubMed1998

Did archaeal and bacterial cells arise independently from noncellular precursors? A hypothesis stating that the advent of membrane phospholipid with enantiomeric glycerophosphate backbones caused the separation of the two lines of descent.

One of the most remarkable biochemical differences between the members of two domains Archaea and Bacteria is the stereochemistry of the glycerophosphate backbone of phospholipids, which are exclusively opposite. The enzyme responsible to the formation of Archaea-specific glycerophosphate was found to be NAD(P)-linked sn-glycerol-1-phosphate (G-1-P) dehydrogenase and it was first purified from Methanobacterium thermoautotrophicum cells and its gene was cloned. This structure gene named egsA (enantiomeric glycerophosphate synthase) consisted of 1,041 bp and coded the enzyme with 347 amino acid residues. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity except for NAD-binding region with that of NAD(P)-linked sn-glycerol-3-phosphate (G-3-P) dehydrogenase of Escherichia coli which catalyzes the formation of G-3-P backbone of bacterial phospholipids, while the deduced protein sequence of the enzyme revealed some similarity with bacterial glycerol dehydrogenases. Because G-1-P dehydrogenase and G-3-P dehydrogenase would originate from different ancestor enzymes and it would be almost impossible to interchange stereospecificity of the enzymes, it seems likely that the stereostructure of membrane phospholipids of a cell must be maintained from the time of birth of the first cell. We propose here the hypothesis that Archaea and Bacteria were differentiated by the occurrence of cells enclosed by membranes of phospholipids with G-1-P and G-3-P as a backbone, respectively.

Amino Acid Sequence↗
PubMed1996

Sequencing the 500-kb GC-rich symbiotic replicon of Rhizobium sp. NGR234 using dye terminators and a thermostable "sequenase": a beginning.

Genomes of the soil-borne nitrogen-fixing symbionts of legumes [Azo(Brady)Rhizobium species] typically have GC contents of 59-65 mol%. As a consequence, compressions (up to 400 per cosmid) are common using automated dye primer shotgun sequencing methods. To overcome this difficulty, we have exclusively applied dye terminators in combination with a thermostable "sequenase" for shotgun sequencing GC-rich cosmids from pNGR234a, the 500-kbp symbiotic replicon of Rhizobium sp. NGR234. A thermostable sequenase incorporates dye terminators into DNA more efficiently than Taq DNA polymerase, thus reducing the concentrations needed (20- to 250-fold). Unincorporated dye terminators can simply be removed by ethanol precipitation. Here, we present data of pXB296, one of 23 overlapping cosmids representing pNGR234a. We demonstrate that the greatly reduced number of compressions results in a much faster assembly of cosmid sequence data by comparing assembly of the shotgun data from pXB296 and the data from another pNGR234a cosmid (pXB110) sequenced using dye primer methods. Within the 34,010-bp sequence from pXB296, 28 coding regions were predicted. All of them showed significant homologies to known proteins, including oligopeptide permeases, an essential cluster for nitrogen fixation, and the C4-dicarboxylate transporter DctA.

Base Composition↗
PubMed1999

Identification and characterization of a novel fibronectin-binding protein on the surface of group A streptococci.

Understanding the role surface proteins play in the interaction of group A streptococci with epithelial cells is an important step toward the development of new strategies to fight infections. Fibronectin-binding proteins in streptococci and staphylococci have been described as important mediators for adherence to eukaryotic cells. In the present study we describe a new Streptococcus pyogenes fibronectin-binding protein (PFBP). The gene encoding the PFBP protein (pfbp) was identified from an M12 strain genomic library. It encodes a protein of 127.4 kDa which contains the LPXTGX motif characteristic of cell wall-associated proteins in gram-positive organisms and is among the largest surface molecules described for group A streptococci. The pfbp gene is transcribed during cell growth and was present in several class I and II streptococcal strains tested. The deduced amino acid sequence of PFBP exhibits a variable N-terminal region and a conserved C-terminal region when compared to most fibronectin-binding proteins identified from other gram-positive bacteria. The N-terminal region presents a stretch of 105 amino acids with no homology with N-terminal regions of previously described fibronectin-binding molecules, while the C-terminal region contains three repeat domains that share significant similarity with the repeat regions of fibronectin-binding proteins from S. pyogenes, S. dysgalactiae, and S. equisimilis. The PFBP repeated region, when expressed on the surface of S. gordonii, a commensal organism, binds to soluble and immobilized fibronectin. This study also shows that, in addition to pfbp, a second gene homologous with that of protein F1 (which also codes for a fibronectin-binding protein) is transcribed during cell growth in the same S. pyogenes strain.

Adhesins, Bacterial↗
PubMed1998

Isolation and characterization of an oligodendrocyte precursor-derived B-cell epitope in multiple sclerosis.

In a search for possible central nervous system-specific autoantigens in multiple sclerosis (MS), a lambda-phage protein expression library was constructed from an oligodendrocyte-precursor cell line. The library was screened with pooled cerebrospinal fluid (CSF) from 54 patients with definite MS according to the criteria of Poser. Pooled CSF samples from 44 patients with other neurological diseases including bacterial meningitis and viral encephalitis were used as control. A total of 1,000,000 colonies were screened and 6 positive clones were detected. At the DNA level none of the sequences showed significant homology to a known coding sequence. All 6 clones contained an open reading frame for small peptides ranging from 14 to 38 amino acids. It was noteworthy that 5 clones contained a common sequence of 7 amino acids, which was highly homologous to a translated consensus Alu repeat epitope. Screening of sera and CSF from patients with MS showed that approximately 44% reacted with these so-called Alu peptides, end-point antibody titers in their sera ranging from 1:1,000 to 1:25,000. In addition, some samples selected by their reactivity with Alu peptides stained intensively the cytoplasm of oligodendrocyte precursors but not of astrocytes ex vivo. We postulate that autoantibodies to a hitherto unknown oligodendrocyte precursor-derived B-cell epitope could contribute to the pathogenesis in a subgroup of MS patients.

Adolescent↗
PubMed1992

Characterization of the genes coding for the F1F0 subunits of the sodium dependent ATPase of Propionigenium modestum.

The DNA coding for the eight structural genes and uncI of the sodium dependent ATPase of Propionigenium modestum has been cloned and sequenced. Based on sequence homology, the genes were determined to appear in the order uncBEFHAGDC as in several other bacterial species. Minicell experiments revealed that plasmids containing the P. modestum DNA expressed those ATPase polypeptides in Escherichia coli. These were very similar in molecular mass to those obtained from the purified ATPase of P. modestum. No membrane-bound ATPase activity was observed in E. coli unc deletion strains containing the P. modestum ATPase genes. Amino acid alignments which were done with the Fo subunits revealed only a few conservative changes in the highly conserved regions of the polypeptides.

Adenosine Triphosphatases↗
PubMed2004

Structural analysis of the eukaryotic initiation factor 4E gene controlling potyvirus resistance in pepper: exploitation of a BAC library.

The pvr2 locus in pepper codes for a eukaryotic translation initiation factor 4E (eIF4E) gene that confers resistance to viruses belonging to the potyvirus genus. In this work, we describe the isolation and characterisation of the genomic sequence carrying the pvr2 locus. A Bacterial Artificial Chromosome (BAC) library that consisted of 239,232 clones with an average insert size of 123 kilobases (kb) was constructed from a Capsicum annuum line with the pvr2(+) allele for susceptibility to potato virus Y (PVY) and tobacco etch virus (TEV). Based on a polymerase chain reaction (PCR) screen with single-copy markers, three to seven positive BAC clones per markers were identified, indicating that the BAC library is suitable for pepper genome analysis. To determine the genomic organization of the pepper eIF4E gene, the library was screened with primers designed from the cDNA sequence and four positive BAC clones carrying the pvr2 locus were identified. A 7-kb DNA fragment containing the complete eIF4E gene was sub-cloned from the positive BAC clones and analysed. The eIF4E gene is organised into five exons and four introns and showed a strictly conserved exon/intron structure with eIF4E genes from Arabidopsis thaliana and rice. Moreover, the splice sites between plant exons 1/2 and 2/3 are conserved among eukaryotes including human, Drosophila and yeast. Several potential binding sites for MADS box transcription factors within the 5' flanking region of eIF4E genes from the three plant species were also predicted.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of the fosB gene conferring fosfomycin resistance in Staphylococcus epidermidis.

We have determined the nucleotide sequence of gene fosB of plasmid pIP1842, which confers resistant to fosfomycin in Staphylococcus epidermidis BM2641. The resistance gene was identified as a coding sequence of 417 bases pairs corresponding to a protein with a calculated Mw of 16,345 daltons. This value is in good agreement with that of 15,000, estimated by SDS-polyacrylamide gel electrophoresis of a bacterial cell-free coupled transcription-translation system. The fosB gene product exhibits 48% sequence homology with the fosfomycin resistance protein FOSA of Enterobacteriaceae. The substitutions are scattered throughout indicating that the two corresponding genes have diverged from a common ancestor. Downstream from fosB is located an open reading frame that was partially sequenced. The deduced amino-acid sequence was 50% homologous to REP alpha 1, a protein involved in the replication of the Enterococcus faecalis plasmid pAM alpha 1.

Amino Acid Sequence↗