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PubMed2005

REP code: defining bacterial identity in extragenic space.

Through the analysis of 57 bacterial genomes we have detected repetitive extragenic palindromic DNA sequences (REPs) in 11 species. For a sequence to be considered as REP, the following criteria should be met: (i) It should be extragenic, (ii) palindromic, (iii) of a length between 21 and 65 bases and (iv) should constitute more than 0.5% of the total extragenic space. Species-specific REPs have been found in human pathogens such as Escherichia coli, Salmonella enterica, Neisseria meningitidis, Mycobacterium tuberculosis, Rickettsia conorii and Pseudomonas aeruginosa, the plant pathogen Agrobacterium tumefaciens and the soil bacteria Deinococcus radiodurans, Pseudomonas putida and Sinorhizobium meliloti.

Agrobacterium tumefaciens↗

PubMed1984

Evidence for a coding pattern on the non-coding strand of the E. coli genome.

Analysis of codon usage frequency for the combined coding sequences of 52 E. coli genes, taken from the European Molecular Biology Laboratory Nucleotide Sequence Data Library, Release 2, shows that there is a significant positive correlation between the frequency with which a given codon appears on the coding strand and the frequency with which it appears, in phase, on the non-coding strand.

Amino Acid Sequence↗

PubMed1999

Cloning, sequence, and transcriptional regulation of the operon encoding a putative N-acetylmannosamine-6-phosphate epimerase (nanE) and sialic acid lyase (nanA) in Clostridium perfringens.

Clostridium perfringens can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates. After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and N-acetylmannosamine. The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts N-acetylmannosamine-6-phosphate to N-acetylglucosamine-6-phosphate. A 4.0-kb DNA fragment from C. perfringens NCTC 8798 that contains the nanE and nanA genes has been cloned. The identification of the nanA gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a nanA Escherichia coli strain, EV78. The nanA gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source. By using Northern blot experiments, it was demonstrated that the nanE and nanA genes comprise an operon and that transcription of the operon in C. perfringens is inducible by the addition of sialic acid to the growth medium. The Northern blot experiments also showed that there is no catabolite repression of nanE-nanA transcription by glucose. With a plasmid construct containing a promoterless cpe-gusA gene fusion, in which beta-glucuronidase activity indicated that the gusA gene acted as a reporter for transcription, a promoter was localized to the region upstream of the nanE gene. Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the nanE coding sequence.

Bacterial Proteins↗

PubMed1988

Structural analysis of the human glucocerebrosidase genes.

Two different genomic clones containing the entire coding sequence of human glucocerebrosidase were isolated from a fetal liver library using a cDNA probe previously cloned by us. These clones correspond to two human glucocerebrosidase genes, designated 6-1 and 10-2. Clone 6-1 contains sequences homologous to the cDNA we cloned previously. The promoter regions of the genes were identified by S1 analysis and sequenced. They contain TATA- and CAAT-like boxes, but lack a GGCGGG motif. When coupled to the bacterial gene coding for chloramphenicol acetyl transferase (CAT) and transfected to Gaucher skin fibroblast lines, both promoter fragments enhanced CAT activity. The promoter of gene 6-1 was eight times more efficient than the promoter of gene 10-2. Northern blot analysis revealed three human glucocerebrosidase RNA species of 6, 2.6, and 2.2 kb in size. The 6-kb transcript is probably a nuclear transcript whereas the 2.6-kb and 2.2-kb transcripts are cytoplasmic species which emerge from polyadenylation at different sites.

Base Sequence↗

PubMed1992

The N-terminal 22 amino acids encoded by the gene specifying the major secreted protein of vaccinia virus, strain Lister, can function as a signal sequence to direct the export of a foreign protein.

Cells infected with vaccinia virus strain Lister secrete a polypeptide of approximate molecular weight 35,000 (35K) into the medium. Previous studies identified a cleavable, hydrophobic region of 17 amino acids in the 35K protein which could potentially function as a signal peptide to target the protein to the secretory pathway. Here we report the use of the expression-secretion signals derived from the 35K gene to direct export and secretion of a foreign protein. Vaccinia virus recombinants carrying the bacterial chloramphenicol acetyl transferase gene (cat) immediately downstream from the promoter and the N-terminal coding sequences of the 35K gene were constructed. Our studies show that the N-terminal 22 or 42 amino acids of the 35K protein direct efficient secretion of the CAT protein. However, due to a cryptic glycosylation site within CAT, glycosylated protein was secreted, which reduced enzymatic activity. Activity was restored in the presence of tunicamycin. Removal of the glycosylation site by site-directed mutagenesis abolished glycosylation with no effect on secretion, although CAT activity was again reduced, possibly due to an effect on the active site. The results presented here demonstrate the feasibility of using the promoter and the signal sequence of the 35K gene to generate recombinant viruses for overexpression and secretion of foreign proteins.

Animals↗

PubMed2004

Higher mutation rate helps to rescue genes from the elimination by selection.

Directional mutation pressure associated with replication processes is the main cause of the asymmetry between the leading and lagging DNA strands in bacterial genomes. On the other hand, the asymmetry between sense and antisense strands of protein coding sequences is a result of both mutation and selection pressures. Thus, there are two different ways of superposition of the sense strand, on the leading or lagging strand. Besides many other implications of these two possible situations, one seems to be very important - because of the asymmetric replication-associated mutation pressure, the mutation rate of genes depends on their location. Using Monte Carlo methods, we have simulated, under experimentally determined directional mutation pressure, the divergence rate and the elimination rate of genes depending on their location in respect to the leading/lagging DNA strands in the asymmetric prokaryotic genome. We have found that the best survival strategy for the majority of genes is to sometimes switch between DNA strands. Paradoxically, this strategy results in higher substitution rates but remains in agreement with observations in bacterial genomes that such inversions are very frequent and divergence rate between homologs lying on different DNA strands is very high.

Amino Acid Substitution↗

PubMed1983

Isolation of cDNA sequences coding for a part of human tissue plasminogen activator.

We have isolated a cDNA sequence coding for a part of human tissue plasminogen activator. mRNA coding for tissue plasminogen activator was partially purified, copied into double-stranded cDNA, and cloned into Escherichia coli. Two sets of partially overlapping oligodeoxynucleotide mixtures corresponding to all possible coding sequences for a known portion of the tissue plasminogen activator gene were prepared. One set was used as a probe to screen cDNA containing bacterial clones and both were used as probes in hybridization against purified plasmid DNA. Of 4,200 bacterial clones examined, 1 carried a plasmid that hybridized to both sets of oligonucleotides. This plasmid contained a 370-base-pair cDNA insert, which was shown by nucleotide sequence analysis to code for the cleavage site region in the one-chain form of the human tissue plasminogen activator.

Amino Acid Sequence↗

PubMed1991

Structural and functional analysis of the rcsA gene from Erwinia stewartii.

RcsA is a positive regulator of genes for extracellular polysaccharide biosynthesis in the Enterobacteriaceae. The nucleotide sequence of the rcsA gene from Erwinia stewartii was determined and compared to rcsA sequences from E. amylovora, Escherichia coli, and Klebsiella pneumoniae. Three highly conserved regions of the gene were identified. The C-terminal end of the open reading frame (ORF) shared significant amino acid homology to the LuxR class of bacterial activator proteins. Insertion and deletion mutagenesis of the 5' non-coding region indicated that maximal expression of rcsA was dependent upon cis-acting regulatory sequences located more than 300 bp upstream of the translational start site.

Amino Acid Sequence↗

PubMed2002

Characterisation of silent and active genes for a variable large protein of Borrelia recurrentis.

BACKGROUND: We report the characterisation of the variable large protein (vlp) gene expressed by clinical isolate A1 of Borrelia recurrentis; the agent of the life-threatening disease louse-borne relapsing fever. METHODS: The major vlp protein of this isolate was characterised and a DNA probe created. Use of this together with standard molecular methods was used to determine the location of the vlp1B. recurrentis A1 gene in both this and other isolates. RESULTS: This isolate was found to carry silent and expressed copies of the vlp1B. recurrentis A1 gene on plasmids of 54 kbp and 24 kbp respectively, whereas a different isolate, A17, had only the silent vlp1B. recurrentis A17 on a 54 kbp plasmid. Silent and expressed vlp1 have identical mature protein coding regions but have different 5' regions, both containing different potential lipoprotein leader sequences. Only one form of vlp1 is transcribed in the A1 isolate of B. recurrentis, yet both 5' upstream sequences of this vlp1 gene possess features of bacterial promoters. CONCLUSION: Taken together these results suggest that antigenic variation in B. recurrentis may result from recombination of variable large and small protein genes at the junction between lipoprotein leader sequence and mature protein coding region. However, this hypothetical model needs to be validated by further identification of expressed and silent variant protein genes in other B. recurrentis isolates.

Antigens, Bacterial↗

PubMed1994

Characterization of the herpes simplex virus type 1 strain 17+ neurovirulence gene RL1 and its expression in a bacterial system.

The DNA sequence of herpes simplex virus type 1 (HSV-1) strain 17+ in the region coding for the polypeptide ICP34.5 predicts a protein of 248 amino acids with a proposed M(r) of 26,158. The entire RL1 open reading frame was cloned into the expression vector pET8c to enable over-expression of ICP34.5 in Escherichia coli. The expressed protein was partially purified and used as an immunogen to produce a polyclonal antiserum in rabbits. Construction of an ICP34.5 null mutant (1771), demonstrated that the predicted open reading frame for ICP34.5 in strain 17+ is correct and confirmed that HSV-1 strain 17+ ICP34.5 specifically determines neurovirulence. The specificity of the anti-serum directed against the E. coli-expressed ICP34.5 was defined by Western blotting of wild-type and RL1-negative infected cell extracts.

Animals↗

PubMed1996

The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas.

A chimeric gene composed of the coding sequence of the ble gene from Streptoalloteichus hindustanus fused to the 5' and 3' untranslated regions of the Chlamydomonas reinhardtii nuclear gene RBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome of C. reinhardtii by co-transformation with the ARG7 marker yields Arg+ transformants of which approximately 80% possess the ble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (PmR) phenotype. Western blot analysis using antibodies against the ble gene product confirms the presence of the protein in the PmR transformants and genetic analysis demonstrates the co-segregation of the ble gene with the phenotype in progeny arising from the mating of a PmR transformant to wild-type strains. Direct selection of PmR transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome of C. reinhardtii and provides a useful dominant marker for nuclear transformation.

Animals↗

PubMed2003

Genomics of oral bacteria.

Advances in bacterial genetics came with the discovery of the genetic code, followed by the development of recombinant DNA technologies. Now the field is undergoing a new revolution because of investigators' ability to sequence and assemble complete bacterial genomes. Over 200 genome projects have been completed or are in progress, and the oral microbiology research community has benefited through projects for oral bacteria and their non-oral-pathogen relatives. This review describes features of several oral bacterial genomes, and emphasizes the themes of species relationships, comparative genomics, and lateral gene transfer. Genomics is having a broad impact on basic research in microbial pathogenesis, and will lead to new approaches in clinical research and therapeutics. The oral microbiota is a unique community especially suited for new challenges to sequence the metagenomes of microbial consortia, and the genomes of uncultivable bacteria.

Aggregatibacter actinomycetemcomitans↗

PubMed2004

The impact of prophages on bacterial chromosomes.

Prophages were automatically localized in sequenced bacterial genomes by a simple semantic script leading to the identification of 190 prophages in 115 investigated genomes. The distribution of prophages with respect to presence or absence in a given bacterial species, the location and orientation of the prophages on the replichore was not homogeneous. In bacterial pathogens, prophages are particularly prominent. They frequently encoded virulence genes and were major contributors to the genetic individuality of the strains. However, some commensal and free-living bacteria also showed prominent prophage contributions to the bacterial genomes. Lysogens containing multiple sequence-related prophages can experience rearrangements of the bacterial genome across prophages, leading to prophages with new gene constellations. Transfer RNA genes are the preferred chromosomal integration sites, and a number of prophages also carry tRNA genes. Prophage integration into protein coding sequences can lead to either gene disruption or new proteins. The phage repressor, immunity and lysogenic conversion genes are frequently transcribed from the prophage. The expression of the latter is sometimes integrated into control circuits linking prophages, the lysogenic bacterium and its animal host. Prophages are apparently as easily acquired as they are lost from the bacterial chromosome. Fixation of prophage genes seems to be restricted to those with functions that have been co-opted by the bacterial host.

Bacteria↗

PubMed1999

A new subfamily of short bacterial adenylate kinases with the Mycobacterium tuberculosis enzyme as a model: A predictive and experimental study.

The adk gene from Mycobacterium tuberculosis codes for an enzyme of 181 amino acids. A sequence comparison with 52 different forms of adenylate kinases (AK) suggests that the enzyme from M. tuberculosis belongs to a new subfamily of "short" bacterial AKs. The recombinant protein, overexpressed in Escherichia coli, exhibits a low catalytic activity and an unexpectedly high thermal stability (Tm = 64.8 degrees C). Based on various spectroscopic data, on the known three-dimensional structure of the AK from E. coli and on secondary structure predictions for various sequenced AKs, we propose a structural model for AK from M. tuberculosis (AKmt). Proteins 1999;36:238-248.

Adenylate Kinase↗

PubMed1981

Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site.

With the premise that mRNAs transcribed in Escherichia coli from cloned eukaryotic DNA inserts do not possess the necessary regulatory signals for recognition by prokaryotic ribosomes, we have constructed a general plasmid vector carrying a chemically synthesized prokaryotic ribosome binding site that will ensure the efficient expression of eukaryotic proteins in E. coli. In addition to the regulatory signals necessary for ribosome recognition, the synthetic segment contains, at one end, a Pst I cleavage site which will direct its insertion to pBR322 DNA and, at the other end, a HindIII site to facilitate attachment of the passenger eukaryotic gene. Using simian virus 40 (SV40) tumor (t) antigen as a model system, we have ligated the SV40 DNA fragment containing the entire t antigen gene in tandem with the synthetic ribosome binding site to pBR322 DNA at the Pst I site, which lies within the coding sequence of the beta-lactamase gene. Initiation of transcription at the beta-lactamase promoter would produce a chimeric mRNA with the synthetic ribosome binding signals and the SV40 sequence flanked by beta-lactamase coding sequences. Utilization of the synthetic regulatory signals for initiation of translation is demonstrated by the efficient synthesis, in bacterial transformants, of authentic SV40 t antigen. Excision of the entire SV40 insert by HindIII from those clones that have retained intact HindIII sites at the junction between the ribosome binding site and the SV40 sequence would allow insertion of other heterologous DNAs by using HindIII linkers. The efficient expression of any DNA insert would require that the entire coding sequence be contiguous and that its termini be randomized by treatment with exonuclease III and nuclease S1 to vary the distance between the translational initiation codon and the synthetic ribosome binding site.

Antigens, Neoplasm↗

PubMed1983

Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope.

The nucleotide sequence of the leader peptidase structural gene from Escherichia coli has been determined. The gene codes for a protein of 323 amino acid residues with a calculated Mr = 35,994, in agreement with the apparent molecular weight of leader peptidase (37,000) determined from gel electrophoresis in sodium dodecyl sulfate. In addition, the amino acid composition predicted from the DNA sequence matches that of the purified enzyme. Leader peptidase synthesized in a cell-free transcription-translation system and isolated from cell membranes have identical apparent molecular weights on sodium dodecyl sulfate-polyacrylamide gels. The presence of methionine and cysteine in the NH2-terminal tryptic peptide shows that no more than three amino acid residues have been removed from the primary translation product predicted from the DNA sequence. These results indicate that leader peptidase is synthesized and assembled into the membrane without proteolytic removal of a leader peptide. Leader peptidase is shown to be largely found in the inner membrane of overproducing strains. The majority of the polypeptide chain is exposed on the outer surface of the inner membrane. It is anchored by a membrane-spanning segment near the NH2 terminus. This orientation agrees with the functional orientation of leader peptidase observed in artificial membrane vesicles.

Amino Acid Sequence↗

PubMedDate not supplied

[Molecular organization of plasmid R906 (Inc P-1)].

Genetic and restriction (for enzymes EcoRI, BamHI and HindIII) maps of the relatively broad host range plasmid R906 are constructed. There are two non-essential regions on the R906 DNA which can be deleted and cloned. Non-essential regions confer a resistance to different agents and restriction sites are clustered in these regions. Essential and conjugativity genes are located in two other DNA regions approximately at 0-23 and 29-44 kb of the R906 map. These large regions share a high level of homology with Inc-1 group plasmids R751 and RP4 according to Southern-blot hybridization and heteroduplex analyses. A transposon-like structure is found on the R751 DNA among R751/R906 heteroduplex molecules. This transposon of total length 5.1 kb has 1.4 kb inverted repeats at the ends. Bla genes of R906 and RP4 plasmids do not have homologous sequences. Data evidence that IncP-1 group plasmids irrespective to their original bacterial source and range of coded antibiotic resistance have very similar molecular organization. The role of possible factors which are responsible for the broad host range property of the IncP-1 group plasmids is discussed.

Bordetella↗

PubMed1997

Cloning and expression of bovine corneal antigen cDNA.

PURPOSE: A cornea-associated antigen (CO-Ag) has been found to be the target for autoantibodies in patients with Mooren's ulcer. The study goals were to isolate a full-length clone encoding CO-Ag from a bovine corneal cDNA library and to express this clone in Escherichia coli (E. coli). METHODS: A DNA fragment of CO-Ag was generated, using unique oligonucleotide primers and reverse transcription polymerase chain reaction. This fragment was used as a probe to obtain cDNA clones from a bovine corneal cDNA library. The clone with the longest cDNA insert was selected for sequence analysis. Expression of the CO-Ag protein in E. coli was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). The bacterially-produced CO-Ag was partially purified by calcium (Ca2+)-dependent hydrophobic interaction chromatography. RESULTS: The cDNA insert sequence was 273 nucleotides in length for the entire mRNA coding region, 212 nucleotides in the 5' untranslated region, 83 nucleotides in the 3' untranslated region and a poly(A) tail. The DNA base sequence of this clone also contained a standard initiation codon, termination codon, and the polyadenylation signal. This cDNA predicts a protein which contains 91 amino acids with a molecular weight of 10,584 daltons. The cDNA and deduced amino acid sequence of CO-Ag are completely identical to a S-100 protein, bovine calgranulin C. The cDNA was expressed in E. coli as a fusion protein consisting of 583 N-terminal amino acids of beta-galactosidase (beta-gal), 91 amino acids of CO-Ag, and possibly a number of additional N-terminal and C-terminal residues. The bacterially produced CO-Ag was fully functional with respect to hydrophobic interaction with phenyl-Sepharose matrix for its isolation. The fusion protein was recognized by antiserum raised against bovine CO-Ag protein on Western blots. CONCLUSIONS: The isolation and analysis of a cDNA clone containing the complete coding sequence of the CO-Ag protein and the expression of the CO-Ag protein in E. coli is reported. The availability of a CO-Ag cDNA probe and larger quantities of the CO-Ag protein should aid in elucidating the possible pathogenic role of CO-Ag in Mooren's ulcer.

Amino Acid Sequence↗