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PubMed1995

Restriction-modification systems in Lactococcus lactis.

Several restriction-modification (R-M) systems have been identified in Lactococcus lactis. Most of the systems have been plasmid encoded and function as phage-resistance mechanisms. At least five different type-II R-M systems, LlaAI, LlaBI, LlaCI, LlaDI and LlaEI, were identified in isolates from a mixed Cheddar starter culture. LlaAI and LlaBI recognized the DNA sequences 5'- decreases GATC-3' and 5'-C decreases TRYAG-3', respectively. The genes coding for the LlaAI and LlaBI R-M systems have been cloned and sequenced. The LlaAI R-M system had two genes coding for methyltransferases (MTases) and one gene coding for a restriction endonuclease (ENase). The MTases showed high homology to the MTases from DpnII. The LlaBI R-M system had one gene coding for a MTase and one gene coding for an ENase.

Base Sequence↗
PubMed1995

Promoter structure and transcriptional activation of the murine TSG-14 gene encoding a tumor necrosis factor/interleukin-1-inducible pentraxin protein.

Human TNF-stimulated gene 14 (TSG-14) encodes a secreted 42-kDa glycoprotein that shows significant homology to proteins of the pentraxin family, which includes the acute phase reactants C-reactive protein and serum amyloid P component. Levels of TSG-14 protein (also termed PTX-3) become elevated in the serum of mice and humans after injection with bacterial lipopolysaccharide, but in contrast to conventional acute phase proteins, the bulk of TSG-14 synthesis in the intact organism occurs outside the liver. In the present study we cloned and partially sequenced murine genomic TSG-14 DNA. Analysis of the coding region predicts a high degree of amino acid sequence homology between murine and human TSG-14 (88 and 75% identity in the first and second exons, respectively). The promoter of the TSG-14 gene lacks consensus sequences for either a TATA box or CCAAT box. Primer extension analysis and S1 nuclease protection assay revealed one major transcription start site, situated within a consensus sequence for an initiator element. Sequence analysis of a approximately 1.4-kilobase pair fragment of the 5'-flanking region of the TSG-14 gene revealed the presence of numerous potential enhancer binding elements, including six NF-IL6-like sites, four AP-1, one AP-2, one NF-kB, two Sp1, two interferon-gamma-activated sites (GAS), one Hox-1.3, and five binding sites for Ets family members. Transfection of BALB/c 3T3 cells with promoter DNA fragments linked to the luciferase reporter gene revealed that the 5'-flanking region of the TSG-14 gene comprises elements that can mediate a basal level of transcription and inducibility by TNF.

Amino Acid Sequence↗
PubMed1996

Compositional similarities between the human immunodeficiency virus and surface antigens of pathogens.

The genome of the human immunodeficiency virus (HIV) is rich in A but not U and deficient in C but not G. This asymmetric nucleotide bias is the major factor in determining the unusual composition of HIV proteins. In this report, we have identified the cellular genes in the GenBank database that are compositionally similar to HIV in order to further understand the significance of the nucleotide bias of the viral genome. A total of 101 genes in the bacterial and invertebrate subdivisions of the database were found to have a base composition that is similar to the composition of the HIV genome. The identified cellular sequences represent a discrete subset of the database since 81 of the 101 entries code for antigens from pathogens and nearly all of these organisms infect humans. The amino acid compositions of these surface antigens are also similar to the unusual composition of HIV proteins, which are deficient in proline and rich in lysine and other polar residues encoded by A-rich codons. The similarities between the HIV proteins and the immunodominant antigens from other pathogens may indicate a common pathogenic strategy for the promotion of immune dysregulation.

Animals↗
PubMed1997

A whole genome shotgun gene fusion method for isolation of translation initiation sites in Escherichia coli: identification of Haemophilus influenzae translation initiation sites in E. coli.

We have developed a new method for isolating translation initiation sites based on the expression of Haemophilus influenzae Rd gene fusions with the Escherichia coli galactokinase (galK) gene. We cloned random DNA fragments of H. influenzae Rd DNA into a plasmid vector containing the galK coding sequence from which the translation initiation site (the ribosome binding site and translation initiation codon) had been removed. A subset of the cloned DNA fragments contained translation initiation sites that, when fused to the galK gene, produced active galactokinase and complemented the host galK mutation. Molecules expressing galactokinase activity were isolated and characterized by DNA sequence analysis, and the sequences were aligned with the recently completed whole genomic sequence of H. influenzae Rd. Translation initiation sites for known, hypothetical, and new genes were identified. Translation initiation sites internal to the coding sequences of a number of genes were identified, suggesting that internal translation initiation sites are common, especially in large genes. This shotgun method provides functional information on translation initiation sites and helps to define gene coding sequences.

Amino Acid Sequence↗
PubMed1995

Transfer of macrolide-lincosamide-streptogramin B (MLS) resistance in Clostridium difficile is linked to a gene homologous with toxin A and is mediated by a conjugative transposon, Tn5398.

An MLS resistance gene designated ermBZ, from a toxigenic Clostridium difficile strain (630) could be transferred between C. difficile strains, and to and from Bacillus subtilis. The intergeneric transfer occurred in the absence of any detectable plasmid DNA and the element responsible for gene transfer entered the recipient's chromosome, behaviour which is characteristic of a conjugative transposon. The element was designated Tn5398 and was found in six C. difficile strains. Tn5398 could be transferred to the non-toxigenic strain C. difficile CD37 which lacks the genes for toxins A and B. Transconjugants from both C. difficile and B. subtilis that had received the ermBZ gene also acquired a sequence of DNA that was homologous to the part of the toxin A gene that coded for the C-terminal repeat region.

Anti-Bacterial Agents↗
PubMed1986

Sequence of the cellulase gene of Clostridium thermocellum coding for endoglucanase B.

The nucleotide sequence of the CelB gene, encoding the extracellular endoglucanase B of Clostridium thermocellum, is reported. The putative start of the 1689 bp coding sequence was assigned to an ATG codon which is preceded by an AGGAGG sequence typical of ribosomal binding sites in Gram-positive bacteria. The amino-terminal end of the deduced protein sequence is similar to signal peptides described for other bacterial secretory proteins. The carboxy-terminal ends of endoglucanases A and B appear to be remarkably homologous. A striking feature of the conserved region is that both proteins contain two reiterated stretches of 23 aminoacids each, separated by 9 residues.

Amino Acid Sequence↗
PubMed1980

A bacterial cell that synthesizes a protein containing the antigenic determinants of rat prolactin.

Bacterial minicells containing three different recombinant plasmids with rat prolactin cDNA sequences inserted at the Pst I site of pBR322 via the poly(dG):poly(dC) joining technique were examined for the expression of rat prolactin antigenic determinants. The three prolactin coding sequences were in the same orientation as the coding sequence of the ampicillin-resistance gene of pBR322. The presence of each of the three recombinant plasmids induced some prolactin synthesis by the bacteria as measured by immunoprecipitation with anti-prolactin antisera. About 10% of the protein synthesized from one of the plasmids, prl 3, precipitated with the antisera. These prolactin antigenic determinants were part of a larger fused protein.

Animals↗
PubMed1989

The class 1 outer membrane protein of Neisseria meningitidis: gene sequence and structural and immunological similarities to gonococcal porins.

The class 1 protein is a major protein of the outer membrane of Neisseria meningitidis, and an important immunodeterminant in humans. The complete nucleotide sequence for the structural gene of a class 1 protein has been determined. The sequence predicts a protein of 374 amino acids, preceded by a typical signal peptide of 19 residues. The hydropathy profile of the predicted protein sequence resembles that of the Escherichia coli and gonococcal porins. The predicted protein sequence of the class 1 protein exhibits considerable structural similarity to the gonococcal porins PIA and PIB. Western blot studies also reveal immunologically conserved domains between the class 1 protein, PIA and PIB. A restriction fragment from the class 1 gene hybridizes to gonococcal genomic fragments in Southern blots. In addition to the class 1 gene coding region there is a large open reading frame on the opposite strand.

Amino Acid Sequence↗
PubMed1997

OXA-15, an extended-spectrum variant of OXA-2 beta-lactamase, isolated from a Pseudomonas aeruginosa strain.

Pseudomonas aeruginosa AH, isolated in Ankara, Turkey, was highly resistant to ceftazidime (MIC, 128 microg/ml) and produced a beta-lactamase that gave a doublet of bands at pIs 8.7 and 8.9. beta-Lactamase production was transferable to P. aeruginosa PU21 by conjugation and was determined by a ca. 450-kb plasmid, pMLH54. The transconjugant and Escherichia coli transformed with the cloned gene showed increased resistance to ceftazidime (especially) and to cefpirome, ceftazidime, ceftriaxone, moxalactam, and aztreonam, but not to carbapenems. Resistance was not reversed by clavulanic acid or tazobactam. Sequencing revealed that the beta-lactamase responsible for this resistance was identical to OXA-2 except that glycine replaced aspartate at position 150. Compared to OXA-2, the new enzyme, named OXA-15, had greater cephalosporinase activity, with increased relative hydrolysis rates for cephaloridine and cephalothin and, most dramatically, for ceftazidime. Cefotaxime and carbapenems remained stable to hydrolysis. Thus, as in the TEM, SHV, and OXA-10 (PSE-2) beta-lactamase families, a minor sequence change in OXA-2 gave a major extension of cephalosporinase activity and contingent resistance. The gene encoding the new beta-lactamase, bla(OXA-15), lay close to the highly conserved 3' end of an integron and had flanking sequences typical of an integron-associated gene cassette. Restriction mapping and partial sequence data indicated that pMLH54 carries an integron with three putative gene cassettes: bla(OXA-15) itself, aadB [coding aminoglycoside nucleotidyltransferase (2")-1a], and an uncharacterized cassette.

Amino Acid Sequence↗
PubMed1994

Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum.

Pyruvate kinase activity is an important element in the flux control of the intermediate metabolism. The purified enzyme from Corynebacterium glutamicum demonstrated a marked sigmoidal dependence of the initial rate on the phosphoenolpyruvate concentration. In the presence of the negative allosteric effector ATP, the phosphoenolpyruvate concentration at the half-maximum rate (S0.5) increased from 1.2 to 2.8 mM, and cooperation, as expressed by the Hill coefficient, increased from 2.0 to 3.2. AMP promoted opposite effects: the S0.5 was decreased to 0.4 mM, and the enzyme exhibited almost no cooperation. The maximum reaction rate was 702 U/mg, which corresponded to an apparent kcat of 2,540 s-1. The enzyme was not influenced by fructose-1,6-diphosphate and used Mn2+ or Co2+ as cations. Sequence determination of the C. glutamicum pyk gene revealed an open reading frame coding for a polypeptide of 475 amino acids. From this information and the molecular mass of the native protein, it follows that the pyruvate kinase is a tetramer of 236 kDa. Comparison of the deduced polypeptide sequence with the sequences of other bacterial pyruvate kinases showed 39 to 44% homology, with some regions being very strongly conserved.

Amino Acid Sequence↗
PubMed1986

Cloning and expression in Escherichia coli of the Streptococcus pneumoniae gene encoding pneumolysin.

A gene bank of Sau3A1-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. Clones expressing the pneumolysin determinant were selected by testing for hemolytic activity which could be inhibited by antibody to purified pneumolysin and by cholesterol. Restriction analysis of pneumolysin-positive recombinant cosmid DNA indicated that the coding sequence for the toxin was located within a 2.9-kilobase-pair (kbp) ClaI DNA fragment. This fragment, which included 0.35 kbp of vector pHC79 DNA, was subcloned into the plasmid pBR322. E. coli cells harboring this recombinant plasmid (designated pJCP20) produced approximately one-third of the amount of pneumolysin found in the donor S. pneumoniae strain. Plasmid pJCP20 was stably maintained in E. coli and resulted in the accumulation of active pneumolysin in the cytoplasm. Western blot analysis showed that E. coli harboring pJCP20 produced two forms of the toxin with molecular weights of 54,000 and 52,000. The lower-molecular-weight form was indistinguishable from native pneumolysin. Subcloning the 2.9-kbp DNA fragment into the expression vector pEV31 allowed the determination of the direction of transcription of the pneumolysin gene. The pneumolysin-coding sequence (approximately 1.5 kbp) has been localized to within a 1.75-kbp segment of pneumococcal DNA.

Bacterial Proteins↗
PubMed2006

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants.

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.

Animals↗
PubMed1996

A novel member of the cspA family of genes that is induced by cold shock in Escherichia coli.

Escherichia coli contains a major cold shock protein, CspA (or CS7.4), whose production is predominantly induced at low temperatures. This bacterium is known to possess five additional genes, each encoding a protein highly similar to CspA (referred to as the CspA family). Here we identified a gene that encodes a cold-shock-inducible analog of CspA and CspB. This newly cloned cspG gene is located at 22 min on the E. coli genetic map, apart from the other cspA family genes. Its gene product (70 amino acids) is 73 and 77% identical to CspA (70 amino acids) and CspB (71 amino acids), respectively. Analyses of a cspG-lacZ transcriptional fusion and Northern (RNA) hybridization revealed that cspG is a low-temperature-responsive gene. Its low-temperature-inducible promoters were determined, and the results indicated that the cspG sequence is highly similar to both the cspA and cspB sequences not only in the coding regions but also in the 5'-upstream noncoding regions surrounding their own promoters.

Amino Acid Sequence↗
PubMed2002

Correlations between Shine-Dalgarno sequences and gene features such as predicted expression levels and operon structures.

This work assesses relationships for 30 complete prokaryotic genomes between the presence of the Shine-Dalgarno (SD) sequence and other gene features, including expression levels, type of start codon, and distance between successive genes. A significant positive correlation of the presence of an SD sequence and the predicted expression level of a gene based on codon usage biases was ascertained, such that predicted highly expressed genes are more likely to possess a strong SD sequence than average genes. Genes with AUG start codons are more likely than genes with other start codons, GUG or UUG, to possess an SD sequence. Genes in close proximity to upstream genes on the same coding strand in most genomes are significantly higher in SD presence. In light of these results, we discuss the role of the SD sequence in translation initiation and its relationship with predicted gene expression levels and with operon structure in both bacterial and archaeal genomes.

Archaeal Proteins↗
PubMed1992

The product of the cellular crk gene consists primarily of SH2 and SH3 regions.

We have cloned and sequenced a complementary DNA encoding the cellular homologue of the transforming oncogene v-crk of avian sarcoma virus CT10. This complementary DNA contains an open reading frame of 915 base pairs that encodes a polypeptide of 305 amino acids. The first 205 amino acids of this c-Crk protein were identical to those of the CT10 encoded v-Crk protein, with the exception of 5 amino acids. Like v-Crk, this portion of c-Crk contained one each of the Src homology domains SH2, SH2', and SH3. The 100 carboxy-terminal amino acids of c-Crk protein, which are not coded for in the CT10 viral genome, contain another SH3 region. We found limited sequence homology between c-crk and the avian retrovirus genome, which explains recombination events in the transduction of this protooncogene. Using a polyclonal antiserum made against bacterially expressed v-crk, we identified a 35-kilodalton protein in normal chicken embryo fibroblasts and in all embryonic chicken tissues examined. This 35-kilodalton protein was indistinguishable from a polypeptide made by in vitro translation of c-crk complementary DNA.

Amino Acid Sequence↗