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B Jaurin

Publications and source records attributed to B Jaurin.

At least 19 recordsLinked to original sources
PubMed1990

Nucleotide sequence and T cell epitopes of a membrane protein of Francisella tularensis.

A 17-kDa major membrane protein of the facultative intracellular bacterium Francisella tularensis is recognized by T cells from individuals previously undergoing tularemia or tularemia vaccination. Here the nucleotide sequence of the encoding gene is presented. The A + T rich sequence deduced a protein of 149 amino acids with a predicted m.w. of 15,772. Evidence is presented suggesting that the 17-kDa protein is a lipoprotein. Also, homology was found to a peptidoglycan-associated lipoprotein of Escherichia coli. Thirteen overlapping synthetic peptides encompassing the entire deduced protein were tested on lymphocytes from F. tularensis-primed individuals. Three peptides induced a T cell response, as disclosed by lymphocyte proliferation and production of IL-2 and IFN-gamma. Out of five responding individuals, only one recognized more than one of the peptides. A murine mAb recognized an epitope separate from the T cell-reactive peptides. The identified T cell epitopes are presumably relevant to the host defense against F. tularensis, especially because IFN-gamma is known to enhance the antimicrobial activity of the host against intracellular bacteria.

Amino Acid Sequence↗
PubMed1990

Molecular analysis of beta-lactamases from four species of Streptomyces: comparison of amino acid sequences with those of other beta-lactamases.

Genes encoding extracellular beta-lactamases of Streptomyces badius, Streptomyces cacaoi, Streptomyces fradiae and Streptomyces lavendulae were cloned and mapped in Streptomyces lividans. DNA sequence analysis of the beta-lactamase genes revealed a high overall G + C content, ranging from 71 to 75 mol%, with a G + C content of 95 mol% at the third position of the codons for all four genes. The primary structure of the beta-lactamases including their signal peptides was deduced. The four beta-lactamases exhibited homology to each other and to class A beta-lactamases from other bacterial genera. We suggest that Streptomyces beta-lactamases are representatives of a superfamily of genes, from which class A beta-lactamases of Gram-negative bacteria may have evolved.

Amino Acid Sequence↗
PubMed1990

Streptococcus pneumoniae possesses canonical Escherichia coli (sigma 70) promoters.

Seventeen DNA fragments from Streptococcus pneumoniae were randomly cloned in Escherichia coli with selection for promoter activity. The fragments were sequenced and the promoter locations were determined by primer extension analysis. Examination for sites similar to the E. coli major consensus promoter sequence revealed such a site in each of the seventeen fragments, located five to eight base pairs upstream of the point at which transcription was initiated in the E. coli host. Thus, the abundance of promoter activity found in pneumococcal DNA cloned in E. coli hosts arises primarily from sigma-70-type promoter structures. Combined with the observation that such sequences are usually found just upstream of, but not within, pneumococcal genes, this implies that one class (perhaps the major class) of pneumococcal promoters closely resembles the canonical E. coli promoter consensus.

Base Sequence↗
PubMed1990

Identification of Francisella species and discrimination of type A and type B strains of F. tularensis by 16S rRNA analysis.

Tularemia is a zoonotic disease, occurring throughout the Northern Hemisphere. The causative agent, the bacterium Francisella tularensis, is represented by two main types. Type A is found in North America, whereas type B is mainly found in Asia and Europe and to a minor extent in North America. No routine technique for rapid diagnosis of tularemia has been generally applied. We have partially sequenced 16S rRNAs of two F. tularensis strains, as well as the closely related Francisella novicida. Of 550 nucleotides analyzed, only one difference in 16S rRNA primary sequence was found. This 16S rRNA analysis enabled the construction of oligonucleotides to be used as genus- and type-specific probes. Such probes were utilized for the establishment of a method for rapid and selective detection of the organism. This method allowed identification of Francisella spp. at the level of genus and also discrimination of type A and type B strains of F. tularensis. The analysis also permitted the detection of F. tularensis in spleen tissue from mice infected with the bacterium. The results presented will enable studies on the epizootiology and epidemiology of Francisella spp.

Animals↗
PubMed1989

Molecular cloning and expression of a T-cell stimulating membrane protein of Francisella tularensis.

The isolation and expression in Escherichia coli of a gene encoding a T-cell stimulating 17 kiloDalton (kDa) membrane protein of Francisella tularensis is described. A genomic library of DNA from the live vaccine strain LVS of F. tularensis was constructed in the E. coli expression vector phage lambda gt11. The library was probed with antibodies directed against the 17 kDa protein. One recombinant phage was isolated, containing a 2.8 kilobase (kb) DNA insert. The insert was cleaved and a resulting 1.2 kb fragment was found to express the 17 kDa protein. The 1.2 kb fragment was inserted in the high copy number plasmid pUC18 and expressed in E. coli. Membrane preparations of these bacteria induced a response in T cells from F. tularensis-primed individuals but not in T cells from non-primed individuals. The cloned gene may become useful in studies on host interaction with F. tularensis and enable a precise identification of bacterial structures involved in the T-cell response.

Adult↗
PubMed1989

Transcriptional induction of Streptomyces cacaoi beta-lactamase by a beta-lactam compound.

The soil bacterium Streptomyces cacaoi produces an extracellular beta-lactamase. The beta-lactamase expression could be induced by the beta-lactam compound 6-amino penicillinoic acid (6-APA). In liquid cultures, a 50-fold increase in beta-lactamase expression was observed within the first three hours after addition of 6-APA. Using the cloned beta-lactamase gene as a probe, it was shown that this increase was mediated at the level of transcriptional initiation. The start point of the induced beta-lactamase transcript was determined, and the nucleotide sequence of the promoter region was analysed. No noticeable homology was found to control regions of inducible beta-lactamase genes of other bacteria. A striking feature was the presence of six direct repeats (ten base pairs each) upstream of the promoter region. Thus, an example of an inducible regulatory gene system in this Gram-positive microorganism is presented. Also, the primary structure of the beta-lactamase was deduced, showing a high degree of homology with class A beta-lactamases.

Amino Acid Sequence↗
PubMed1988

Beta-lactamase genes of Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae: cloning and expression in Streptomyces lividans.

Genes encoding extracellular beta-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The beta-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi beta-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The beta-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the beta-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three beta-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi beta-lactamases exhibited a 10-100-times lower activity in S. lividans, whereas the S. fradiae beta-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.

Biological Transport↗
PubMed1987

Chromogenic identification of promoters in Streptomyces lividans by using an ampC beta-lactamase promoter-probe vector.

A promoter-probe system, based on the ampC beta-lactamase gene of Escherichia coli, has been developed for the isolation and characterization of transcriptional signals in the gram-positive bacterium Streptomyces lividans. The promoter-probe vector, denoted pJAS14, has the SLP1.2 replicon with a copy number of four-five plasmids per cell. It contains a unique BamHI site just in front of the ampC ribosome-binding site, and upstream of this BamHI site a transcriptional terminator signal that prevents readthrough transcription from plasmid-borne promoters has been inserted. Using pJAS14, we have shot-gun cloned chromosomal DNA from S. lividans and S. lavendulae into the BamHI site, and isolated a number of promoter containing DNA fragments by the use of the chromogenic cephalosporin nitrocefin. On plates, we identified promoters of varying strengths and also with differences in nutritional and temporal expression. Using liquid cultures of S. lividans, it has been demonstrated that one promoter, denoted P1 (SEP8), as well as the ampC gene of E. coli, show activity corresponding to the vegetative growth of the cells. The P1 (SEP8) promoter was shown to be expressed also in E. coli, and it initiates RNA synthesis at exactly the same nucleotides in both S. lividans and E. coli. The promoter shows good homology to the E. coli promoter consensus sequence in both the -35 and -10 regions. Thus, this promoter is a representative of the SEP (Streptomyces E. coli-type promoter) class of promoters recently described (Jaurin and Cohen 1985). This indicates that an S. lividans RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding E. coli enzyme.

Base Sequence↗
PubMed1985

Streptomyces contain Escherichia coli-type A + T-rich promoters having novel structural features.

We describe here the isolation and characterization of a class of A + T-rich transcriptionally active sequences in the filamentous antibiotic-producing Gram-positive bacterial genus Streptomyces. These regions, which digress dramatically in base composition from the 73% G + C composition characteristic of the Streptomyces genome, promote gene expression in both Escherichia coli and Streptomyces lividans and contain the major elements that determine promoter strength in E. coli. The Streptomyces-E. coli-type promoters (SEP) also show novel structural features that include multiple direct repeats within the promoter region as well as a specific hexameric sequence in the vicinity of the mRNA start point.

Base Composition↗
PubMed1984

Streptomyces lividans RNA polymerase recognizes and uses Escherichia coli transcriptional signals.

We report here the use of a novel genetic approach for the study of transcriptional control in Streptomyces lividans. Using up-promoter mutants of the ampC beta-lactamase gene of Escherichia coli, we have shown that mutations in each of the regions that define the major determinants of promoter strength in E. coli-i.e., the -35 region, the -10 region, and the intervening "spacer" region-lead to increased synthesis of ampC mRNA in S. lividans, just as they do in their host of origin. Results are also presented showing that the ampC transcriptional terminator is functional in S. lividans. Taken together, these findings indicate that Streptomyces have an RNA polymerase that recognizes and uses the various components of E. coli transcriptional signals, and imply that, notwithstanding the high GC content (i.e., 73%) of the Streptomyces genome, these organisms have indigenous promoters similar to those found in E. coli.

Bacterial Proteins↗
PubMed1983

The inhibition of class C beta-lactamases by boronic acids.

Aromatic boronic acids are reversible inhibitors of the recently classified class C beta-lactamases. The boronic acids studied include ortho-, meta- and para-methyl-, -hydroxymethyl- and -formyl-phenylboronic acid. The beta-lactamases were chromosomally-encoded enzymes, one from Pseudomonas aeruginosa, and the other specified by the ampC gene of Escherichia coli. The inhibition may be correlated with our finding that these beta-lactamases are serine enzymes, i.e. their function entails the hydroxy group of a serine residue acting as a nucleophile.

Binding Sites↗
PubMed1983

Insertion of IS2 creates a novel ampC promoter in Escherichia coli.

A class of ampC beta-lactamase-hyperproducing mutants of Escherichia coli were shown to have the insertion element IS2 inserted into the ampC promoter. The insertion of IS2 in orientation II created a novel promoter in which the -35 region and the 17 bp long spacing sequence between the two consensus sequences are present in IS2 DNA, whereas the -10 region from the original ampC promoter is retained. In vitro transcription revealed that the transcription initiation site in the ampC::IS2 mutants was identical with that of ampC wild-type promoter. The novel promoter exhibited a 20-fold increase in promoter strength relative to the original ampC promoter, presumably due to the increase in the spacing sequence from 16 to 17 bp. The evolution of transposable elements and of control elements such as promoters are discussed on the basis of the findings described herein.

Base Sequence↗
PubMed1983

Overlapping genes.

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Base Sequence↗
PubMed1982

The acyl-enzyme mechanism of beta-lactamase action. The evidence for class C Beta-lactamases.

Methanol or ethanol can replace water in the action of certain chromosomal beta-lactamases on benzylpenicillin: the products are alpha-methyl or alpha-ethyl benzylpenicilloate. The beta-lactamases were from a mutant of Pseudomonas aeruginosa 18S that produces the enzyme constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol. 128, 155-159] and from Escherichia coli K12 (the ampC beta-lactamase) [Lindström, Boman & Steele (1970) J. Bacteriol. 101, 218-231]. The variation of the rates of alcoholysis and hydrolysis with concentration of alcohol show that the rate-determining step is breakdown of an intermediate. This intermediate is likely to be the acyl-enzyme. The esters, alpha-methyl or alpha-ethyl benzylpenicilloate, are themselves substrates for the Pseudomonas beta-lactamase, benzylpenicilloic acid being formed. Thus this beta-lactamase can be an esterase. The kinetics for the hydrolysis of cloxacillin by the Pseudomonas beta-lactamase are consistent with the acyl-enzyme, formed by acylation of serine-80, being an intermediate in the overall hydrolysis.

Anti-Bacterial Agents↗
PubMed1982

Active sites of beta-lactamases. The chromosomal beta-lactamases of Pseudomonas aeruginosa and Escherichia coli.

An acyl-enzyme was isolated from certain chromosomal beta-lactamases and a penicillin. The penicillin was cloxacillin which, although it is a substrate for these enzymes, has such a low kcat. that it functions as an inhibitor. The enzymes were from the mutant of Pseudomonas aeruginosa 18 S that produces the beta-lactamase constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol., in the press] and from Escherichia coli K-12 (the ampC beta-lactamase) [Boman, Nordström & Normak (1974) Ann. N.Y. Acad. Sci. 235, 569-586]. The acyl-enzymes have been degraded to determine the residue labelled, and the sequence around it. The residue labelled is serine. The sequences around the labelled serine in these two beta-lactamases are exceedingly similar. However, the sequences are quite different from those around the active site serine in the beta-lactamases previously studied. There is thus more than one class of serine beta-lactamases.

Amino Acid Sequence↗
PubMed1982

Sequence elements determining ampC promoter strength in E. coli.

A number of spontaneous up-promoter mutations have been isolated in the ampC beta-lactamase gene of Escherichia coli. The mutants were analyzed by DNA sequencing, and the level of ampC gene expression was determined. Six mutants with a 21-fold increase in promoter strength compared with the wild-type were mutated in the -35 promoter region from TTGTCA to the consensus sequence TTGACA . The -10 region sequence TACAAT was mutated to the consensus sequence TATAAT in three mutants exhibiting an ampC promoter seven times stronger than the wild-type. We have previously described a 1-bp insertion mutant ( Jaurin et al., 1981) that changes the inter-region distance to the consensus 17 bp. Thus, all the up-mutations found in the ampC promoter represent corrections of the three major discrepancies between the ampC promoter and the consensus E. coli promoter. We conclude that the three consensus elements of E. coli promoters, the -35 and -10 regions and an optimal inter-region distance of 17 bp, are the main elements determining the promoter strength.

Ampicillin↗
PubMed1982

Overlap between ampC and frd operons on the Escherichia coli chromosome.

The promoter for the Escherichia coli ampC beta-lactamase gene is shown to be located within the last gene of the fumarate reductase (frd) operon. Evidence is presented that the ampC attenuator serves as the terminator for transcription of this preceding operon. The nucleotide sequence was determined for two proteins that were shown to be encoded by a DNA segment preceding the ampC gene. The two proteins consisted of 131 and 119 triplets and had molecular weights of 15,000 and 13,100, respectively. The twelve COOH-terminal amino acids of the 13,100 molecular weight protein were found to overlap the ampC promoter. Accordingly, a G.C insertion in the promoter gave both increased transcription of ampC and a frameshift in this overlapping gene, resulting in readthrough proteins. Thus, we describe a type of very compact genetic organization of operons in prokaryotes.

Base Sequence↗