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R C Levesque

Publications and source records attributed to R C Levesque.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of a PSE-4 mutant with different properties in relation to penicillanic acid sulfones: importance of residues 216 to 218 in class A beta-lactamases.

Class A beta-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and tazobactam. An examination of multiple alignments indicated that amino acids 216 to 218 differed among class A enzymes. By random replacement mutagenesis of codons 216 to 218 in PSE-4, a complete library consisting of 40,864 mutants was created. The library of mutants with mutations at positions 216 to 218 in PSE-4 was screened on carbenicillin and ampicillin with the inactivator sulbactam; a collection of 14 mutants was selected, and their bla genes were completely sequenced. Purified wild-type and mutant PSE-4 beta-lactamases were used to measure kinetic parameters. One enzyme, V216S:T217A:G218R, was examined for its peculiar pattern of inhibition. There was an increase in the Km from 68 microM for the wild type to 271 microM for the mutant for carbenicillin and 33 to 216 microM for ampicillin. Relative to the wild-type PSE-4 enzyme, 37- and 30-fold increases in Ki values were observed for the mutant enzyme for sulbactam and tazobactam, respectively. The results that were obtained suggested that positions 216 to 218 are important for interactions with penicillanic acid sulfone inhibitors. In contrast, V216 and A217 in the TEM-1 class A beta-lactamase do not tolerate amino acid residue substitutions. However, for the PSE-4 beta-lactamase, 11 of 14 mutants from the library of mutants with mutations at positions 216 to 218 whose sequences were determined had substitutions at position 216 (G, R, A, S) and position 217 (A, S). The data showed the importance of residues 216 to 218 in their atomic interactions with inactivators in the PSE-4 beta-lactamase structure.

Binding Sites↗
PubMed1998

ASD-GFP vectors for in vivo expression technology in Pseudomonas aeruginosa and other gram-negative bacteria.

We describe the construction of promoter probe vectors designed for identification of bacterial genes induced in vitro and/or in vivo and for measurement of gene expression levels for in vivo expression technology. These plasmids use the Pseudomonas aeruginosa aspartate beta-semialdehyde dehydrogenase (asd) gene as a selectable marker and beta-galactosidase (pIVPRO, 10.88 kb) or mutant green fluorescent protein with enhanced fluorescence properties (mut3GFP, pIVET-GFP, 5.48 kb) as reporter gene systems. The proposed strategies can be adapted for use in most Gram-negative bacteria.

Aspartate-Semialdehyde Dehydrogenase↗
PubMed1996

Secondary structures and features of the 18S, 5.8S and 26S ribosomal RNAs from the Apicomplexan parasite Toxoplasma gondii.

The two major subunits of the ribosomal RNA (rRNA) of Toxoplasma gondii, 18S and 26S, as well as 5.8S, have been sequenced and folded according to known consensus and established secondary structures. Conserved and variable nucleotide (nt) regions were identified using multiple alignments with rRNA sequences of selected organisms. The 18S rRNA showed a well conserved core structure of 48 stems and a hypervariable V4 region identified four additional stems including a pseudoknot. The 18S rRNA contained an additional helix in the V2 region located between nt 204 to 258. We noted that T. gondii 18S does not have a true V6 region, but was organized as a motif of a simple stem. T. gondii 26S had a conserved core structure of 83 stems and its expansion segments, so-called divergent domains, demonstrated a high degree of similarity with secondary structures from rRNA of dinoflagellates and ciliates. For the T. gondii 26S sequence, we found two additional stems, D3d and D3e, composed of 140 nt having a higher deltaG value. These segments are absent from the prokaryotic rRNA structures, whereas the hypervariable V4 region of the small subunit is not as variable. The well preserved structures could indicate an additional function for the eukaryotic ribosome.

Animals↗
PubMed1996

Toxoplasma gondii: structure and characterization of the 26S ribosomal RNA and peptidyl transferase domain.

The 26S, 5.8S, and the intergenic spacer ribosomal DNAs (rDNA) of Toxoplasma gondii have been cloned and completely sequenced from both DNA strands. The length of the large subunit was found to be 3487 nucleotides and the 5.8S was 153 nucleotides long. These formed the large rRNA subunit of Toxoplasma and were mapped in the rDNA unit known to be repeated 110 times in a head-to-tail fashion in the genome. Primer extension analysis and multiple alignments localized the 5' end point of the two rRNAs. Comparisons with Toxoplasma rDNA by nucleic acid homology studies gave 76% similarity with the dinoflagellate Prorocentrum micans, 66% with yeast Saccharomyces cerevisiae, and 64% with the ciliate Tetrahymena thermophila. Similarity was apparent in the conserved core structure of the large subunit rRNA and divergent sequences were identified in the so-called divergent domains. Construction of a secondary structure model of the peptidyl transferase center of the large rRNA revealed similarities with the same domain from other life forms.

Animals↗
PubMed1995

Primary structure of OXA-3 and phylogeny of oxacillin-hydrolyzing class D beta-lactamases.

We determined the nucleotide sequence of the blaOXA-3(pMG25) gene from Pseudomonas aeruginosa. The bla structural gene encoded a protein of 275 amino acids representing one monomer of 31,879 Da for the OXA-3 enzyme. Comparisons between the OXA-3 nucleotide and amino acid sequences and those of class A, B, C, and D beta-lactamases were performed. An alignment of the eight known class D beta-lactamases including OXA-3 demonstrated the presence of conserved amino acids. In addition, conserved motifs composed of identical amino acids typical of penicillin-recognizing proteins and specific class D motifs were identified. These conserved motifs were considered for possible roles in the structure and function of oxacillinases. On the basis of the alignment and identity scores, a dendrogram was constructed. The phylogenetic data obtained revealed five groups of class D beta-lactamases with large evolutionary distances between each group.

Amino Acid Sequence↗
PubMed1995

Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species.

Bacillus thuringiensis spacer regions between the 16S and 23S rRNAs were amplified with conserved primers, designated 19-mer and 23-mer primers. A spacer region of 144 bp was determined for all of 6 B. thuringiensis strains, 7 B. thuringiensis subspecies, and 11 B. thuringiensis field isolates, as well as for the closely related species Bacillus cereus and Bacillus anthracis. Computer analysis and alignment of nucleotide sequences identified three mutations and one deletion in the intergenic spacer region (ISR) of B. thuringiensis subsp. kurstaki HD-1 when compared with ISR sequences from other subspecies. The same differences were identified between the ISR of B. thuringiensis strains and the ISR of B. cereus and B. anthracis. These minor differences do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe.

Bacillus anthracis↗
PubMed1995

Comparative Analysis of the 16S to 23S Ribosomal Intergenic Spacer Sequences of Bacillus thuringiensis Strains and Subspecies and of Closely Related Species.

Volume 61, no. 4, p. 1624, column 2, lines 38-41: The sentence should read "For example, at position 21, the G nucleotide (Fig. 1) was present in all the ISR B. thuringiensis subspecies except for B. thuringiensis subsp. tenebrionis (Te4), which contained an A." Page 1624, column 2, line 45: "Position 62" should read "position 11." Page 1624, column 2, line 47: "Position 90" should read "position 39." Page 1624, column 2, line 49: "Position 83" should read "position 32." Page 1625, column 1, line 3: "Position 83" should read "position 32." Page 1626, column 1, line 1: "Positions 62, 90, and 165, and one deletion at position 83" should read "positions 11, 39, and 114, and one deletion at position 32." [This corrects the article on p. 1623 in vol. 61.].

Journal Article↗
PubMed1993

Role of Ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type beta-lactamases probed by site-directed mutagenesis and three-dimensional modeling.

A growing number of extended spectrum SHV-type beta-lactamases capable of hydrolyzing third-generation cephalosporins such as cefotaxime and ceftazidime have been reported. These new enzymes differ by a few amino acids from SHV-1, an enzyme incapable of hydrolyzing these drugs. Two of these substitutions, Gly-238-->Ser and Glu-240-->Lys, are in a key beta-strand of the catalytic site of class A beta-lactamases. To understand the structural basis of these new activities, we first subcloned the DNA region coding for SHV-1 and SHV-2 and did site-directed mutagenesis to create two mutant SHV-1 proteins containing Ser and Glu or Gly and Lys and two mutant SHV-2 proteins containing Gly and Glu or Ser and Lys in positions 238 and 240, respectively. Phenotypic analysis (antibiograms and minimum inhibitory concentrations) and activity spectra of mutant enzymes showed that Ser-238 is critical for cefotaxime hydrolysis whereas both Ser-238 and Lys-240 are needed for strong ceftazidime hydrolysis. A three-dimensional model for SHV beta-lactamase complexes was constructed using the crystallographic structure of the homologous Bacillus licheniformis beta-lactamase, the complex of cefotaxime with the Streptomyces sp. R61 D-alanyl-D-alanine peptidase, and the complex of aztreonam with the Citrobacter freundii beta-lactamase. The modeling of SHV beta-lactamase complexes showed that factors which are most likely to correlate with binding and kinetic data are the size of the relatively buried amino acid at position 238 and the electrostatic charge of the exposed group at position 240.

Amino Acid Sequence↗
PubMed1993

Multiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis.

A rapid identification of Bacillus thuringiensis strains was established by using multiplex polymerase chain reaction (PCR). Primers of high homology specific to regions within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each strain of B. thuringiensis subsp. kurstaki. Differentiation among these strains was made on the basis of the electrophoretic pattern of the PCR products. Known B. thuringiensis subsp. kurstaki strains as well as unidentified strains isolated from insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a two-step PCR containing five primers capable of distinguishing between the strains giving products of 1,500, 858, and 653 bp for the CryIA(a) CryIA(b), and CryIA(c) genes, respectively. The method can be applied to rapidly detect the strains of B. thuringiensis subsp. kurstaki in commercial formulations and in the field.

Animals↗
PubMed1993

Detection of the pathogenic parasite Toxoplasma gondii by specific amplification of ribosomal sequences using comultiplex polymerase chain reaction.

Amplification of DNA sequences from ribosomal DNA (rDNA) was tested as a specific and sensitive method for the detection of small numbers of Toxoplasma gondii tachyzoite cells. We applied the polymerase chain reaction (PCR) on the basis of detection of the 110-fold repetitive rDNA as a target by using (i) DNA sequences within the small ribosomal subunit known to be universal and conserved in all eukaryotes and (ii) small ribosomal subunit and intergenic spacer rDNA sequences known to be T. gondii species specific. The level of sensitivity obtained from a crude cell lysate allowed the detection of as few as one parasite visualized directly as a specific PCR product in agarose gels. By using a combination of universal and T. gondii species-specific primers, we propose a comultiplex-based PCR approach as a new diagnostic tool. The combination of sensitivity, specificity, and built-in positive and negative PCR controls should make detection of the rDNA sequences by comultiplex PCR a useful clinical test for the diagnosis of toxoplasmosis and for epidemiological studies. Finally, the idea of a built-in positive control to support or counter the T. gondii-specific PCR result is novel and is a notable advance.

Animals↗
PubMed1992

Cloning, sequencing and expression in Escherichia coli of the ptsI gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system from Streptococcus salivarius.

We present the cloning and sequencing of the ptsI gene, encoding enzyme I (EI) of the phosphoenolpyruvate (PEP): sugar phosphotransferase (PTS) transport system from Streptococcus salivarius. The ptsI gene corresponds to an open reading frame of 1731 nucleotides, which translates into a putative 577-amino acid (aa) protein with a M(r) of 62,948 and a pI of 4.49. The EI was produced in Escherichia coli under the control of its own promoter located immediately upstream of ptsI, a situation never previously reported for any other gene coding for an EI. The deduced aa sequence of the S. salivarius EI shows a high degree of similarity with the E. coli EI and the EI moiety of the multiphosphoryl transfer protein from Rhodobacter capsulatus. The S. salivarius EI also shares a highly conserved aa cluster with a non-PTS protein, the maize pyruvate:orthophosphate dikinase. The conserved cluster is located in a domain which is hypothesized to be the PEP-binding site.

Amino Acid Sequence↗
PubMed1992

Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene.

The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.

Animals↗
PubMed1992

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.

Ampicillin↗
PubMed1992

Phylogeny of LCR-1 and OXA-5 with class A and class D beta-lactamases.

The nucleotide sequences of blaLCR-1 and blaOXA-5 beta-lactamase genes have been determined. Polypeptide products of 260 and 267 amino acids with estimated molecular masses of 27 120 Da and 27,387 Da were obtained for the mature form of LCR-1 and OXA-5 proteins. A progressive alignment was used to evaluate the extent of identity between LCR-1 and OXA-5 with 29 other beta-lactamase amino acid sequences. The data showed that both belong to class D. We identified amino acids conserved in 24 positions for class A beta-lactamases and in 28 positions for five class D enzymes. The structural similarities between class A and class D beta-lactamases are more extensive than indicated by earlier biochemical studies with overall 16% identity between both classes. From the alignment, dendograms were constructed with a distance-matrix and parsimony methods which defined three major groups of proteins subdivided into clusters giving insight on beta-lactamase phylogeny and evolution.

Amino Acid Sequence↗
PubMed1992

Identification of a ROB-1 beta-lactamase in Haemophilus ducreyi.

A collection of 100 clinical isolates of Haemophilus ducreyi from Thailand were all found to harbor a 5.4-kb plasmid, designated pTH126, which was shown to contain the bla ROB-1 gene. Restriction enzyme analysis and DNA-DNA hybridization studies confirmed that pTH126 was similar to the ROB-1 beta-lactamase plasmid pVM105 from Actinobacillus pleuropneumoniae. In approximately one-half of the isolates, pTH126 was found together with pHD131, which mediates TEM-1 beta-lactamase production.

Centrifugation, Density Gradient↗
PubMed1991

Characterization of the blaCARB-3 gene encoding the carbenicillinase-3 beta-lactamase of Pseudomonas aeruginosa.

We have isolated the blaCARB-3 structural gene encoding the CARB-3 carbenicillinase of Pseudomonas aeruginosa strain Cilote, tested the specificity of blaCARB-3 DNA probes and determined the nucleotide sequence of blaCARB-3. Three restriction fragment probes internal or delimiting the blaCARB-3 structural gene were hybridized with purified plasmid DNA coding for 18 other beta-lactamases (Blas). Under high-stringency conditions, only blaPSE-1, blaPSE-4, and blaCARB-4 sequences cross-hybridized with blaCARB-3. Sequencing of blaCARB-3 identified the structural gene which encodes a polypeptide product of 268 amino acids with a calculated estimated Mr of 29,246 for the mature form of the protein. Homology studies and computer analysis of primary structures confirmed that CARB-3 is a class-A Bla. The CARB-3 carbenicillinase differs from PSE-4 at two positions: Phe (PSE-4) instead of Leu188 (CARB-3), and Glu (PSE-4) instead of Ala266 (CARB-3), which changes the isoelectric value from (PSE-4) 5.4 to 5.75 (CARB-3). The possible effects of these two mutations were examined by comparisons on the 2 A crystal structure of the Staphylococcus aureus penicillinase, and they were shown to be silent substitutions causing no changes in the phenotype. The nucleic acid hybridization studies and sequence data confirmed that carbenicillinase-encoding bla genes are closely related and that blaCARB-3 is a variant of blaPSE-4.

Amino Acid Sequence↗