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R C Levesque

Publications and source records attributed to R C Levesque.

54 records · Page 3Linked to original sources
PubMed1991

Molecular characterization of the class II multiresistance transposable element Tn1403 from Pseudomonas aeruginosa.

Transposon Tn1403 is a 19.9-kb multiresistance class II transposable element originally found on the RPL11 plasmid from a clinical isolate of Pseudomonas aeruginosa. It encodes resistance to ampicillin (PSE-1 beta-lactamase), streptomycin and spectinomycin (aadA and aphC), and chloramphenicol (cat). It has structural homology with the tnpM and tnpI sequences of Tn21 and inverted repeats and res and tnpR sequences of Tn501, but it has no structural homology nor functional complementation with the resolvase gene of Tn21 or Tn3. Sequence analysis revealed long inverted repeats at each extremity of Tn1403 containing 38-bp inverted repeats that were 97.4% similar to those of Tn1721 and 5-bp direct repeats. Transposition assays showed a low frequency of transposition (3.5 x 10(-6)) compared with that of Tn3 (3.3 x 10(-3)) and no resolution of cointegrates.

Base Sequence↗
PubMed1991

Molecular distribution of ROB-1 beta-lactamase in Actinobacillus pleuropneumoniae.

The plasmid content and molecular distribution of the ROB-1 beta-lactamase was investigated in 31 swine isolates of Actinobacillus pleuropneumoniae. Three types of plasmid patterns were observed in ampicillin-resistant isolates: a predominant one consisting of 2.6-, 2.9-, and 5.0-kb plasmids and two other patterns consisting of 4.2- and 5.5-kb plasmids and of a 6.8-kb plasmid. Plasmid DNA preparations were hybridized with a blaROB-1 intragenic fragment from the Haemophilus influenzae Rrob plasmid. Positive hybridizations were observed with all ampicillin-resistant isolates. The blaROB-1 gene was found on 3 plasmids of 2.6, 5.5, and 6.8 kb. One swine isolate of Pasteurella multocida also had a 2.6-kb plasmid bearing blaROB-1. ROB-1 was confirmed in typical isolates by isoelectric focusing and blaROB-1 sequences were confirmed by polymerase chain reaction amplification with an intragenic set of primers. Plasmids bearing blaROB-1 were successfully electro-transformed in a susceptible A. pleuropneumoniae isolate. These results emphasize the importance of ROB-1 in A. pleuropneumoniae and identify a possible reservoir of beta-lactam resistance.

Actinobacillus↗
PubMed1991

Construction of a DNA probe and detection of Actinobacillus pleuropneumoniae by using polymerase chain reaction.

A 1.5-kb Actinobacillus pleuropneumoniae 4074 DNA fragment from a genomic library was found to hybridization. No cross-hybridization hybridization. No cross-hybridization was detected with DNAs from hemolytic members of the family Pasteurellaceae. From the nucleotide sequence of the putative genomic probe, three primers were synthesized for use in polymerase chain reactions (PCRs), with 31 strains tested by using purified and crude DNA targets. PCR amplification products of 610 and 985 bp were observed in nucleic acids extracted from the 12 known serotypes and a biotype 2 strain. Template DNAs from other gram-negative and gram-positive bacteria, some of them found in the normal flora of swine and the upper respiratory tract, were not amplified by PCR. The only exception was an amplification of a similar 610- or 985-bp sequence in Actinobacillus lignieresii, a species that is closely related to A. pleuropneumoniae but that has never been isolated from swine. Amplification of specific A. pleuropneumoniae sequences by PCR directly from clinical specimens may find applications in the identification of asymptomatic carriers as well as in efforts to eradicate porcine pleuropneumonia.

Actinobacillus↗
PubMed1990

Nucleotide sequence of the PSE-4 carbenicillinase gene and correlations with the Staphylococcus aureus PC1 beta-lactamase crystal structure.

The nucleotide sequence of the PSE-4 beta-lactamase gene from Pseudomonas aeruginosa strain Dalgleish has been determined. The structural gene encodes a polypeptide product of 252 amino acids with an estimated molecular mass of 29,246 Da for the mature form of the protein. The PSE-4 gene has limited homology with other beta-lactamases at the DNA level. An alignment of all known class A beta-lactamases permitted as to identify specific residues important for enzyme structure and function. To confirm observations based on the linear sequences, we designed a new molecular model for PSE-4 beta-lactamase based on x-ray data from the Staphylococcus aureus PC1 beta-lactamase at 2.0-A resolution. The structural similarities between PSE-4 and class A beta-lactamases are more extensive than indicated by earlier biochemical studies. The combined structural and sequence information now available for a series of beta-lactamases identifies conserved residues in these molecules, giving insight of their divergence and ancestry. Analysis of the PSE-4 flanking DNA sequences revealed an integration site common to antibiotic resistance genes inserted into transposons of the Tn21 family with the target integration sequence AAGTT.

Amino Acid Sequence↗
PubMed1990

Sequence analysis and evolutionary perspectives of ROB-1 beta-lactamase.

The nucleotide sequence of the ROB-1 beta-lactamase gene from Haemophilus influenzae plasmid RRob was determined. The structural gene encodes a polypeptide of 305 amino acids, with an estimated molecular mass of 30,424 for the mature form of the protein. The ROB-1 gene showed low homologies with other beta-lactamases at the nucleic acid level. By using two statistical computer methods, assessment of the extent of similarity between ROB-1 and other known beta-lactamase amino acid sequences suggested that ROB-1 is a class A enzyme. Alignment of class A beta-lactamases with ROB-1 identified conserved residues. The use of a mutation matrix for detecting distance relationships indicated that ROB-1 has higher values and homologies with beta-lactamases of gram-positive bacteria, giving insight into its ancestry and divergence.

Amino Acid Sequence↗
PubMed1990

Cloning of SHV-2, OHIO-1, and OXA-6 beta-lactamases and cloning and sequencing of SHV-1 beta-lactamase.

Molecular cloning of DNA fragments permitted the isolation of structural genes coding for SHV-1, SHV-2, OHIO-1, and OXA-6 beta-lactamases. DNA probes were constructed for SHV-1, and under conditions of high stringency, hybridization was observed only between SHV-1 and SHV-2. Oligonucleotide typing with a 15-mer SHV-1 probe was capable of discriminating between SHV-1 and SHV-2 but not OHIO-1. The nucleotide sequence of the SHV-1 beta-lactamase gene from plasmid R974 has been determined. The structural gene encodes a polypeptide product which differs by 9 residues from the p453 (SHV-1) PIT-2 enzyme determined by peptide sequencing. The significance of each mutation was assessed by alignment of amino acid sequences and comparisons with the Staphylococcus aureus PC1 penicillinase crystal structure. Structural similarities between SHV-1 and class A beta-lactamases are extensive, with amino acid identities of 88.9% between SHV-1 and LEN-1, 91.8% between SHV-1 and OHIO-1, and 63.7% between SHV-1 and TEM-1.

Base Sequence↗
PubMed1990

Nucleotide sequence and phylogeny of SHV-2 beta-lactamase.

We determined the nucleotide sequence of the blaSHV-2(pBP60-1) gene from Klebsiella ozaenae which confers resistance to broad-spectrum cephalosporins. The structural gene encodes a polypeptide product of 286 amino acids, and the estimated molecular weight of the mature protein is 28,900. Amino acid sequence comparison of the SHV-2pBP60-1 enzyme with all known class A beta-lactamases and homology studies showed that the residues were highly conserved. Furthermore, SHV-2pBP60-1 was clearly related to SHV-1, LEN-1, and OHIO-1. The SHV-2pBP60-1 enzyme differed from SHV-1 isolated from Klebsiella pneumoniae by seven amino acid substitutions. One of these substitutions, the Gly----Ser substitution at position 234, is probably a key region for the novel activity of cefotaxime hydrolysis. A phylogenetic tree was constructed by using all class A beta-lactamases of known sequences by a progressive alignment method. The data suggested that the beta-lactamases of gram-positive Streptomyces, Staphylococcus, and Bacillus species appeared early in evolution, followed by the PSE and CARB enzymes of Pseudomonas species and, more recently, by the SHV-type and TEM-type enzymes found in enteric bacteria. Larger evolutionary distances separated clusters of the gram-positive beta-lactamases than separated clusters of the gram-negative enzymes. Results of this phylogenetic study suggested that extended-spectrum enzymes are recent derivatives that are selected by the use of new cephalosporins.

Amino Acid Sequence↗
PubMed1990

Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons.

A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.

Amino Acid Sequence↗
PubMed1989

Development of gene probes and evolutionary relationships of the PSE-4 bla gene to plasmid-mediated beta-lactamases of gram-negative bacteria.

Six types of plasmid-mediated carbenicillinases can be distinguished on the basis of their substrate profiles, molecular mass isoelectric values and immunological properties. As yet, no structural classification has been attempted for these enzymes at the molecular level. We have isolated the PSE-4 structural gene responsible for carbenicillinase production in Pseudomonas aeruginosa strain Dalgleish and studied its expression in E. coli. A detailed physical map of the cloned fragment and the construction of deletion mutants permitted the precise localization of the PSE-4 structural gene. Various restriction endonuclease fragments known to be flanking or internal to the PSE-4 bla gene were used as DNA probes and tested for homologous sequences in other beta-lactamase genes. A collection of three restriction fragment probes internal or delimiting the PSE-4 structural gene were hybridized with purified plasmid DNA coding for 18 other beta-lactamases. Under high stringency conditions, only the PSE-1, CARB-3 and CARB-4 genes cross-hybridized with PSE-4; while one of the probes tested hybridized solely with CARB-3. Further analysis indicated that the PSE-1, PSE-4, CARB-3 and CARB-4 bla genes are related and could presumably have evolved from a common progenitor.

Biological Evolution↗
PubMed1989

Decreased outer membrane permeability in imipenem-resistant mutants of Pseudomonas aeruginosa.

The outer membrane of imipenem-resistant mutants of Pseudomonas aeruginosa was shown to have decreased permeability to imipenem but not to cephaloridine. These experiments were performed with intact cells and liposomes containing imipenem-hydrolyzing beta-lactamase derived from Pseudomonas maltophilia, in both cases utilizing an imipenem concentration of 50 microM. In contrast, liposome swelling assays using imipenem at 8 mM detected no significant difference between the imipenem-resistant mutants and their parents. It was found that the outer membrane of P. aeruginosa has a saturable specific channel through which imipenem travels mainly at low concentrations, whereas at high concentrations this pathway is obscured by diffusion through nonspecific porin channels.

Cell Membrane Permeability↗
PubMed1989

Evolutionary perspectives on multiresistance beta-lactamase transposons.

A series of intragenic DNA probes, encoding the major part of the transposase resolvase and inverted repeats of transposons Tn3, Tn21, and Tn2501, were used in hybridization assays for homologous DNA sequences in 18 transposons studied. The tnpA and tnpR probes detected extensive homology with Tn3-like and Tn21-like elements for 11 transposons. This high degree of homology was confirmed with the 38- and 48-base-pair inverted-repeat oligonucleotide probes of Tn3, Tn21, and Tn2501. The Southern-type gel hybridization experiments localized the tnpA-homologous sequences on the physical DNA maps constructed. The genetic and physical maps of the transposons were compared, as were their nucleic acid sequence homologies. These comparisons suggested a subfamily of mobile elements distinct from but related to the Tn21 group. Based on these results, an evolutionary model is proposed and a pedigree is presented for the genesis of multiresistance beta-lactamase transposons.

Base Sequence↗
PubMed1988

Molecular structure and interrelationships of multiresistance beta-lactamase transposons.

Transposons coding for beta-lactamases OXA-3, OXA-4, OXA-5, LCR-1, and CARB-3 have been isolated and compared functionally and structurally with transposons for TEM-1, OXA-1, PSE-1, PSE-2, and PSE-4 enzymes. Each beta-lactamase gene type occurred in a unit together with resistance to other antibiotics, particularly streptomycin and sulfonamide but also chloramphenicol, mercuric ion, or gentamicin, kanamycin, and tobramycin. Restriction mapping, gene cloning, and DNA hybridization were used to compare the transposons and to localize their functional components. Although the multiresistance beta-lactamase transposons varied in size from 8 to 25 kb, the similarity of some of their restriction maps suggested a common derivation. Six of 12 transposons contained DNA segments homologous to the tnpR gene of transposon Tn21 and could complement a tnpR- Tn21 derivative. Consequently, these six transposons appear to have evolved from a common progenitor by acquisition of DNA coding for various beta-lactamases and other resistance genes.

DNA Restriction Enzymes↗
PubMed1988

Cloning and expression of the imipenem-hydrolyzing beta-lactamase operon from Pseudomonas maltophilia in Escherichia coli.

The L-1 penicillinase structural gene, blaS, from Pseudomonas maltophilia has been cloned into the vector pACYC184. The pMON01 recombinant plasmid selected by ampicillin resistance carried a 2.6-kilobase Sau3A fragment of P. maltophilia DNA and was confirmed to express L-1 beta-lactamase by comparative isoelectric focusing. A detailed physical map was constructed, and the blaS structural gene was localized with a 17-mer oligonucleotide mixed probe encoding the L-1 N-terminal amino acid sequence. Induction studies confirmed constitutive expression. Isolation of a complete beta-lactamase operon was attempted by construction of a P. maltophilia genomic library into phage lambda 2001. A recombinant phage was selected by DNA hybridization, and the 13.4-kilobase DNA insert was physically mapped and subcloned into plasmid vectors. Expression and L-1 beta-lactamase synthesis were studied in Escherichia coli.

Cloning, Molecular↗
PubMed1987

Molecular cloning and DNA homology of plasmid-mediated beta-lactamase genes.

Molecular cloning of DNA fragments between 1.5 and 8 kb from BamHI, EcoRI, HindIII, SalI, or Sau3A digests permitted the isolation of structural genes coding for TEM-1, ROB-1, OXA-1, OXA-3, OXA-4, OXA-5, PSE-1, PSE-2, PSE-3, PSE-4, CARB-3, CARB-4, AER-1, and LCR-1 beta-lactamases. Ampicillin-resistant clones were selected and it was confirmed that they contained the respective beta-lactamase genes by isoelectric focusing. Detailed physical maps of 14 different recombinant plasmids were constructed using 8 restriction endonucleases. Plasmid deletions and lacZ fusions were used to localize the beta-lactamase structural genes. DNA probes were constructed for the TEM-1, ROB-1, OXA-1, and PSE-1 genes. Under conditions of high stringency, hybridization was observed between the genes for TEM-1 and TEM-2 or TLE-1, OXA-1 and OXA-4, and PSE-1 and PSE-4 or CARB-3, while the ROB-1 gene probe showed no cross-hybridization. Such bla gene probes should facilitate studies of beta-lactamase molecular epidemiology.

Cloning, Molecular↗
PubMed1987

Monoclonal antibodies to TEM-1 plasmid-mediated beta-lactamase.

At least 28 plasmid-mediated beta-lactamases have been described in gram-negative bacteria. To assess the relationship among these enzymes, we produced and characterized 28 murine monoclonal antibodies to the TEM-1 plasmid-mediated beta-lactamase. Radial immunodiffusion identified 3 monoclonal antibodies as immunoglobulin M (IgM), 18 as subclass IgG1, 2 as IgG2a, and 5 as IgG2b. Using a newly described enzyme immunoassay, cross-reactivity of 16 of these monoclonal antibodies was tested against 24 plasmid-determined beta-lactamases. The 16 monoclonal antibodies cross-reacted with TEM-2 and TLE-1 and, to a certain extent, SHV-1. Different levels of cross-reactivity were also observed with OXA-3 (11 of 16), OXA-7 (8 of 16), OXA-1 (2 of 16), OXA-6 (2 of 16), and AER-1 (2 of 16). Six monoclonal antibodies demonstrated partial neutralization of beta-lactamase activity. This study suggests that common epitopes are shared by nine biochemically distinct plasmid-mediated beta-lactamases. On the basis of cross-reactivities with these monoclonal antibodies, we identified four epitopes on TEM-1, TEM-2, TLE-1, and SHV-1 beta-lactamases.

Animals↗
PubMed1987

Development of natural and synthetic DNA probes for OXA-2 and TEM-1 beta-lactamases.

Cloning of a 6.3-kilobase BglII DNA fragment from plasmid R46 permitted the isolation of the OXA-2 beta-lactamase gene. Selected DNA fragments internal and adjacent to the OXA-2 beta-lactamase structural gene were used as probes in homology studies with other plasmid-mediated beta-lactamases. Under conditions of high stringency, no cross hybridization could be detected with DNA probes from within the open reading frame of the OXA-2 structural gene. At a lower stringency, one of two DNA fragments used as probes cross hybridized weakly with the OXA-3 bla gene. Other DNA fragments tested and known to contain sequences flanking the OXA-2 determinant cross hybridized with OXA-3 and PSE-4 plasmid DNA. From the known nucleotide sequence of OXA-2 and TEM-1, we synthesized a series of oligonucleotides corresponding to sequences internal to their respective structural genes. A 12-mer oligonucleotide containing the OXA-2-active-site nucleotide sequences cross hybridized only with OXA-3. All other oligonucleotides tested were found to be specific for their respective OXA-2 or TEM-1 gene. Such beta-lactamase gene probes should facilitate studies of beta-lactamase molecular epidemiology and beta-lactamase gene polymorphism.

Base Sequence↗
PubMedDate not supplied

Molecular cloning and expression of the imipenem-hydrolyzing beta-lactamase gene from Pseudomonas maltophilia in Escherichia coli.

The L-1 penicillinase structural gene, blaS, from Pseudomonas maltophilia was cloned into the vector pACYC184. The pMON01 recombinant plasmid selected by ampicillin resistance carried a 2.6-kilobase (kb) Sau3A fragment of P. maltophilia DNA and was confirmed to express L-1 beta-lactamase by comparative isoelectric focusing. A detailed physical map was constructed, and the blaS structural gene was localized with a 17-mer oligonucleotide mixed probe encoding the L-1 NH2-terminal amino acid sequence. Induction studies confirmed constitutive expression. Isolation of a complete beta-lactamase operon was attempted by construction of a P. maltophilia genomic library into phage lambda 2001. A recombinant phage was selected by DNA hybridization; the 13.4-kb DNA insert was physically mapped and subcloned into the high-copy-number plasmid pACYC184 and into the low-copy-number vector pLG338. The expression of the cloned blaS L-1 structural gene and levels of beta-lactamase synthesis were studied in Escherichia coli. The protein synthesized was found to be similar to the L-1 beta-lactamase of the prototype P. maltophilia, although expression levels were gene dosage dependent for beta-lactamase synthesis.

Amino Acid Sequence↗