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1.
Curr Opin Microbiol ; 4(5): 515-9, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11587926

RESUMO

Transposon-based approaches are very powerful for identification of essential and infection-related genes in bacteria, particularly in the context of microbial genomics. We describe recent progress in several of these approaches, and their underlying principles. The essential gene test (EGT) is a transposon-based technique that can rapidly identify a nucleotide sequence from a database as essential or dispensable. Also, variations of in vitro transposon mutagenesis applications, such as genomic analysis and mapping by in vitro transposition (GAMBIT), are described. The development of techniques including PCR-based signature-tagged mutagenesis is now used to find essential virulence genes in different bacterial hosts. These approaches form the basis for the identification of microbial targets in development of novel antimicrobials and vaccines by the biotechnology and pharmaceutical industry.


Assuntos
Bactérias/genética , Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Genes Bacterianos , Genes Essenciais , Biotecnologia/métodos , Elementos de DNA Transponíveis/genética , Indústria Farmacêutica/métodos , Humanos , Mutagênese Insercional/métodos , Virulência/genética
2.
FEMS Microbiol Lett ; 201(2): 229-35, 2001 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-11470366

RESUMO

Bacterial peptidoglycan is the cell wall component responsible for maintaining cell integrity against osmotic pressure. Biosynthesis of the cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is catalyzed by the Mur enzymes. Genomic analysis of the three regions encoding Mur proteins was achieved. We have cloned and over-expressed the murA, -B, -D, -E and -F genes of Pseudomonas aeruginosa in pET expression system by adding a His-Tag to the C-termini of the proteins. Mur proteins were purified to homogeneity by a single chromatographic step on affinity nickel columns. Protein identities were verified through N-terminal sequencing. Enzyme activity was proved by the identification of the pathway's final product.


Assuntos
Genes Bacterianos/genética , Peptidoglicano/biossíntese , Pseudomonas aeruginosa/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cromatografia de Afinidade , Clonagem Molecular , Dados de Sequência Molecular , Peptidoglicano/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Análise de Sequência de Proteína
3.
Biochemistry ; 40(2): 395-402, 2001 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-11148033

RESUMO

PSE-4 is a class A beta-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determined to 1.95 A resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition of the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 A for 263 Calpha atoms. Most carbenicillinases are unique among class A beta-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k(cat)/K(m) and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 A resolution with the wild-type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A beta-lactamase structures. Our molecular modeling studies suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin alpha-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 may be involved in accommodating this shift in the bound substrate position.


Assuntos
Penicilinase/química , beta-Lactamases/química , Alanina/genética , Arginina/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Ativação Enzimática , Hidrólise , Cinética , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Penicilinase/metabolismo , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/genética , beta-Lactamases/metabolismo
4.
FEMS Microbiol Lett ; 190(1): 141-6, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10981704

RESUMO

Novel putative pyoverdine synthetase pvdIJK genes were found upstream of pvdD in the 6.2-Mb chromosome of Pseudomonas aerugilosa strain PAO1. These genes formed a locus implicated in pyoverdine biosynthesis. Sequence analysis showed that the product of these genes shared 43%, 60% and 57% identity with PvdD. PvdIJK are thought to be implicated in synthesis of pyoverdine, a siderophore chelating Fe3+. A pvdI mutant was obtained by gene disruption mutagenesis and confirmed by Southern hybridization. The pvdl mutant produced gave no significant growth on solid media supplemented with the iron chelator 2,2-dipyridyl; while the PvdI- phenotype abolished pyoverdine fluorescence. The role of PvdI in pathogenicity was tested by measuring the in vivo growth of P. aeruginosa wild-type and mutant strains in a chronic lung infection rat model, and by measuring the competitive infectivity index into a neutropenic mice model. The data obtained confirmed the importance of PvdI in virulence and iron uptake.


Assuntos
Proteínas de Bactérias , Genoma Bacteriano , Oligopeptídeos , Óperon/genética , Peptídeo Sintases/genética , Pigmentos Biológicos/biossíntese , Pseudomonas aeruginosa/genética , Sequência de Aminoácidos , Animais , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Pigmentos Biológicos/genética , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/fisiopatologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Ratos , Ratos Sprague-Dawley , Análise de Sequência de DNA
5.
Protein Eng ; 13(4): 267-74, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10810158

RESUMO

We extracted maximum information for structure-function analysis of the PSE-4 class A beta-lactamase by random replacement mutagenesis of three contiguous codons in the H4 alpha-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129. These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model. Structure-function studies on positions 125-129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127. The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 alpha-helix structure together, thus maintaining the important Ser130-Asp131-Asn132 motif positioned towards the active site. At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4. The Tyr 125 of the mutant YAM formed an edge to face pi-pi interaction with Phe 124 which also interacts with the Trp 210 with the same interactions. Antibiotic susceptibilities showed that amino acid changes in the the H4 alpha-helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors. However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 alpha-helix region than clavulanic acid, an inhibitor of the oxypenam class. The analysis of H4 alpha-helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes.


Assuntos
Modelos Moleculares , beta-Lactamases/química , Dicroísmo Circular , Ácido Clavulânico/farmacologia , Simulação por Computador , Inibidores Enzimáticos/farmacologia , Cinética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/farmacologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Sulbactam , Tazobactam , Resistência beta-Lactâmica , Inibidores de beta-Lactamases , beta-Lactamases/biossíntese , beta-Lactamases/genética
6.
J Antimicrob Chemother ; 45(4): 517-20, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10747830

RESUMO

The class A PSE-4 beta-lactamase was used for studying the importance of amino acids in the omega (Omega) loop and its interactions for hydrolysis of beta-lactam antibiotics. By cassette mutagenesis, we replaced the amino acids 163-179 Omega loop in PSE-4 with TEM-1, SHV-1 and Streptomyces albus G beta-lactamase Omega loops. Phenotypic analysis of Escherichia coli recombinants expressing the Omega loop PSE-4 mutant enzymes gave MICs and kinetic data similar to those of wild-type PSE-4.


Assuntos
Streptomyces/enzimologia , Streptomyces/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Substituição de Aminoácidos/genética , Escherichia coli/genética , Cinética , Mutagênese Insercional/genética , Fenótipo
7.
FEBS Lett ; 470(3): 285-92, 2000 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-10745083

RESUMO

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.


Assuntos
Carbenicilina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pseudomonas aeruginosa/enzimologia , Inibidores de beta-Lactamases , beta-Lactamases/química , Acilação/efeitos dos fármacos , Sítios de Ligação , Ácido Clavulânico/química , Ácido Clavulânico/metabolismo , Ácido Clavulânico/farmacologia , Simulação por Computador , Inibidores Enzimáticos/metabolismo , Escherichia coli/enzimologia , Ligação de Hidrogênio , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/química , Ácido Penicilânico/metabolismo , Ácido Penicilânico/farmacologia , Resistência às Penicilinas , Penicilinase/química , Penicilinase/metabolismo , Sulbactam/química , Sulbactam/metabolismo , Sulbactam/farmacologia , Tazobactam , Termodinâmica , beta-Lactamases/metabolismo
8.
Infect Immun ; 68(4): 2359-62, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10722644

RESUMO

In vivo expression technology was used for testing Pseudomonas aeruginosa in the rat lung model of chronic infection and in a mouse model of systemic infection. Three of the eight ivi proteins found showed sequence identity to known virulence factors involved in iron acquisition via an open reading frame (called pvdI) implicated in pyoverdine biosynthesis, membrane biogenesis (FtsY), and adhesion (Hag2).


Assuntos
Oligopeptídeos , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa/genética , Animais , Aderência Bacteriana/genética , Proteínas de Bactérias/biossíntese , Membrana Celular/genética , Membrana Celular/metabolismo , Contagem de Colônia Microbiana , Biblioteca Gênica , Fígado/microbiologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fases de Leitura Aberta , Pigmentos Biológicos/biossíntese , Pigmentos Biológicos/genética , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Virulência
9.
FEMS Microbiol Lett ; 183(2): 281-8, 2000 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-10675598

RESUMO

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.


Assuntos
Peptídeo Sintases/genética , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeo Sintases/química , Peptídeo Sintases/isolamento & purificação , Filogenia
10.
Angew Chem Int Ed Engl ; 39(1): 143-145, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10649355

RESUMO

Natural peptide spine and artificial crown ether are blended together to form a hybrid structure. A dicationic benzylammonium guest threads through the crown ether pendants and the resulting noncovalent, self-assembled pseudorotaxane complex, shown schematically in the picture, is both stable and has optical properties dependant on temperature.

11.
Biotechniques ; 26(3): 473-8, 480, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10090989

RESUMO

We describe a fast and simple method for signature-tagged mutagenesis (STM) using defined oligonucleotides for tag construction into mini-Tn5 and PCR instead of hybridization for rapid screening of bacterial mutants in vivo. A collection of 12 unique 21-mers were synthesized as complementary DNA strands to tag bacterial mutants constructed by insertional mutagenesis using pUTmini-Tn5Km2 plasmids. Tags were tested in a combination of assays by PCR and compared to hybridization for specificity and for large-scale screening. Each defined tag has the same melting temperature, an invariable region to optimize PCRs and a variable region for specific amplification by PCR. A series of "suicide" plasmids carrying mini-Tn5s, each with a specific tag, were transferred into Pseudomonas aeruginosa, giving 12 libraries of mutants; groups of 12 mutants were pooled and arrayed into 96-well microplates, representing approximately one-sixth of the P. aeruginosa 5.9-Mb genome. This simple STM method can be adapted to any bacterial system and used for genome scanning in various growth conditions.


Assuntos
Genes Bacterianos/genética , Mutagênese , Oligonucleotídeos/genética , Sequência de Bases , Elementos de DNA Transponíveis/genética , Genes Essenciais/genética , Mutação , Oligonucleotídeos/síntese química , Plasmídeos/genética , Reação em Cadeia da Polimerase , Pseudomonas aeruginosa/genética
12.
Antimicrob Agents Chemother ; 43(3): 543-8, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10049265

RESUMO

Site-directed mutagenesis of Ser-289 of the class C beta-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in beta-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant beta-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC beta-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the kcat of four of the five beta-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant beta-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.


Assuntos
Proteínas de Bactérias , Enterobacter cloacae/enzimologia , Enterobacter cloacae/genética , Serina/genética , beta-Lactamases/genética , Catálise , Cefaclor/farmacologia , Cefazolina/farmacologia , Cefalosporinas/farmacologia , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Plasmídeos , Conformação Proteica , Serina/química , Relação Estrutura-Atividade , beta-Lactamases/química
13.
Antimicrob Agents Chemother ; 42(10): 2576-83, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9756758

RESUMO

The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Omega loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Omega loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.


Assuntos
Ceftazidima/farmacologia , Cefalosporinas/farmacologia , beta-Lactamases/química , Sequência de Aminoácidos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Relação Estrutura-Atividade , Especificidade por Substrato , beta-Lactamases/metabolismo
14.
Antimicrob Agents Chemother ; 42(9): 2319-25, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9736556

RESUMO

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.


Assuntos
Inibidores Enzimáticos/farmacologia , Ácido Penicilânico/análogos & derivados , Sulbactam/farmacologia , Inibidores de beta-Lactamases , Sítios de Ligação , Testes de Sensibilidade Microbiana , Ácido Penicilânico/farmacologia , Relação Estrutura-Atividade , Tazobactam , beta-Lactamases/química
15.
Microb Comp Genomics ; 3(2): 105-17, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9697095

RESUMO

Pseudomonas aeruginosa is an opportunistic bacterial pathogen frequently found in nosocomial infections and is a major cause of morbidity and mortality in patients with cystic fibrosis. To facilitate molecular studies of this organism, we have generated a bacterial artificial chromosome (BAC) library. Genomic DNA was isolated from the prototype strain PAO1, partially digested with HindIII, size selected after pulsed-field gel electrophoresis, and used to construct a BAC library using the pBeloBAC11 vector. DNAs from approximately 850 clones, representing more than 9.5-fold physical coverage of the 5.9-Mb PAO1 genome, were analyzed after SpeI and HindIII digestions and agarose gel electrophoresis. The BAC library had clones with insert fragments ranging from 20 to more than 290 kb. A subset of 264 BACs having inserts > 80 kb, representing > 4 genome equivalents, were rearrayed into 96-well plates, and a clone pooling and PCR screening strategy was developed. The PCR library screening enabled the identification and recovery of BACs containing genes implicated in cell division and in cell wall biosynthesis, as well as a series of known genes mapping to different regions of the PAO1 chromosome. A physical and genetic map was constructed for the 98-kb pMOC5 BAC clone, which spans the entire fts-mur locus. Chromosome walking from each end of the pMOC5 clone placed it within a contig spanning 243 kb. The BAC library and screening resources now allow a PCR-based screening of a P. aeruginosa genomic library for any gene of interest. The restriction fragment analysis of overlapping clones indicated that BAC clones stably maintain and propagate Pseudomonas DNA, providing evidence that the PAO1 BAC library is an appropriate reagent for genome sequencing.


Assuntos
Mapeamento Cromossômico/métodos , Clonagem Molecular/métodos , Proteínas do Citoesqueleto , Proteínas de Escherichia coli , Genoma Bacteriano , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/genética , Sequência de Bases , Passeio de Cromossomo , Cromossomos Bacterianos/genética , Primers do DNA , Biblioteca Gênica , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mapeamento por Restrição , Seleção Genética , Análise de Sequência de DNA
16.
Antimicrob Agents Chemother ; 42(8): 1966-72, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9687391

RESUMO

We determined the nucleotide sequences of blaCARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing beta-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between the bla genes for these beta-lactamases varied from 98.7 to 99.9%, while that between these genes and blaCARB-4 encoding CARB-4 was 86.3%. The blaCARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that blaAER-1 shared 60.8% DNA identity with blaPSE-3 encoding PSE-3. The deduced AER-1 beta-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A beta-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flanking blaCARB-4 and blaAER-1 confirmed the importance of gene cassettes acquired via integrons in bla gene distribution.


Assuntos
Carbenicilina/metabolismo , Penicilinas/metabolismo , beta-Lactamases/química , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
17.
Antimicrob Agents Chemother ; 42(5): 1245-8, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9593158

RESUMO

We have determined the nucleotide sequence of the blaS gene encoding the carbapenem-hydrolyzing L-1 beta-lactamase from Stenotrophomonas maltophilia GN12873. Analysis of the DNA and deduced amino acid sequences identified a product of 290 amino acids. Comparisons of the L-1 amino acid sequence with those of other zinc beta-lactamases showed 88.6% identity with the L-1 enzyme from S. maltophilia IID1275 and less than 20% identity with other class B metalloenzymes.


Assuntos
Genes Bacterianos/genética , Xanthomonas/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Sequência de Aminoácidos , Sequência de Bases , Variação Genética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Xanthomonas/enzimologia
18.
Biotechniques ; 24(2): 261-4, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9494727

RESUMO

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.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Vetores Genéticos/genética , Bactérias Gram-Negativas/genética , Pseudomonas aeruginosa/genética , Aspartato-Semialdeído Desidrogenase/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/genética , Genes Reporter/genética , Marcadores Genéticos/genética , Bactérias Gram-Negativas/enzimologia , Proteínas de Fluorescência Verde , Óperon Lac/genética , Proteínas Luminescentes/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Pseudomonas aeruginosa/enzimologia , Homologia de Sequência de Aminoácidos , beta-Galactosidase/genética
19.
Antimicrob Agents Chemother ; 39(4): 887-93, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7785990

RESUMO

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.


Assuntos
beta-Lactamases/química , Sequência de Aminoácidos , Sequência de Bases , Genes Bacterianos , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Homologia de Sequência de Aminoácidos , beta-Lactamases/genética
20.
Mol Cell Probes ; 8(1): 23-37, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8028605

RESUMO

The use of new molecular typing methods for the characterization of Haemophilus influenzae strains is reported. Sixty-four isolates of H. influenzae originating from different types of infection and obtained from eight hospitals across Canada were first analysed for restriction polymorphism. Chromosomal DNA fragments generated by two different combinations of restriction endonucleases were electrophoresed and transferred to nylon membranes before hybridization with a species specific 32P-labelled DNA fragment (5 kb) used as a probe. The combinations Bg/II/PstI led to 11 typing groups (A-K) and BamHI/Bg/II/PstI to 14 sub-groups, respectively. Most of the isolates retrieved from cerebrospinal fluids (10/13; 76.9%) were classified in two groups (A and B) and two sub-groups. Isolates from respiratory tract infections were mostly found in groups C and E (24/32; 75.0%), and divided into seven sub-groups. Selected ampicillin-resistant, beta-lactamase-negative strains were also found in groups C and E (11/14; 78.6%). Isolates from conjunctivitis and acute otitis media were classified in various groups. All biotypes (I-VIII) and serotypes (none, a-f) were spread among the typing groups although biotype I prevailed in groups A, B, and G; II in group E (sub-group 6); and III in group C. A PCR approach derived from the typing system was also tested. A set of 25-mer primers was selected from the 5-kb DNA probe for the amplification of a 317-bp region. This set of primers was used concomitantly in a PCR multiplex assay with a set of primers selected from the nucleotide sequence of the gene encoding the H. influenzae P1 protein. This multiplex assay was also able to discriminate the clonal origin of some H. influenzae strains because size polymorphism was observed in PCR products. The PCR approach was then used to determine the genetic relatedness of H. influenzae strains found persistently in sputa of some patients with cystic fibrosis. Genetically related strains could be isolated from some patients even after antibiotherapy and months between visits, whereas other patients showed distinct strains. In summary, our typing system is able to provide new characteristics for strains having identical biotype or serotype. The rapid PCR alternative may prove useful for specific epidemiological and strain-tracking studies.


Assuntos
Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Haemophilus influenzae/classificação , Haemophilus influenzae/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Criança , Pré-Escolar , Fibrose Cística/genética , Fibrose Cística/microbiologia , Sondas de DNA , DNA Bacteriano/análise , Feminino , Genótipo , Infecções por Haemophilus/genética , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/isolamento & purificação , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase
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