Cloning of a Chryseobacterium(Flavobacterium) meningosepticum Chromosomal Gene (blaACME) Encoding an Extended-Spectrum Class A β-Lactamase Related to the BacteroidesCephalosporinases and the VEB-1 and PER β-Lactamases

1999 ◽  
Vol 43 (9) ◽  
pp. 2193-2199 ◽  
Author(s):  
Gian Maria Rossolini ◽  
Nicola Franceschini ◽  
Laura Lauretti ◽  
Berardo Caravelli ◽  
Maria Letizia Riccio ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Genomic Library ◽  
Expression System ◽  
Plasmid Vector ◽  
Gel Permeation Chromatography ◽  
Exchange Chromatography ◽  
Class A ◽  
Extended Spectrum ◽  
Cloned Gene ◽  
Chromatography Step

ABSTRACT In addition to the BlaB metallo-β-lactamase,Chryseobacterium (Flavobacterium)meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine β-lactamase, named CME-1, with an alkaline isoelectric pH. The blaA CME gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A β-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER β-lactamases and the Bacteroides chromosomal cephalosporinases. TheblaA CME determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum β-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of β-lactams, with both Km andk cat values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (k cat/Km ratios) for all types of β-lactam substrates.

scholarly journals Purification and Biochemical Characterization of the VIM-1 Metallo-β-Lactamase

2000 ◽  
Vol 44 (11) ◽  
pp. 3003-3007 ◽  
Author(s):  
Nicola Franceschini ◽  
Berardo Caravelli ◽  
Jean-Denis Docquier ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Metal Removal ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Mediterranean Area ◽  
Exchange Chromatography ◽  
Amino Terminal ◽  
Chromatography Step

ABSTRACT VIM-1 is a new group 3 metallo-β-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla VIM-1 gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of β-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-β-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/Km ratios (>106M−1 · s−1) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different β-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these β-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-β-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.

scholarly journals The Legionella (Fluoribacter)gormanii Metallo-β-Lactamase: a New Member of the Highly Divergent Lineage of Molecular-Subclass B3 β-Lactamases

2000 ◽  
Vol 44 (6) ◽  
pp. 1538-1543 ◽  
Author(s):  
Letizia Boschi ◽  
Paola Sandra Mercuri ◽  
Maria Letizia Riccio ◽  
Gianfranco Amicosante ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Genomic Library ◽  
Plasmid Vector ◽  
Gel Permeation Chromatography ◽  
Structural Similarity ◽  
E Coli ◽  
Molecular Subclass ◽  
Class B ◽  
Marked Preference ◽  
Isoelectric Ph ◽  
T7 Phage

ABSTRACT A metallo-β-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter)gormanii ATCC 33297T constructed in the plasmid vector pACYC184 and transformed into Escherichia coliDH5α, by screening for clones showing a reduced susceptibility to imipenem. The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6. Sequencing revealed that FEZ-1 is a molecular-class B β-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage. All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1. Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem. The properties of FEZ-1 overall resembled those of a β-lactamase previously purified from the same strain of L. gormanii (T. Fujii, K. Sato, K. Miyata, M. Inoue, and S. Mitsuhashi, Antimicrob. Agents Chemother. 29:925–926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class. A system for overexpression of thebla FEZ-1 gene in E. coli, based on the T7 phage promoter, was also developed.

scholarly journals Cloning and Characterization of blaVIM, a New Integron-Borne Metallo-β-Lactamase Gene from a Pseudomonas aeruginosa Clinical Isolate

1999 ◽  
Vol 43 (7) ◽  
pp. 1584-1590 ◽  
Author(s):  
Laura Lauretti ◽  
Maria Letizia Riccio ◽  
Annarita Mazzariol ◽  
Giuseppe Cornaglia ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Isolate ◽  
Genomic Library ◽  
Plasmid Vector ◽  
Gene Cassette ◽  
University Hospital ◽  
Class 1 Integron ◽  
E Coli ◽  
Class B ◽  
Cloned Gene

ABSTRACT Production of a metallo-β-lactamase activity was detected in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate (isolate VR-143/97) from an Italian inpatient at the Verona University Hospital (northern Italy). The metallo-β-lactamase determinant was isolated from a genomic library of VR-143/97, constructed in an Escherichia coli plasmid vector, by screening for clones with reduced susceptibility to imipenem. Sequencing of the cloned gene revealed that it encoded a new class B β-lactamase that was named VIM-1. At the sequence level VIM-1 was rather divergent from the other class B enzymes (16.4 to 38.7% identity), overall being more similar to members of subclass B1 including the β-lactamase II of Bacillus cereus (Bc-II), the Bacteroides fragilis CcrA, the Chryseobacterium meningosepticum BlaB, and the cassette-encoded IMP-1 enzymes. Among these, VIM-1 showed the highest degree of similarity to Bc-II. Similarly to bla IMP,bla VIM was also found to be carried on a gene cassette inserted into a class 1 integron. Thebla VIM-containing integron was located on the chromosome of P. aeruginosa VR-143/97, and the metallo-β-lactamase-encoding determinant was not transferable toE. coli by conjugation. Expression of the integron-bornebla VIM gene in E. coli resulted in a significant decrease in susceptibility to a broad array of β-lactams (ampicillin, carbenicillin, piperacillin, mezlocillin, cefotaxime, cefoxitin, ceftazidime, cefoperazone, cefepime, and carbapenems), revealing a very broad substrate specificity of the VIM-1 enzyme.

scholarly journals Enzymatic assay of d-mannose in serum

1997 ◽  
Vol 43 (3) ◽  
pp. 533-538 ◽  
Author(s):  
James R Etchison ◽  
Hudson H Freeze
Keyword(s):  
Bacillus Stearothermophilus ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enzymatic Assay ◽  
Enzymatic Reactions ◽  
Reaction Products ◽  
Serum Samples ◽  
Healthy Human ◽  
Human Volunteers ◽  
Chromatography Step

Abstract We describe a new and improved enzymatic assay for determining the concentration of d-mannose in sera. Serum d-glucose is selectively converted to glucose-6 phosphate with the highly specific thermostable glucokinase (EC 2.7.1.2) from Bacillus stearothermophilus. The anionic reaction products and excess substrates are removed by a rapid and simple anion-exchange chromatography step in microcentrifuge spin columns. d-Mannose in the glucose-depleted sample is then assayed spectrophotometrically by using coupled enzymatic reactions. The quantitative elimination of glucose from the serum samples allowed the accurate and reproducible assay of serum mannose in the 0–200 μmol/L range. Recovery of mannose added to serum (5–200 μmol/L) was 94% ± 4.4%. The intraassay CV was 6.7% at 40 μmol/L mannose (n = 5; 39.6 ± 1.6 μmol/L) and 4.4% at 80 μmol/L (n = 11; 75.0 ± 1.8 μmol/L); the interassay CV at these concentrations was 12.2% (n = 7; 36.9 ± 2.1 μmol/L) and 9.8% (n = 7; 74.2 ± 2.7 μmol/L), respectively. Sera from 11 healthy human volunteers contained an average of 54.1 ± 11.9 μmol/L mannose (range 36–81 μmol/L).

scholarly journals Ragged N-termini and other variants of class A β-lactamases analysed by chromatofocusing

1991 ◽  
Vol 273 (3) ◽  
pp. 503-510 ◽  
Author(s):  
A Matagne ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Frère
Keyword(s):  
Experimental Error ◽  
Catalytic Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Gram Positive Bacteria ◽  
Class A ◽  
Partial Inactivation ◽  
Asparagine Residue

Four beta-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analysed by chromatofocusing or ion-exchange chromatography. Ragged N-termini were in part responsible for the charge variants, but deamidation of an asparagine residue was also involved, at least for the Bacillus licheniformis enzyme. The activity of a contaminating proteinase could also be demonstrated in the case of Actinomadura R39 beta-lactamase. With that enzyme, proteolysis resulted in partial inactivation, but the inactivated fragments were easily separated from the active forms. With these, as with the other enzymes, the kinetic parameters of the major variants were identical with those of the mixture within the limits of experimental error, so that the catalytic properties of these enzymes can be determined with the ‘heterogeneous’ preparations.

scholarly journals The dps Gene of Symbiotic “Candidatus Legionella jeonii” in Amoeba proteus Responds to Hydrogen Peroxide and Phagocytosis

2006 ◽  
Vol 188 (21) ◽  
pp. 7572-7580 ◽  
Author(s):  
Miey Park ◽  
Seong Tae Yun ◽  
Sue-Yun Hwang ◽  
Choong-Ill Chun ◽  
Tae In Ahn
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Host Cell ◽  
Genomic Library ◽  
Amoeba Proteus ◽  
Host Cells ◽  
Intracellular Pathogens ◽  
Protective Mechanisms ◽  
Dps Protein ◽  
Cloned Gene

ABSTRACT To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for DNA-binding protein from starved cells) gene of the symbiotic “Candidatus Legionella jeonii” organism (called the X bacterium) (dps X) that grows in Amoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. DpsX proteins purified from E. coli transformed with the dps X gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H2O2-mediated damage. The expression of the dps X gene in “Candidatus Legionella jeonii” was enhanced when the host amoeba was treated with 2 mM H2O2 and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps X expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.

scholarly journals Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase.

1996 ◽  
Vol 40 (3) ◽  
pp. 616-620 ◽  
Author(s):  
A Bauernfeind ◽  
I Stemplinger ◽  
R Jungwirth ◽  
P Mangold ◽  
S Amann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Beta Lactamase ◽  
Reading Frame ◽  
Beta Lactamases ◽  
Class A ◽  
Extended Spectrum ◽  
The Family ◽  
Extended Spectrum Beta Lactamases ◽  
Lactamase Gene

Plasmidic extended-spectrum beta-lactamases of Ambler class A are mostly inactive against ceftibuten. Salmonella typhimurium JMC isolated in Argentina harbors a bla gene located on a plasmid (pMVP-5) which confers transferable resistance to oxyiminocephalosporins, aztreonam, and ceftibuten. The beta-lactamase PER-2 (formerly ceftibutenase-1; CTI-1) is highly susceptible to inhibition by clavulanate and is located at a pI of 5.4 after isoelectric focusing. The blaPER-2 gene was cloned and sequenced. The nucleotide sequence of a 2.2-kb insert in vector pBluescript includes an open reading frame of 927 bp. Comparison of the deduced amino acid sequence of PER-2 with those of other beta-lactamases indicates that PER-2 is not closely related to TEM or SHV enzymes (25 to 26% homology). PER-2 is most closely related to PER-1 (86.4% homology), an Ambler class A enzyme first detected in Pseudomonas aeruginosa. An enzyme with an amino acid sequence identical to that of PER-1, meanwhile, was found in various members of the family Enterobacteriaceae isolated from patients in Turkey. Our data indicate that PER-2 and PER-1 represent a new group of Ambler class A extended-spectrum beta-lactamases. PER-2 so far has been detected only in pathogens (S. typhimurium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) isolated from patients in South America, while the incidence of PER-1-producing strains so far has been restricted to Turkey, where it occurs both in members of the family Enterobacteriaceae and in P. aeruginosa.

scholarly journals Frequency and diversity of Class A extended-spectrum β-lactamases in hospitals of the Auvergne, France: a 2 year prospective study

2004 ◽  
Vol 54 (3) ◽  
pp. 634-639 ◽  
Author(s):  
C. De Champs ◽  
C. Chanal ◽  
D. Sirot ◽  
R. Baraduc ◽  
J. P. Romaszko ◽  
...  
Keyword(s):  
Prospective Study ◽  
Class A ◽  
Extended Spectrum
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