INVESTIGADORES
WOLMAN Federico Javier
congresos y reuniones científicas
Título:
Kinetic characterization and genetic context of the metallo‐β‐lactamase LRA‐12 from an Alaskan soil metagenome.
Autor/es:
M. M. RODRÍGUEZ; B. GHIGLIONE; J. DI CONZA; L. MOE; M. PÉREZ GARÓFALO; E. BLATEZKY; FEDERICO JAVIER WOLMAN; J. HANDELSMAN; G. GUTKIND; PABLO POWER
Lugar:
CABA
Reunión:
Congreso; XIII Congreso Argentino de Microbiología 2013, II Congreso de Microbiología Agrícola y Ambiental; 2013
Institución organizadora:
Asociación Argentina de Microbiología
Resumen:
Background: Metagenomics includes methodologies aimed at the direct recovery of genes or proteins from uncultured microorganisms, generally involving the construction of metagenomic libraries in Escherichia coli, and avoiding the use of culturing techniques. One of the most convenient screening methods is based on the use of selective media for allowing the growth of those clones producing a specific enzyme or protein (activity‐driven metagenomics). By this means, antimicrobial resistance genes for different families of antibiotics from environmental soil samples have been disclosed. Among them, several β‐lactamase‐encoding genes from the four Ambler classes have been recovered from Alaskan soil samples, being most of them metallo‐β‐lactamases (MBLs). Objectives: From all the metagenomic‐derived β‐lactamases, we focused on the kinetic characterization of one of the MBLs, named LRA‐12. We also analyzed the genetic context of the blaLRA‐12 gene. Methods: The blaLRA‐12 gene was cloned in suitable vectors for both antimicrobial susceptibility tests (performed according to CLSI?s recommendations), and protein production. LRA‐12 was overexpressed in Escherichia coli BL21(DE3) cells containing the recombinant plasmid pET28/blaLRA‐ 12. The MBL was purified to homogeneity by affinity and ion exchange chromatography. Steady‐state kinetic parameters were determined for β‐lactam antibiotics and β‐lactamase inhibitors. In silico analysis of the genetic context of blaLRA‐12 was performed using bioinformatics tools. Results: LRA‐12 has 61% amino acid identity with GOB metallo‐β‐lactamases from Elizabethkingia meningoseptica. The MBL also showed 41% and 33% identity with FEZ‐1 (Fluoribacter gormanii) and BJP‐1 (Bradyrhizobium japonicum), respectively, two class B3 enzymes for which the crystallographic structure is available. LRA‐12‐producing clones were resistant to all tested β‐lactams (except aztreonam) and showed inhibition by EDTA. The purified enzyme displayed high catalytic efficiencies towards all β‐lactams except monobactams, in good agreement with the behavior of other metallo‐ β‐lactamases. The overall GC content of the whole insert containing blaLRA‐12 (33,920 bp) is 41%. It includes 4 putative islets between 204‐356 bp with an atypical GC% 50.5‐56.2%. No putative IS, transposons or other transferable elements (or signals of transpositions) were found associated with the bla gene. Conclusions: These results support the hypothesis of the origin of many β‐lactamases, residing in the chromosome of the ?environmental resistome? with native wide‐range activity spectrum towards most β‐lactams, even before being recruited and disseminated among human pathogens. Conservation of the DNA surrounding the bla gene and the lack of putative recombination/transposition signals is in agreement with the origin of the sample, which comes from an ?antibiotic‐free? area and therefore the possibility of occurrence of gene exchanges is low.
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