title: Natural insertion of the bro-1 β-lactamase gene into the gatCAB operon affects Moraxella catarrhalis aspartyl-tRNA(Asn) amidotransferase activity.
abstract: Only about half of bacterial species use an asparaginyl-tRNA synthetase (AsnRS) to attach Asn to its cognate tRNA(Asn). Other bacteria, including the human pathogen Moraxella catarrhalis, a causative agent of otitis media, lack a gene encoding AsnRS, and form Asn-tRNA(Asn) by an indirect pathway catalysed by two enzymes: first, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) catalyses the formation of aspartyl-tRNA(Asn) (Asp-tRNA(Asn)); then, a tRNA-dependent amidotransferase (GatCAB) transamidates this 'incorrect' product into Asn-tRNA(Asn). As M. catarrhalis has a Gln-tRNA synthetase, its GatCAB functions as an Asp-tRNA(Asn) amidotransferase. This pathogen rapidly evolved to about 90 % ampicillin resistance worldwide by insertion of a bro-1 β-lactamase gene within the gatCAB operon. Comparison of the GatCAB subunits from bro-1 β-lactamase-positive and bro-negative strains showed that the laterally transferred bro-1 gene, inserted into the gatCAB operon, affected the C-terminal sequence of GatA. The identity between the C-terminal sequences of GatA(wt) (residues 479-491) and of GatA(BRO-1) (residues 479-492) was about 36 %, whereas the rest of the GatA sequence was relatively conserved. The characterization of these two distinct GatCABs as well as the hybrid GatCAB containing GatA(1-478)(wt)(479-492)(BRO-1) and truncated GatCAB enzymes of M. catarrhalis showed that the substitution in GatA(wt) of residues 479-492 of GatA(BRO-1) causes increased specificity for glutamine, and decreased specificity for Asp-tRNA(Asn) in the transamidation reaction. We conclude that the bro gene insertion has altered the kinetic parameters of Asp-tRNA(Asn) amidotransferase, and we propose a model for gatA evolution after the insertion of bro-1 at the carboxyl end of gatA.
authors: Centre de Recherche en Infectiologie, CHUQ Pavillon CHUL, 2705 boul. Laurier, RC-709, QC G1V 4G2, Canada.|Institut Pasteur de Côte d'Ivoire, 01 BP 490 Abidjan, Côte d'Ivoire.|Département de Biochimie, de Microbiologie et de Bio-informatique, Université Laval, QC G1V 0A6, Canada.
authors: Pierre-Marie
authors: PM
authors: Akochy
authors: Institut de biologie intégrative et des systèmes (IBIS), Pavillon Charles-Eugène-Marchand, G1V 0A6, Canada.|Département de Biochimie, de Microbiologie et de Bio-informatique, Université Laval, QC G1V 0A6, Canada.
authors: Jacques
authors: J
authors: Lapointe
authors: Centre de Recherche en Infectiologie, CHUQ Pavillon CHUL, 2705 boul. Laurier, RC-709, QC G1V 4G2, Canada.|Département de Biochimie, de Microbiologie et de Bio-informatique, Université Laval, QC G1V 0A6, Canada.
authors: Paul H
authors: PH
authors: Roy
mesh_terms: D022761:Gene Transfer, Horizontal; D006801:Humans; D007700:Kinetics; D001936:Moraxella catarrhalis / Q000201:enzymology* / Q000235:genetics*; D016254:Mutagenesis, Insertional; D019884:Nitrogenous Group Transferases / Q000235:genetics* / Q000378:metabolism*; D009876:Operon; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: C412016:Asp-tRNA(Asn) amidotransferase; D019884:Nitrogenous Group Transferases; C408134:BRO beta-lactamase; D001618:beta-Lactamases