title: Genetic, biochemical characterization and mutagenesis of the chromosomal class A β-lactamase of Raoultella (formerly Klebsiella) terrigena.
abstract: BACKGROUND
  Chromosomal class A β-lactamases have been characterized in Raoultella ornithinolytica and Raoultella planticola. The purpose of this study was to characterize that of Raoultella terrigena.
  
  
  METHODS
  The blaTER-1 gene of R. terrigena strain ATCC33257(T) was cloned (pACter-1) and sequenced. It was then used to detect the bla gene of strains BM 85 01 095 and SB2796. The hypermutable Escherichia coli strain AB1157 mutS::Tn10 was transformed with pACter-1 and mutants growing on plates containing>2mg/L ceftazidime were studied. Notably, the impact of mutations only observed in the promoter region on β-lactam resistance was assessed by site-directed mutagenesis experiments.
  
  
  RESULTS
  R. terrigena strains ATCC33257(T) and BM 85 01 095 had the same bla gene and deduced protein (TER-1) whereas there were 3 substitutions in those of strain SB2796 (TER-2). Class A β-lactamases TER showed 78%, 69.9% and 38.7% identity with PLA or ORN, TEM-1 and KOXY, respectively. Compared with TEM-1, TER-1 and TER-2 showed 2 particular substitutions, Leu75Pro and Glu240Asn demonstrated to be involved in the inherent β-lactam resistance profile of R. terrigena. TER-1 (pI of 7.6) had a high activity against penicillin G and a significantly low one against amoxicillin. Substitution G/T observed in the -35 region of the blaTER gene harbored by strains growing in the presence of≥2mg/L ceftazidime was shown to be responsible for this growth.
  
  
  CONCLUSIONS
  TER is a new class A β-lactamase belonging to functional group 2b.
authors.affiliation: Service de microbiologie, faculté de médecine D.-Diderot, université Paris-VII, 16, rue Henri-Huchard, 75018 Paris, France.
authors.forename: E
authors.initials: E
authors.lastname: Walckenaer
authors.affiliation: Laboratoire de bactériologie clinique, CHU de Clermont-Ferrand, EA3844, UFR de médecine université Clermont-1, 58, rue Montalembert, 63000 Clermont-Ferrand, France.
authors.forename: J
authors.initials: J
authors.lastname: Delmas
authors.affiliation: Service de microbiologie, hôpital Beaujon, AP-HP, 100, boulevard du Général-Leclerc, 92110 Clichy, France.
authors.forename: V
authors.initials: V
authors.lastname: Leflon-Guibout
authors.affiliation: Laboratoire de bactériologie clinique, CHU de Clermont-Ferrand, EA3844, UFR de médecine université Clermont-1, 58, rue Montalembert, 63000 Clermont-Ferrand, France.
authors.forename: R
authors.initials: R
authors.lastname: Bonnet
authors.affiliation: Service de microbiologie, faculté de médecine D.-Diderot, université Paris-VII, 16, rue Henri-Huchard, 75018 Paris, France; Service de microbiologie, hôpital Beaujon, AP-HP, 100, boulevard du Général-Leclerc, 92110 Clichy, France; CRI, centre de recherche sur l'inflammation, UMR 1149, Inserm, faculté de médecine, université Paris-Diderot, site Bichat, 16, rue Henri-Huchard, 75018 Paris, France. Electronic address: mhn.chanoine@bjn.aphp.fr.
authors.forename: M-H
authors.initials: MH
authors.lastname: Nicolas-Chanoine
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002442:Ceftazidime / Q000494:pharmacology; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D004755:Enterobacteriaceae / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D047090:beta-Lactams; D002442:Ceftazidime; D001618:beta-Lactamases
keywords: Gène bla; Promoteur fort; Raoultella terrigena; Strength promoter; bla gene