Search tips
Search criteria 


Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
Antimicrob Agents Chemother. 1997 November; 41(11): 2374–2382.
PMCID: PMC164131

Characterization of a new TEM-derived beta-lactamase produced in a Serratia marcescens strain.


A natural TEM variant beta-lactamase was isolated from an epidemic strain of Serratia marcescens. Nucleotide gene sequencing revealed multiple point mutations located in the 42-to-44 tripeptide and positions 145 to 146, 178, and 238. In addition, a glutamic acid 212 deletion was also found. The purified enzyme was studied from a kinetic point of view, revealing the highest catalytic efficiency (k[cat]/Km) values for ceftazidime and aztreonam compared with the TEM-1 prototype enzyme. The in vitro resistance correlated with kinetic parameters, and the enzyme also mediated resistance to some penicillins and an ampicillin-clavulanic acid combination. The mutational and kinetic changes are discussed in relation to the three-dimensional crystallographic structure of the wild-type TEM-1 enzyme.

Full Text

The Full Text of this article is available as a PDF (330K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ambler RP, Coulson AF, Frère JM, Ghuysen JM, Joris B, Forsman M, Levesque RC, Tiraby G, Waley SG. A standard numbering scheme for the class A beta-lactamases. Biochem J. 1991 May 15;276(Pt 1):269–270. [PubMed]
  • Blazquez J, Baquero MR, Canton R, Alos I, Baquero F. Characterization of a new TEM-type beta-lactamase resistant to clavulanate, sulbactam, and tazobactam in a clinical isolate of Escherichia coli. Antimicrob Agents Chemother. 1993 Oct;37(10):2059–2063. [PMC free article] [PubMed]
  • Bonomo RA, Dawes CG, Knox JR, Shlaes DM. Complementary roles of mutations at positions 69 and 242 in a class A beta-lactamase. Biochim Biophys Acta. 1995 Feb 22;1247(1):113–120. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Bradford PA, Urban C, Jaiswal A, Mariano N, Rasmussen BA, Projan SJ, Rahal JJ, Bush K. SHV-7, a novel cefotaxime-hydrolyzing beta-lactamase, identified in Escherichia coli isolates from hospitalized nursing home patients. Antimicrob Agents Chemother. 1995 Apr;39(4):899–905. [PMC free article] [PubMed]
  • Bush K. Characterization of beta-lactamases. Antimicrob Agents Chemother. 1989 Mar;33(3):259–263. [PMC free article] [PubMed]
  • Chanal C, Poupart MC, Sirot D, Labia R, Sirot J, Cluzel R. Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 beta-lactamase genes. Antimicrob Agents Chemother. 1992 Sep;36(9):1817–1820. [PMC free article] [PubMed]
  • De Meester F, Frère JM, Waley SG, Cartwright SJ, Virden R, Lindberg F. 6-beta-Iodopenicillanate as a probe for the classification of beta-lactamases. Biochem J. 1986 Nov 1;239(3):575–580. [PubMed]
  • Dideberg O, Charlier P, Wéry JP, Dehottay P, Dusart J, Erpicum T, Frère JM, Ghuysen JM. The crystal structure of the beta-lactamase of Streptomyces albus G at 0.3 nm resolution. Biochem J. 1987 Aug 1;245(3):911–913. [PubMed]
  • Fonzé E, Charlier P, To'th Y, Vermeire M, Raquet X, Dubus A, Frère JM. TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant. Acta Crystallogr D Biol Crystallogr. 1995 Sep 1;51(Pt 5):682–694. [PubMed]
  • Hall A, Knowles JR. Directed selective pressure on a beta-lactamase to analyse molecular changes involved in development of enzyme function. Nature. 1976 Dec 23;264(5588):803–804. [PubMed]
  • Herzberg O. Refined crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.0 A resolution. J Mol Biol. 1991 Feb 20;217(4):701–719. [PubMed]
  • Huletsky A, Knox JR, Levesque RC. Role of Ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type beta-lactamases probed by site-directed mutagenesis and three-dimensional modeling. J Biol Chem. 1993 Feb 15;268(5):3690–3697. [PubMed]
  • Jacoby GA, Medeiros AA. More extended-spectrum beta-lactamases. Antimicrob Agents Chemother. 1991 Sep;35(9):1697–1704. [PMC free article] [PubMed]
  • Jelsch C, Mourey L, Masson JM, Samama JP. Crystal structure of Escherichia coli TEM1 beta-lactamase at 1.8 A resolution. Proteins. 1993 Aug;16(4):364–383. [PubMed]
  • Jones TA. Diffraction methods for biological macromolecules. Interactive computer graphics: FRODO. Methods Enzymol. 1985;115:157–171. [PubMed]
  • Kado CI, Liu ST. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. [PMC free article] [PubMed]
  • Knox JR. Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure. Antimicrob Agents Chemother. 1995 Dec;39(12):2593–2601. [PMC free article] [PubMed]
  • Knox JR, Moews PC, Escobar WA, Fink AL. A catalytically-impaired class A beta-lactamase: 2 A crystal structure and kinetics of the Bacillus licheniformis E166A mutant. Protein Eng. 1993 Jan;6(1):11–18. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lamotte-Brasseur J, Dive G, Dideberg O, Charlier P, Frère JM, Ghuysen JM. Mechanism of acyl transfer by the class A serine beta-lactamase of Streptomyces albus G. Biochem J. 1991 Oct 1;279(Pt 1):213–221. [PubMed]
  • Lamotte-Brasseur J, Knox J, Kelly JA, Charlier P, Fonzé E, Dideberg O, Frére JM. The structures and catalytic mechanisms of active-site serine beta-lactamases. Biotechnol Genet Eng Rev. 1994;12:189–230. [PubMed]
  • Lee KY, Hopkins JD, O'Brien TF, Syvanen M. Gly-238-Ser substitution changes the substrate specificity of the SHV class A beta-lactamases. Proteins. 1991;11(1):45–51. [PubMed]
  • Linström EB, Boman HG, Steele BB. Resistance of Escherichia coli to penicillins. VI. Purification and characterization of the chromosomally mediated penicillinase present in ampA-containing strains. J Bacteriol. 1970 Jan;101(1):218–231. [PMC free article] [PubMed]
  • Mabilat C, Goussard S, Sougakoff W, Spencer RC, Courvalin P. Direct sequencing of the amplified structural gene and promoter for the extended-broad-spectrum beta-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae. Plasmid. 1990 Jan;23(1):27–34. [PubMed]
  • Matagne A, Frère JM. Contribution of mutant analysis to the understanding of enzyme catalysis: the case of class A beta-lactamases. Biochim Biophys Acta. 1995 Jan 19;1246(2):109–127. [PubMed]
  • Moews PC, Knox JR, Dideberg O, Charlier P, Frère JM. Beta-lactamase of Bacillus licheniformis 749/C at 2 A resolution. Proteins. 1990;7(2):156–171. [PubMed]
  • Pagani L, Luzzaro F, Ronza P, Rossi A, Micheletti P, Porta F, Romero E. Outbreak of extended-spectrum beta-lactamase producing Serratia marcescens in an intensive care unit. FEMS Immunol Med Microbiol. 1994 Nov;10(1):39–46. [PubMed]
  • Palzkill T, Botstein D. Identification of amino acid substitutions that alter the substrate specificity of TEM-1 beta-lactamase. J Bacteriol. 1992 Aug;174(16):5237–5243. [PMC free article] [PubMed]
  • Palzkill T, Le QQ, Venkatachalam KV, LaRocco M, Ocera H. Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta-lactamase. Mol Microbiol. 1994 Apr;12(2):217–229. [PubMed]
  • Raquet X, Lamotte-Brasseur J, Fonzé E, Goussard S, Courvalin P, Frère JM. TEM beta-lactamase mutants hydrolysing third-generation cephalosporins. A kinetic and molecular modelling analysis. J Mol Biol. 1994 Dec 16;244(5):625–639. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Shlaes DM, Currie-McCumber C. Mutations altering substrate specificity in OHIO-1, and SHV-1 family beta-lactamase. Biochem J. 1992 Jun 1;284(Pt 2):411–415. [PubMed]
  • Sowek JA, Singer SB, Ohringer S, Malley MF, Dougherty TJ, Gougoutas JZ, Bush K. Substitution of lysine at position 104 or 240 of TEM-1pTZ18R beta-lactamase enhances the effect of serine-164 substitution on hydrolysis or affinity for cephalosporins and the monobactam aztreonam. Biochemistry. 1991 Apr 2;30(13):3179–3188. [PubMed]
  • Strynadka NC, Adachi H, Jensen SE, Johns K, Sielecki A, Betzel C, Sutoh K, James MN. Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 A resolution. Nature. 1992 Oct 22;359(6397):700–705. [PubMed]
  • Venkatachalam KV, Huang W, LaRocco M, Palzkill T. Characterization of TEM-1 beta-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime. J Biol Chem. 1994 Sep 23;269(38):23444–23450. [PubMed]
  • Viadiu H, Osuna J, Fink AL, Soberón X. A new TEM beta-lactamase double mutant with broadened specificity reveals substrate-dependent functional interactions. J Biol Chem. 1995 Jan 13;270(2):781–787. [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)