Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in enterobacteriaceae

We have determined the nucleotide sequence of the plasmid genes blaT-4 and blaT-5 which encode the broad-substrate-range beta-lactamases TEM-4 and TEM-5, respectively. The TEM-4 enzyme, which confers high-level resistance to cefotaxime (Ctx) and ceftazidime (Caz), differed from the TEM-1 penicillinase by four amino acid substitutions. Two of the mutations are identical to those responsible for the wide substrate range of the TEM-3 beta-lactamase which hydrolyses Ctx and Caz. The amino acid sequence of TEM-5, which confers higher levels of resistance to Caz than to other recently developed cephalosporins, differed from that of TEM-1 by three mutations distinct from those of TEM-4. Analysis of the location of the mutations in the primary and tertiary structures of class A beta-lactamases suggests that interactions between the substituted residues and beta-lactam antibiotics non-hydrolysable by TEM-1 and TEM-2 allow TEM-4 and TEM-5 to hydrolyse efficiently novel broad-spectrum cephalosporins such as Ctx and Caz.     

A novel sequence framework (bla(TEM-1G)) encoding the parental TEM-1 beta-lactamase

A novel parental bla(TEM) gene (bla(TEM-1G)), encoding a TEM-1 beta-lactamase (pI of 5.4) produced by the uropathogenic Escherichia coli strain FMV194 was isolated from a dog. We report PCR-restriction fragment length polymorphism analysis and nucleotide sequencing of this gene. The bla(TEM-1G) sequence was identical to the bla(TEM-1C) gene framework in the coding and promoter (P3) regions, except for a silent G(604)-->T mutation in the coding region. Molecular phylogenetic analysis of parental bla(TEM) genes indicated two distinct groups, one comprising bla(TEM-1F) and bla(TEM-2). The other group comprises bla(TEM-1C) which is the probable ancestor of bla(TEM-1A), bla(TEM-1D) and bla(TEM-1G). The bla(TEM-1G) gene has the same framework as a gene encoding an inhibitor-resistant TEM beta-lactamase produced by an E. coli strain of human origin. Thus, parental bla(TEM) genes encoding beta-lactamases in E. coli strains isolated from different host species, in this case human and canine, may be phylogenetically very close.     

An IS1-like element is responsible for high-level synthesis of extended-spectrum beta-lactamase TEM-6 in Enterobacteriaceae.

Resistance of Escherichia coli strain HB251 to the newer beta-lactam antibiotics, in particular ceftazidime and aztreonam, results from production of the extended-spectrum beta-lactamase TEM-6. The corresponding structural gene, bla(T)-6, and its promoter region were amplified by the polymerase chain reaction. Analysis of the sequence of the amplification product showed that bla(T)-6 differed by two nucleotide substitutions from bla(T)-1, the gene encoding TEM-1 penicillinase in plasmid pBR322. The mutations led to the substitution of a lysine for a glutamic acid at position 102 and of a histidine for an arginine at position 162 of the unprocessed TEM-1 protein. The presence of a 116 bp DNA insert upstream from bla(T)-6 resulted in the creation of hybrid promoter P6 in which the -10 region was that of TEM-1 promoter P3 whereas the -35 canonical sequence TTGACA was provided by the right end of the insert. P6 was found to be 10 times more active than P3 and to confer higher levels of antibiotic resistance upon the host. Analysis of the sequence of the insert indicated that the 116 bp fragment is related to insertion sequence IS1 but differs from it by three internal deletions that removed regions encoding the transposase. The distribution of the IS1-like element in clinical isolates of Enterobacteriaceae was studied by the polymerase chain reaction and by DNA-DNA hybridization. The element appeared to be widespread and was detected in strains producing TEM-6 or other TEM variants.     

The TEM-3 β-lactamase, which hydrolyzes broad-spectrum cephalosporins, is derived from the TEM-2 penicillinase by two amino acid substitutions

Abstract We have determined the nucleotide sequence of the blaT -3 gene of plasmid pCF04 which confers resistance to penicillins and most cephalosporins by mediating the production of TEM-3 β-lactamase. The deduced amino acid sequence of TEM-3 differed in two positions from that of the TEM-2 penicillinase: Lys (TEM-3) for Glu (TEM-2) at position 102, and Ser (TEM-3) for Gly (TEM-2) at position 236 of the unprocessed protein. Examination of the location of the two modified amino acids of TEM-3 in the tertiary structure of class A β-lactamases suggested that they both are part of the substrate binding site. Analysis, by in vitro recombination, indicated that each mutation contributes to the extented substrate range of the enzyme, compared to that of the TEM-type penicillinase, and that the strength of the promoter of blaT -3 is responsible for high-level resistance towards broad-spectrum cephalosporins of strains producing TEM-3.     

Oligonucleotide probes for the detection of TEM-1 and TEM-2 beta-lactamase genes and their transposons

We describe the use of molecular probes to detect the TEM-type beta-lactamase genes. As a general probe, we prepared a 656 base pair restriction fragment, entirely within the TEM structural gene. This probe was specific for the TEM family, hybridizing only with TEM-1 and TEM-2. The TEM-1 and TEM-2 beta-lactamases differ by only one amino acid. We synthesized two oligonucleotides whose central bases correspond to this difference. The use of these oligonucleotides enables us to discriminate between TEM-1 and TEM-2 genes. Using oligonucleotides homologous to parts of Tn3, we also monitored the presence of TnA-like transposons in bacteria harboring different beta-lactamase genes. Only the TEM-1 and TEM-2 genes were found to be on transposons with terminal sequences identical to those of Tn3. All hybridization experiments were performed with both dot-blot and colony-hybridization techniques, and the suitability of these two methods for epidemiological studies is compared.     

The structural bases of antibiotic resistance in the clinically derived mutant beta-lactamases TEM-30, TEM-32, and TEM-34

Widespread use of beta-lactam antibiotics has promoted the evolution of beta-lactamase mutant enzymes that can hydrolyze ever newer classes of these drugs. Among the most pernicious mutants are the inhibitor-resistant TEM beta-lactamases (IRTs), which elude mechanism-based inhibitors, such as clavulanate. Despite much research on these IRTs, little is known about the structural bases of their action. This has made it difficult to understand how many of the resistance substitutions act as they often occur far from Ser-130. Here, three IRT structures, TEM-30 (R244S), TEM-32 (M69I/M182T), and TEM-34 (M69V), are determined by x-ray crystallography at 2.00, 1.61, and 1.52 A, respectively. In TEM-30, the Arg-244 --> Ser substitution (7.8 A from Ser-130) displaces a conserved water molecule that usually interacts with the beta-lactam C3 carboxylate. In TEM-32, the substitution Met-69 --> Ile (10 A from Ser-130) appears to distort Ser-70, which in turn causes Ser-130 to adopt a new conformation, moving its O gamma further away, 2.3 A from where the inhibitor would bind. This substitution also destabilizes the enzyme by 1.3 kcal/mol. The Met-182 --> Thr substitution (20 A from Ser-130) has no effect on enzyme activity but rather restabilizes the enzyme by 2.9 kcal/mol. In TEM-34, the Met-69 --> Val substitution similarly leads to a conformational change in Ser-130, this time causing it to hydrogen bond with Lys-73 and Lys-234. This masks the lone pair electrons of Ser-130 O gamma, reducing its nucleophilicity for cross-linking. In these three structures, distant substitutions result in accommodations that converge on the same point of action, the local environment of Ser-130.     

Direct sequencing of the amplified structural gene and promoter for the extended-broad-spectrum beta-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae

Extended-broad-spectrum beta-lactamase TEM-9, detected in a clinical isolate of Klebsiella pneumoniae, confers high-level resistance to recent cephalosporins, in particular ceftazidime, and to the monobactam aztreonam. Using oligonucleotide probes, we found that the plasmid gene blaT-9 encoding TEM-9 differs from characterized blaT genes by a new combination of already known mutations. Gene blaT-9 was further studied by direct sequencing of an amplified 1.1-kb DNA fragment which contained the open reading frame and its promoter. Analysis of the nucleotide and of the deduced amino acid sequence confirmed the hybridization results and indicated that TEM-9 differs from TEM-1 by four amino acid substitutions: Phe at position 19 and Met at position 261, which have been found in TEM-4 and are known not to expand the enzyme substrate range; Lys 102, detected in TEM-3 and TEM-4, and Ser 162, present in TEM-5 and TEM-7. Each of the latter substitutions enlarges the substrate spectrum of the enzymes and they are found associated for the first time in TEM-9.     

Phenotypic and molecular characterization of TEM-116 extended-spectrum beta-lactamase produced by a Shigella flexneri clinical isolate from chickens.

Extended-spectrum beta-lactamases (ESBLs) produced by a clinical isolate of Shigella flexneri from chickens were detected with confirmatory phenotypic tests of the Clinical and Laboratory Standards Institute, and minimum inhibitory concentrations of several antibacterial drugs against the isolate were determined by the twofold dilution method. The genotype and subtype of the ESBL-producing S. flexneri isolate were identified by PCR amplifying of ESBL genes and DNA sequencing analysis. The results revealed that the isolate was able to produce ESBLs. They were resistant to third-generation cephalosporins such as ceftiofur and ceftriaxone and showed characteristics of multidrug resistance. The ESBL gene from the S. flexneri isolate was of the TEM type. Sequence analysis indicated that the TEM-type gene had 99.1% and 99.2% identity to TEM-1D ESBL and TEM-1 beta-lactamase, respectively, at the nucleotide level. The amino acid sequence inferred from the TEM-type gene revealed three substitutions compared with the TEM-1 and TEM-1D enzymes: Ser51Gly, Val82Ila and Ala182Val. When it was compared with TEM-116 (99.8% identity), there were only two mutations (A(151)G and T(403)C) in the TEM-type gene, resulting in the substitution of Ser to Gly at position 51 in the amino acid sequence. The TEM type was a TEM-116 derivative.     

An additional ionic bond suggested by molecular modelling of TEM-2 might induce a slight discrepancy between catalytic properties of TEM-1 and TEM-2 β-lactamases

Abstract The plasmid-mediated TEM-1 and TEM-2 β-lactamases are the most commonly encountered among Gram-negative bacteria. They belong to molecular class A, and differ by one amino acid at position 39: TEM-1 have a glutamine and TEM-2 a lysine. Kinetic parameters ( k _cat and K _m) and catalytic efficiency ( k _cat/ K _m) of TEM-1 and TEM-2 β-lactamases are slightly, but significantly different. For all antibiotics except methicillin and cefazolin, the catalytic efficiency values of TEM-2 are clearly greater than that of TEM-1. Molecular modelling of TEM-2, when compared to that of TEM-1, showed an additional ionic bond between Lys-39 and Glu-281.     

Phenotypic and molecular characterization of TEM-116 extended-spectrum β-lactamase produced by a Shigella flexneri clinical isolate from chickens

Extended-spectrum β-lactamases (ESBLs) produced by a clinical isolate of Shigella flexneri from chickens were detected with confirmatory phenotypic tests of the Clinical and Laboratory Standards Institute, and minimum inhibitory concentrations of several antibacterial drugs against the isolate were determined by the twofold dilution method. The genotype and subtype of the ESBL-producing S. flexneri isolate were identified by PCR amplifying of ESBL genes and DNA sequencing analysis. The results revealed that the isolate was able to produce ESBLs. They were resistant to third-generation cephalosporins such as ceftiofur and ceftriaxone and showed characteristics of multidrug resistance. The ESBL gene from the S. flexneri isolate was of the TEM type. Sequence analysis indicated that the TEM-type gene had 99.1% and 99.2% identity to TEM-1D ESBL and TEM-1 β-lactamase, respectively, at the nucleotide level. The amino acid sequence inferred from the TEM-type gene revealed three substitutions compared with the TEM-1 and TEM-1D enzymes: Ser51Gly, Val82Ila and Ala182Val. When it was compared with TEM-116 (99.8% identity), there were only two mutations (A¹⁵¹G and T⁴⁰³C) in the TEM-type gene, resulting in the substitution of Ser to Gly at position 51 in the amino acid sequence. The TEM type was a TEM-116 derivative.     

Biochemical and genetic characteristics of TEM-29B, a novel extended spectrum beta-lactamase

A clinical strain of Escherichia coli (strain Ec 41553) that was resistant to ceftazidime produced a TEM-type beta-lactamase with a pI of 5.4. Clavulanic acid restored the ceftazidime activity, thus suggesting an extended spectrum beta-lactamase (ESBL). The gene encoding ESBL was located in a plasmid of 57 kb. After cloning and sequencing, the ESBL (TEM-29B) showed one amino acid replacement with respect to the TEM-1 sequence, Arg-164 to His. This change increased mainly the rate of hydrolysis of ceftazidime but not of cefotaxime and aztreonam. The relevance of this substitution in the increase of ceftazidime MIC is thus stressed.     

Molecular characterisation by PCR-restriction fragment length polymorphism of TEM β-lactamases

Abstract To rapidly characterise TEM-derived extended-spectrum β-lactamases a fast and easy method using polymerase chain reaction-restriction fragment length polymorphism was developed. This method was validated with ten reference TEM-type extended-spectrum β-lactamases. The mutations involved in TEM-20 and TEM-21, which were previously reported only with biochemical analysis, were then characterised. TEM-20 differed from TEM-19 by a silent mutation at position 925 (A for G), and TEM-21 differed from TEM-3 and TEM-14 by a single mutation (G for A) in an unreported position 660, involving an amino acid substitution, arginine for histidine, at position 153. Moreover, a new extended-spectrum β-lactamase conferring low resistance to ceftazidime (TEM-29), was described. TEM-29 derived from TEM-1, with an amino acid substitution, his-164. Finally, the combination of polymerase chain reaction-restriction fragment length polymorphism and plasmid analysis allowed us to investigate nosocomial outbreaks due to clinical isolates of multi-resistant Klebsiella pneumoniae in three hospitals.     

Substitution of Thr for Ala-237 in TEM-17, TEM-12 and TEM-26: alterations in β-lactam resistance conferred on Escherichia coli

Non-naturally occurring mutants of TEM-17 (E104K), TEM-12 (R164S) and TEM-26 (E104K:R164S) extended-spectrum (ES) beta-lactamases bearing threonine at position 237 were constructed by site-specific mutagenesis and expressed under isogenic conditions in Escherichia coli. Quantification of beta-lactamase activities and immunoblotting indicated that Ala-237-->Thr did not significantly affect expression levels of these ES enzymes. Minimum inhibitory concentrations of beta-lactam antibiotics showed that the presence of threonine at position 237 exerted a dominant effect increasing the enzymes' preference for various early generation cephalosporins over penicillins. Activity against broad-spectrum oxyimino-beta-lactams was also changed. The effect of Ala-237-->Thr on the activity against ceftazidime, aztreonam, cefepime and cefpirome of all three ES TEM enzymes was detrimental. Introduction of Thr-237 improved activity against cefotaxime and ceftriaxone in TEM-12 and TEM-26, but not in TEM-17.     

Thermodynamic investigation of the role of contact residues of beta-lactamase-inhibitory protein for binding to TEM-1 beta-lactamase

We have determined the thermodynamics of binding for the interaction between TEM-1 beta-lactamase and a set of alanine substituted contact residue mutants ofbeta-lactamase-inhibitory protein (BLIP) using isothermal titration calorimetry. The binding enthalpies for these interactions are highly temperature dependent, with negative binding heat capacity changes ranging from -800 to -271 cal mol(-1) K(-1). The isoenthalpic temperatures (at which the binding enthalpy is zero) of these interactions range from 5 to 38 degrees C. The changes in isoenthalpic temperature were used as an indicator of the changes in enthalpy and entropy driving forces, which in turn are related to hydrophobic and hydrophilic interactions. A contact residue of BLIP is categorized as a canonical residue if its alanine substitution mutant exhibits a change of isoenthalpic temperature matching the change of hydrophobicity because of the mutation. A contact position exhibiting a change in isoenthalpic temperature that does not match the change in hydrophobicity is categorized as an anti-canonical residue. Our experimental results reveal that the majority of residues where alanine substitution results in a loss of affinity are canonical (7 of 10), and about half of the residues where alanine substitutions have a minor effect are canonical. The interactions between TEM-1beta-lactamase and BLIP canonical contact residues contribute directly to binding free energy, suggesting potential anchoring sites for binding partners. The anti-canonical behavior of certain residues may be the result of mutation-induced modifications such as structural rearrangements affecting contact residue configurations. Structural inspection of BLIP suggests that the Lys(74) side chain electrostatically holds BLIP loop 2 in position to bind to TEM-1 beta-lactamase, explaining a large loss of entropy-driven binding energy of the K74A mutant and the resulting anti-canonical behavior. The anti-canonical behavior of the W150A mutant may also be due to structural rearrangements. Finally, the affinity enhancing effect of the contact residue mutant Y50A may be due to energetic coupling interactions between Asp(49) and His(41).     

Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS). Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton. Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains. In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 beta-lactamase. Translocation was detected directly in living host cells by using the fluorescent beta-lactamase substrate CCF2/AM. We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells. Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner. A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation. In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation. In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.     

Val-237 for Ala substitution in the TEM-2 β-lactamase dramatically alters the catalytic efficiencies towards carbenicillin and ticarcillin

Abstract The mutant 554 of TEM-2 β-lactamase was selected for a decrease in the resistance to carbenicillin of an Escherichia coli K12 carrier. The amino acid sequence of the mutant β-lactamase was determined by manual Edman degradation analysis of proteolytic peptides. A single substitution Val for Ala was localized at position 237. The mutant exhibited only 2% of the catalytic efficiency of the wild-type enzyme towards carbenicillin and ticarcillin, whereas it retained 30–60% of the hydrolytic activity towards other penicillin and cephalosporin substrates. Carfecillin, the phenyl ester of the side-chain carboxyl group of carbenicillin, was hydrolysed as a good substrate. This suggests that the behaviour of the mutant enzyme towards carbenicillin may result from ionic rather than steric constraints. A molecular model of the Val-237 TEM-2 mutant suggests possible electrostatic interaction between Glu-171 and the carboxylic group of the side chain of carbenicillin.     

Noncovalent interaction energies in covalent complexes: TEM-1 beta-lactamase and beta-lactams

The class A beta-lactamase TEM-1 is a key bacterial resistance enzyme against beta-lactam antibiotics, but little is known about the energetic bases for complementarity between TEM-1 and its inhibitors. Most inhibitors form a covalent adduct with the catalytic Ser70, making the measurement of equilibrium constants, and hence interaction energies, technically difficult. This study evaluates noncovalent interactions within covalent complexes by examining the differential stability of TEM-1 and its inhibitor adducts. The thermal denaturation of TEM-1 follows a two-state, reversible model with a melting temperature (T(m)) of 51.6C and a van't Hoff enthalpy of unfolding (DeltaH(VH)) of 146.2 kcal/mol at pH 7.0. The stability of the enzyme changes on forming an inhibitor adduct. As expected, some inhibitors stabilize TEM-1; transition-state analogues increase the T(m) by up to 3.7C (1.7 kcal/mol). Surprisingly, all beta-lactam covalent acyl--enzyme complexes tested destabilize TEM-1 significantly relative to the apo-enzyme. For instance, the clinically used inhibitor clavulanic acid and the beta-lactamase-resistant beta-lactams moxalactam and imipenem destabilize TEM-1 by over 2.6C (1.2 kcal/mol) in their covalent adducts. Based on the structure of the TEM-1/imipenem complex (Maveyraud et al., J Am Chem Soc 1998;120:9748--52), destabilization by moxalactam and imipenem is thought to be caused by a steric clash between the side-chain of Asn132 and the 6(7)-alpha group of these beta-lactams. To test this hypothesis, the mutant enzyme N132A was made. In contrast with wild-type, the covalent complexes between N132A and both imipenem and moxalactam stabilize the enzyme, consistent with the hypothesis. To investigate the structural bases of this dramatic change in stability, the structure of N132A/imipenem was determined by X-ray crystallography. In the complex with N132A, imipenem adopts a very different conformation from that observed in the wild-type complex, and the putative destabilizing interaction with residue 132 is relieved. Studies of several enzymes suggest that beta-lactams, and covalent inhibitors in general, can have either net favorable or net unfavorable noncovalent interaction energies within the covalent complex. In the case of TEM-1, such unfavorable interactions convert substrate analogues into very effective inhibitors.     

Characterization of PDR4, a Saccharomyces cerevisiae gene that confers pleiotropic drug resistance in high-copy number: identity with YAP1, encoding a transcriptional activator [corrected

PDR4 is a gene that confers pleiotropic drug resistance (pdr) to the yeast Saccharomyces cerevisiae when present in high copy number [Leppert et al., Genetics 125 (1990) 13-20]. Transposon insertion mutations had identified the active region of the gene as a 3.7-kb SalI-EcoRI restriction fragment of the 8-kb cloned fragment. We have confirmed this by showing that this fragment is sufficient to confer pdr, and have sequenced its entire 3761 bp. It contains a single complete open reading frame (ORF) extending from nucleotide (nt) position 1631-3580, coding for a protein of 650 amino acids (aa). A 2.7-kb fragment containing this ORF is also sufficient to confer pdr. The aa sequence contains no recognizable homologies or consensus sequences, so it is a novel protein of unknown function. It is apparently soluble, since no transmembrane-type sequences were predicted. A second, partial ORF was also found, on the opposite strand, extending from nt position 774 to past the SalI site, which is apparently unrelated to pdr.     

Structural insight into the kinetics and DeltaCp of interactions between TEM-1 beta-lactamase and beta-lactamase inhibitory protein (BLIP)

In a previous study, we examined thermodynamic parameters for 20 alanine mutants in beta-lactamase inhibitory protein (BLIP) for binding to TEM-1 beta-lactamase. Here we have determined the structures of two thermodynamically distinctive complexes of BLIP mutants with TEM-1 beta-lactamase. The complex BLIP Y51A-TEM-1 is a tight binding complex with the most negative binding heat capacity change (DeltaG = approximately -13 kcal mol(-1) and DeltaCp = approximately -0.8 kcal mol(-1) K(-1)) among all of the mutants, whereas BLIP W150A-TEM-1 is a weak complex with one of the least negative binding heat capacity changes (DeltaG = approximately -8.5 kcal mol(-1) and DeltaCp = approximately -0.27 kcal mol(-1) K(-1)). We previously determined that BLIP Tyr51 is a canonical and Trp150 an anti-canonical TEM-1-contact residue, where canonical refers to the alanine substitution resulting in a matched change in the hydrophobicity of binding free energy. Structure determination indicates a rearrangement of the interactions between Asp49 of the W150A BLIP mutant and the catalytic pocket of TEM-1. The Asp49 of W150A moves more than 4 angstroms to form two new hydrogen bonds while losing four original hydrogen bonds. This explains the anti-canonical nature of the Trp150 to alanine substitution, and also reveals a strong long distance coupling between Trp150 and Asp49 of BLIP, because these two residues are more than 25 angstroms apart. Kinetic measurements indicate that the mutations influence the dissociation rate but not the association rate. Further analysis of the structures indicates that an increased number of interface-trapped water molecules correlate with poor interface packing in a mutant. It appears that the increase of interface-trapped water molecules is inversely correlated with negative binding heat capacity changes.