The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors

The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.     

Molecular characterization of a new plasmid-encoded SHV-type beta-lactamase (SHV-2 variant) conferring high-level cefotaxime resistance upon Klebsiella pneumoniae

Between 1986 and 1988, multiresistant Klebsiella pneumoniae strains exhibiting high-level cefotaxime resistance were isolated from patient specimens particularly of the intensive care units of the Aachen Technical University Hospital. The resistance gene responsible was shown to be encoded on a conjugative 66 kb plasmid designated pZMP1. The MIC values for cefotaxime of the original isolates and the transconjugants were greater than 128 mg l-1 and 64 mg l-1, respectively. Isoelectric focusing of protein preparations from the transconjugants showed a beta-lactamase with a pI of 7.6. A 3.6 kb BamHI fragment containing the beta-lactamase gene was cloned into pLG339 resulting in the recombinant plasmid pZMP1-1. A restriction map of the cloned insert was established and PstI subfragments of the insert were further subcloned into pBGS18. The nucleotide sequence of the complete 3.6 kb fragment was determined. Within 3663 bp an open reading frame of 858 kb was found to show 99% homology to the SHV-2 and -3 nucleotide sequences. The deduced amino acid sequence differed in one and two positions, respectively, from these established SHV enzymes. The 3' noncoding sequence exhibited nearly perfect homology to that of SHV-2, but the 5' upstream sequence showed homology of less than 50% to the corresponding SHV-2 sequence, indicating an altered promoter region of the variant SHV-enzyme. Kinetic analysis of the beta-lactamase revealed a 50-100% elevated hydrolytic effectivity on cefotaxime in comparison to other SHV enzymes. Cefoxitin, ceftazidime, aztreonam and imipenem were not hydrolysed by the enzyme. The variant enzyme was inhibited by commonly available beta-lactamase inhibitors. Clavulanic acid had the highest affinity for the enzyme and the greatest effectivity in blocking its action. Based on the genetic and kinetic data we propose to classify the enzyme as a new variant beta-lactamase of the SHV-type and name it SHV-2a.     

Substitution of Met-69 by Ala or Gly in TEM-1 beta-lactamase confer an increased susceptibility to clavulanic acid and other inhibitors

In some inhibitor-resistant TEM-derived beta-lactamases, Met-69 is substituted by Leu, Ile or Val. Residue 69 is located in a region of strong structural constraints, at the beginning of H2 alpha-helix, and in the vicinity of B3 and B4 beta-strands. Analysis of the three-dimensional structure of TEM-1 beta-lactamase suggests that alteration of the substrate-binding site can be produced by changes of the size of residue 69 side chain. Met-69 was substituted by alanine or glycine in TEM-Bs beta-lactamase (a TEM-1-related enzyme) using site-directed mutagenesis. The minimum inhibitory concentrations of the mutants compared with the wild-type revealed an increased susceptibility to beta-lactamase inhibitor-beta-lactam combinations and to first-generation cephalosporins. Comparing the Met69Ala and Met69Gly beta-lactamases with TEM-Bs, K(m) constants of the mutants showed an increased affinity for most beta-lactams but the kcat for most substrates did not change substantially. Mutants also demonstrated lower IC50 for the three inhibitors (clavulanic acid, tazobactam and sulbactam). The two substitutions of the residue 69 by alanine and glycine had a noticeable effect on K(m) values of TEM-Bs beta-lactamase, and on affinity for beta-lactamase inhibitors.     

Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.     

Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.     

Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.     

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by phosphonate monoesters.

The class C serine beta-lactamase of Enterobacter cloacae P99 was inhibited by a series of aryl methylphosphonate monoester monoanions. The effectiveness of these inhibitors was promoted by an acylamido substituent on the methyl group and a good leaving group at phosphorus. The former preference suggests that noncovalent interaction of these inhibitors with the enzyme resembles that of substrates, while the latter suggests that nucleophilic displacement at phosphorus occurs as part of the inhibition mechanism. The truth of the latter proposition was confirmed by observation of release of 1 equiv of phenol concomitant with inhibition and of the presence of an equivalent amount of 14C-label on the enzyme after inhibition by a 14C-labeled phosphonate. The hydrolytically inert nature of the enzyme-inhibitor adduct, and its 31P chemical shift, suggested that O-phosphonylation of the enzyme had occurred. Although, by analogy with substrates, one might expect that the hydroxyl of the active site serine residue would be covalently modified by these inhibitors, successive alkali and acid treatment of the enzyme-inhibitor adduct generated no pyruvate. Instead, 1 equiv of lysinoalanine was found. This product was rationalized to arise through intramolecular capture by an adjacent lysine amine group of the dehydroalanine residue produced by alkali treatment of an O-phosphonylated serine residue. One equivalent of lysinoalanine was also produced by alkali treatment of the enzyme that had been inhibited by 6 beta-bromopenicillanic acid, a mechanism-based inhibitor known to acylate the hydroxyl group of the active site serine residue. It is therefore likely that the aryl phosphonates phosphonylate this residue. These compounds should be useful as beta-lactamase active site titrants and as sources of fresh insight into the chemical properties of the active site. The significant mechanistic features of the inhibition, in particular its strong leaving group dependence and the distinctive ability of the beta-lactamase active site to stabilize a dianionic transition state containing a pentacoordinated phosphorus, are discussed with respect to the active site structure. The comparison with phosph(or/on)yl inhibitors of serine proteinases is made, and the mechanism-based features of inhibition of serine hydrolases by phosph(on)ates are noted.     

Cephalosporinase and penicillinase activities of a β-lactamase from Pseudomonas pyocyanea

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.     

In vivo regulation of chromosomal beta-lactamase in Escherichia coli.

Chromosomal beta-lactamase, a periplasmic enzyme of Escherichia coli, was studied with respect to its regulation in vivo. Both the activity and the amount of beta-lactamase increased with growth rate. During a nutritional shift-down, chromosomal beta-lactamase activity followed stable ribonucleic acid accumulation. After a nutritional shift-up the differential rate of beta-lactamase synthesis did not increase immediately (like stable ribonucleic acid), but did increase after a lag period of 30 min. To determine whether beta-lactamase was under stringent control, strains carrying a temperature-sensitive valyl-transfer ribonucleic acid synthetase and differing only in the allelic state of the relA gene were shifted from a permissive to a semipermissive temperature. No influence by the relA gene product was found on beta-lactamase synthesis. The regulation of this periplasmic enzyme is discussed in relation to that of some components of the translational apparatus.     

On the formation of a dicloxacillin-p-hydroxymercuribenzoate suicide complex mediated by beta-lactamase I from Bacillus cereus

p-Hydroxymercuribenzoate is a non-competitive inhibitor of beta-lactamase I from Bacillus cereus and also, after preliminary preincubation, an inactivator of the enzyme. Submitted to the simultaneous action of PCMB plus dicloxacillin, the enzyme completely loses its activity. Extensive dialysis can restore the enzymatic activity only if preincubation had been carried out with either PCMB or dicloxacillin but not if both inhibitors had been simultaneously present. Mercaptoethanol protects the enzyme from the action of PCMB, but not from the severe inactivation caused by dicloxacillin-PCMB mixtures. All these data suggest the formation of a complex between PCMB and the acyl-enzyme intermediate generated upon hydrolysis of the beta-lactam bond of dicloxacillin.     

Mechanism of inhibition of the beta-lactamase of Enterobacter cloacae P99 by 1:1 complexes of vanadate with hydroxamic acids

The class C beta-lactamase of Enterobacter cloacae P99 is competitively inhibited by low concentrations of 1:1 complexes of vanadate and hydroxamic acids. Structure-activity studies indicated that the hydroxamic acid functional group was essential to this inhibition. Both aryl and alkyl hydroxamic acids form inhibitory ternary complexes with vanadate and the enzyme, although, in certain cases of the latter, the inhibition may not be seen because of the low formation constants of the vanadate-hydroxamic acid complex. After all of the vanadate species present in solution had been taken into account, "real" K(i) values for the vanadate complexes could be determined. The K(i) value of the best of the inhibitors that were investigated, the 1:1 complex of vanadate with 4-nitrobenzohydroxamic acid, was 0.48 microM. Kinetics studies showed that the association and dissociation rate constants of this complex with the enzyme were 1.48 x 10(6) s(-1) M(-1) and 0.73 s(-1), respectively; the magnitude of the latter indicates covalent interaction of the complex with the enzyme. (51)V NMR and UV-vis spectra suggest that the structure of the vanadate complex bound to the enzyme may be very similar to that in solution. A (13)C NMR spectrum of the enzyme complex with 4-nitrobenzo[(13)C]hydroxamic acid and vanadate yields a coordination-induced shift (CIS) of 7.74 ppm. This is significantly larger than that of the vanadate complex in free solution (3.62 ppm), suggesting either, somewhat contrary to the (51)V and UV-vis spectra, greater interaction between vanadium and the hydroxamate carbonyl oxygen in the enzyme complex than in free solution or, more likely, polarization of the hydroxamate by interaction, e.g., hydrogen bonding, with the enzyme. Molecular modeling indicates that a pentacoordinated vanadate complex may well be able to snugly occupy the enzyme active site; Asn 152 is suitably placed to hydrogen bond to the hydroxamic acid oxygen atom. The experimental results are in accord with a model whereby the vanadate-hydroxamate-enzyme complex is a moderately good analogue of the transition state of the reaction of the beta-lactamase with phosphonate inhibitors.     

Interactions between non-classical beta-lactam compounds and the beta-lactamases of Actinomadura R39 and Streptomyces albus G.

6-Aminopenicillanic acid, 7-aminocephalosporanic acid, mecillinam and quinacillin have varying substrate activities for both the R39 beta-lactamase (excreted by Actinomadura R39) and the G beta-lactamase (excreted by Streptomyces albus G). Cefoxitin and quinacillin sulphone are not recognized by the G beta-lactamase and are weak inactivators of the R39 beta-lactamase. N-Formimidoylthienamycin is a poor substrate for the G beta-lactamase and a potent inactivator of the R39 beta-lactamase. The high value of the bimolecular rate constant for enzyme inactivation is mainly due to a very low dissociation constant (1 microM). Clavulanate is an inactivator of both G and R39 beta-lactamases. The reaction with this latter enzyme is a branched pathway where normal turnover and permanent enzyme inactivation occur concomitantly. Between 28 and 43 molecules of clavulanate are hydrolysed before one of them has the opportunity to inactivate one molecule of enzyme.     

Separation, purification and properties of β-lactamase I and β-lactamase II from Bacillus cereus 569/H/9

1. A procedure was devised which is suitable for the isolation of beta-lactamase I and beta-lactamase II from Bacillus cereus 569/H/9 on a large scale. After adsorption on to Celite both enzymes were eluted in good yield and separated by chromatography on Sephadex CM-50. 2. beta-Lactamase I was separated into three main components by isoelectric focusing and into two components by chromatography. 3. The Zn(2+)-requiring beta-lactamase II obtained by this procedure had a lower molecular weight (22000) than beta-lactamase I (28000) and also differed from the latter in containing one cysteine residue. 4. The beta-lactamase II contained no carbohydrate, but showed the thermostability of the enzyme isolated earlier as a protein-carbohydrate complex. 5. Amino acid analyses and tryptic-digest ;maps' indicate that some degree of homology between beta-lactamase I and beta-lactamase II is possible, but that beta-lactamase I is not composed of the entire sequence of beta-lactamase II together with an additional peptide fragment. 6. A 6-methylpenicillin and a 7-methylcephalosporin showed much lower affinities for both enzymes than did penicillins and cephalosporins themselves.     

Use of electrospray mass spectrometry to directly observe an acyl enzyme intermediate in beta-lactamase catalysis

Electrospray mass spectrometry was used to directly observe intact acyl enzyme complexes formed between a class C beta-lactamase (from Enterobacter cloacae P99) and four poor substrates/inhibitors. In each case the molecular weight difference between the unreacted and the reacted beta-lactamase was consistent with the formation of an acyl enzyme.     

Substrate-induced deactivation of penicillinases. Studies of beta-lactamase I by hydrogen exchange.

The conformational motility of beta-lactamase I from Bacillus cereus was studied by hydrogen exhange. The time course of the isotopic replacement of peptide hydrogen atoms was followed by 'exchange-in' or 'exchange-out' experiments. Many of the substrates for this enzyme that have o-substituted aromatic or heterocyclic side chains (e.g. methicillin or cloxacillin) are known to effect a decrease in enzymic activity ('substrate-induced deactivation'). There was a marked discontinuity in the exchange-out curve when methicillin or cloxacillin was diffused into the enzyme solution. About one-half of the hydrogen atoms that were probed were affected by the presence of these substrates, and the change in the reactivity of the hydrogen atoms was also large. Substrates that do not bring about deactivation (benzylpenicillin and cephalosporin C) do not affect the hydrogen exchange, nor do reversible competitive inhibitors such as the penicilloic acid or penilloic acid. On the other hand, Zn2+ ions do affect the hydrogen exchange; their effect is similar to that of methicillin or cloxacillin.     

Characterization of the membrane beta-lactamase in Bacillus cereus 569/H/9

The membrane-bound beta-lactamase from Bacillus cereus, strain 569/H/9, has been purified to apparent homogeneity. Nonionic detergent (0.5% Triton X-100) is required to keep the enzyme (traditionally called gamma-penicillinase and now called beta-lactamase III) in solution. Antibodies to beta-lactamase III have been prepared, and the membrane-bound enzyme is immunochemically distinct from the extracellular enzymes. beta-Lactamase III has a molecular weight of 31 500, in contrast to the extracellular enzymes beta-lactamase I and beta-lactamase II which have molecular weights of 30 000 and 22 000, respectively. The isoelectric point of beta-lactamase III is pH 6.8, whereas beta-lactamase I and beta-lactamase II have isoelectric points about 8.6 and 8.3. The amino acid composition of beta-lactamase III differs from those of beta-lactamase I and beta-lactamase II; however, the difference index between the compositions of beta-lactamase I and beta-lactamase III (52%) suggests relatedness. beta-Lactamase III is inactivated by 6 beta-bromopenicillanic acid and by the sulfone of 6 alpha-chloropenicillanic acid, and cephalosporins are poorer substrates than penicillins. beta-Lactamase III may be a membrane-bound class A beta-lactamase.     

OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors.

Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates. The Arg244Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5alpha expressing the Arg244Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs. 8192 micrograms ml-1; cephaloridine, 4 vs. 32 micrograms ml-1, respectively. The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 micrograms ml-1 respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg244Ser beta-lactamase enzyme demonstrated increased Km and decreased kcat values for benzylpenicillin (Km=17 vs. 50 microM, kcat=345 vs. 234 s-1) and cephaloridine (Km=97 vs. 202 microM, kcat=1023 vs. 202 s-1). Although the Ki and IC50 values were increased for each inhibitor when compared to OHIO-1 beta-lactamase, the turnover numbers (tn) required for inactivation were increased only for clavulanate. For the Arg244Ser mutant enzyme of OHIO-1, the increased Ki, decreased tn for the sulfones, and different partition ratio (kcat/kinact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids. The resistance phenotype of a specific mutations can vary depending on the enzyme.     

Beta-lactamase of Lysobacter enzymogenes: induction, purification and characterization

Lysobacter enzymogenes produces an inducible beta-lactamase and induction with 100 micrograms ampicillin ml-1 resulted in an increase of more than 100-fold in enzyme activity. Various other beta-lactam antibiotics also served as effective inducers. The enzyme was obtained from cells by osmotic shocking to release periplasmic components and it was purified primarily by ion-exchange chromatography and PAGE. The beta-lactamase consists of one polypeptide with a molecular mass of about 28 kDa and an isoelectric point greater than 9.6. It is strongly inhibited by p-chloromercuribenzoate and clavulanic acid but not by EDTA. The enzyme readily hydrolyses several penicillins and cephalosporins, but not oxacillin or cloxacillin. The enzyme therefore belongs to group 2b of the bacterial beta-lactamases.     

Gratuitous Synthesis of β-Lactamase in Staphylococcus aureus

The synthesis of beta-lactamase in response to 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid as inducer was studied in Staphylococcus aureus. The inducer was not detectably hydrolyzed by beta-lactamase and had minimal antibacterial activity. The kinetics of induction showed a lag of 4 to 6 min in a nutrient broth medium and 8 to 12 min in a defined medium, followed by constant differential rates of synthesis of beta-lactamase. The differential rate of beta-lactamase synthesis in nutrient broth was unaltered by supplementing the medium with glucose, galactose, lactose, arabinose, glycerol, or sucrose. Variations in the partial pressure of oxygen did not alter the differential rate of synthesis of beta-lactamase over the range 18 to 50% oxygen in nitrogen. Even when the rate of growth was considerably reduced by high-oxygen tension, the differential rate of synthesis of the enzyme remained the same. The differential rate of beta-lactamase synthesis at low inducer concentration increased after a shift down in growth rate. The effect was observed with several inducers and under different nutritional conditions, but was always preceded by a change in growth rate. It is suggested that the change in growth rate itself causes the increase in differential rate of beta-lactamase synthesis.     

TRC-1: Emergence of a clavulanic acid-resistant TEM β-lactamase in a clinical strain

A novel TEM-derived plasmid-encoded beta-lactamase, resistant to inhibition by clavulanic acid, has been identified in a clinical strain of Escherichia coli found in Scotland. The beta-lactamase gene was carried on an 81-kb plasmid that conferred no other resistances. The novel enzyme conferred resistance to the amoxycillin/clavulanic acid combination on the host bacterium. The beta-lactamase has a pI of 5.25 and lies between the PSE-4 and SAR-1 beta-lactamases on an isoelectric focusing gel. This beta-lactamase has a Mr value of 25,000, similar to the TEM-1 enzyme and a comparable substrate profile. Its most significant difference is that it is inhibited by clavulanic acid 100-fold less efficiently than the TEM-1 enzyme. The enzyme was confirmed to be derived from the TEM enzymes by probing the plasmid DNA with an intragenic gene probe for TEM-1. This is the first report of a clinical bacterium carrying a TEM-enzyme that confers resistance to clavulanic acid combinations and we have designated the beta-lactamase as TRC-1.