Beta lactamase

Antimicrob Agents Chemother, 2000 Jul, 44(7), 1970 - 3
Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-9) from Escherichia coli in Spain; Sabate M et al.; A new CTX-M-type beta-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant Escherichia coli strain . Despite the close identity that exists between the CTX-M-9 and Toho-2 beta-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes . Outside of this region there are only six amino acids substitutions between both proteins.

J Antimicrob Chemother, 2000 Jun, 45(6), 783 - 8
Analysis of the effects of -42 and -32 ampC promoter mutations in clinical isolates of Escherichia coli hyperproducing ampC; Caroff N et al.; Escherichia coli usually produces only very small amounts of a constitutive AmpC beta-lactamase, but clinical strains overproducing this enzyme have been isolated . Three different ampC promoters of E . coli clinical strains were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene in the pKK232-8 reporter plasmid and their relative strengths were compared by two different methods . The strength of the promoters from AmpC hyperproducers was 70- to 120-fold higher than those from a low-level AmpC producer . One of the strong promoters, which differs from strain K12 at bases -88, -82, -42, -18, -1 and +58, was mutated to abolish the -42 mutation . This change resulted in a 43-fold decrease in CAT concentration . In another promoter, with eight different mutations at positions -88, -82, -32, -18, -1, +5, +24 and +58, the -32T-->A transversion, which created perfect homology with the -35 consensus sequence, was reverted; this led to a 13-fold decrease in CAT concentration . The -42 and -32 mutations play an important role in E . coli resistance to beta-lactams by increasing ampC transcription.

Microbiol Res, 2000 Apr, 155(1), 1 - 6
A search for beta-lactamase in chlamydiae, mycoplasmas, planctomycetes, and cyanelles: bacteria and bacterial descendants at different phylogenetic positions and stages of cell wall development; Claus H et al.; Bacteria from different phylogenetic positions such as chlamydiae, mycoplasmas, planctomycetes and also endosymbiotic murein-containing cyanelles were investigated for the production of beta-lactamases . No beta-lactamase activity was found in bacteria lacking murein such as Chlamydia pneumoniae, Mycoplasma pneumoniae, Pirellula marina and Planctomyces maris . In the murein-containing cyanelles of Cyanophora paradoxa no beta-lactamase activity could be detected.

Appl Biochem Biotechnol, 2000 Jan-Mar, 83(1-3), 163 - 9; discussion 169-71, 297-313
Catalytic mechanism of an abzyme displaying a beta-lactamase-like activity; Avalle B et al.; A catalytic IgG (Ab2) displaying a beta-lactamase-like activity was previously obtained by using the antiidiotypic pathway: the particularity of this antibody is that it is a true antiidiotype of the beta-lactamase active site . We have previously demonstrated that this IgG has retained some of the structural information displayed by the beta-lactamase active site, evident from data that polyclonal anti-Ab2 antibodies (Ab3) recognize beta-lactamase . In this article, we investigated the catalytic mechanism of the abzyme compared to that of the enzyme . The experimental data allow us to hypothesize the catalytic residues required for catalysis.

Scand J Infect Dis, 2000, 32(2), 217 - 8
Moraxella catarrhalis endocarditis: case report and review of the literature; Stefanou J et al.; A case of bacterial endocarditis caused by Moraxella catarrhalis in an apparently immunocompetent Greek male is presented, which was diagnosed after a 2-month history of low-grade fever of unknown origin . The agent seems to be a rare pathogen, but due to the high mortality rate, it should always be considered in the differential diagnosis of relevant cases . Beta-lactamase production by many strains complicates the choice of antibiotic.

Antimicrob Agents Chemother, 2000 Jun, 44(6), 1725 - 7
A new SHV-derived extended-spectrum beta-lactamase (SHV-24) that hydrolyzes ceftazidime through a single-amino-acid substitution (D179G) in the -loop; Kurokawa H et al.; A new SHV-derived extended-spectrum beta-lactamase (SHV-24) conferring high-level resistance to ceftazidime but not cefotaxime and cefazolin was identified in Japan . This enzyme was encoded by a transferable 150-kb plasmid from an Escherichia coli clinical isolate . The pI and K(m) for CAZ of this enzyme were 7.5 and 30 microM, respectively . SHV-24 was found to have a D179G substitution in the Omega-loop of the enzyme.

Antimicrob Agents Chemother, 2000 Jun, 44(6), 1538 - 43
The Legionella (Fluoribacter) gormanii metallo-beta-lactamase: a new member of the highly divergent lineage of molecular-subclass B3 beta-lactamases; Boschi L et al.; A metallo-beta-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter) gormanii ATCC 33297(T) constructed in the plasmid vector pACYC184 and transformed into Escherichia coli DH5alpha, by screening for clones showing a reduced susceptibility to imipenem . The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6 . Sequencing revealed that FEZ-1 is a molecular-class B beta-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage . All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1 . Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem . The properties of FEZ-1 overall resembled those of a beta-lactamase previously purified from the same strain of L . gormanii (T . Fujii, K . Sato, K . Miyata, M . Inoue, and S . Mitsuhashi, Antimicrob . Agents Chemother . 29:925-926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class . A system for overexpression of the bla(FEZ-1) gene in E . coli, based on the T7 phage promoter, was also developed.

Proteins, 2000 Jul 1, 40(1), 23 - 8
pK(a) calculations for class C beta-lactamases: the role of Tyr-150; Lamotte-Brasseur J et al.; The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases . In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution . Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH . pK(a) values were also computed in catalytically impaired beta-lactamase mutants . Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation . In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes . These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine . The possible roles of Lys67 and Lys315 during catalysis are also discussed .

Protein Eng, 2000 Apr, 13(4), 267 - 74
Structure-function analysis of alpha-helix H4 using PSE-4 as a model enzyme representative of class A beta-lactamases; Savoie A et al.; We extracted maximum information for structure-function analysis of the PSE-4 class A beta-lactamase by random replacement mutagenesis of three contiguous codons in the H4 alpha-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129 . These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model . Structure-function studies on positions 125-129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127 . The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 alpha-helix structure together, thus maintaining the important Ser130-Asp131-Asn132 motif positioned towards the active site . At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4 . The Tyr 125 of the mutant YAM formed an edge to face pi-pi interaction with Phe 124 which also interacts with the Trp 210 with the same interactions . Antibiotic susceptibilities showed that amino acid changes in the the H4 alpha-helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors . However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 alpha-helix region than clavulanic acid, an inhibitor of the oxypenam class . The analysis of H4 alpha-helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes.

Farmaco, 2000 Feb, 55(2), 134 - 5
An efficient method for the synthesis of sulbactam pivoxil; Changov LS et al.; Sulbactam pivoxil, a prodrug of the beta-lactamase inhibitor sulbactam, was prepared in high yield by reacting the sodium salt of sulbactam with chloromethyl pivalate in a polar solvent, then diluting the reaction mixture with water and isolating the product by filtration . Dimethyl sulfoxide was found to be the solvent of choice among several aprotic organic solvents.

Antimicrob Agents Chemother, 2000 May, 44(5), 1387 - 90
Contributions of the AmpC beta-lactamase and the AcrAB multidrug efflux system in intrinsic resistance of Escherichia coli K-12 to beta-lactams; Mazzariol A et al.; The roles of the AmpC chromosomal beta-lactamase and the AcrAB efflux system in levels of intrinsic resistance and susceptibility of Escherichia coli to beta-lactams were studied with a set of isogenic strains . MICs of ureidopenicillins, carbenicillin, oxacillin, and cloxacillin were drastically reduced by the inactivation of AcrAB, whereas those of the earlier cephalosporins were affected mostly by the loss of AmpC beta-lactamase.

J Infect Dis, 2000 Apr, 181(4), 1376 - 87 Epub 2000 Apr 13.
Analysis of Moraxella catarrhalis by DNA typing: evidence for a distinct subpopulation associated with virulence traits; Bootsma HJ et al.; Two DNA typing methods, probe-generated restriction fragment length polymorphism analysis and single-adapter amplified fragment length polymorphism analysis, were used to study the genetic relationships among 90 Moraxella catarrhalis strains . Both methods were found to be highly concordant, generating a dendrogram with 2 main branches . The division of the M . catarrhalis population into 2 subspecies was supported by analysis of the 16S rRNA sequences . Both beta-lactamase-positive and beta-lactamase-negative strains were found in all main branches, suggesting horizontal transfer of the beta-lactamase gene . In contrast, 2 virulence traits, complement resistance and adherence to epithelial cells, were strongly associated with 1 of the 2 subspecies . The branch depth suggested that complement-resistant adherent strains diverged from a common ancestor more recently than did complement-sensitive nonadherent strains . These findings suggest the existence of subpopulations of M . catarrhalis that differ in virulence, and they may have implications for vaccine development.

Mol Microbiol, 2000 Apr, 36(1), 93 - 104
Genesis of BRO beta-lactamase-producing Moraxella catarrhalis: evidence for transformation-mediated horizontal transfer; Bootsma HJ et al.; The dramatic rise in BRO-producing M . catarrhalis strains observed in the last decades is without precedence . The aim of this study was to elucidate the events that led to the emergence of BRO-1 and BRO-2 beta-lactamases . Previously, we showed bro1 and bro2 to be >99% identical . Data presented here suggested that bro2 was acquired by a fortuitous event and inserted between M . catarrhalis genes orf1 and orf3 . Subsequently, bro1 evolved from bro2 . Promoter-up mutations increased fitness of bro2, explaining its present predominance . The highly conserved nature of bro compared with orf1 and orf3 suggested that acquisition has occurred relatively recently . The random distribution of bro among M . catarrhalis fingerprint types indicated that bro has spread by horizontal transfer . Sequence analysis revealed that 80-200 bp is generally cotransferred with bro, serving as regions of homology that target bro to the same chromosomal locus . A region of 160 bases upstream of bro1 lacked polymorphism, indicating it was derived from the original strain that acquired bro2 . We observed that bro was readily transferred by transformation between M . catarrhalis strains in vitro, suggesting a mechanism by which bro has disseminated . In conclusion, we have been able to reconstruct the steps that led to the emergence of BRO-producing M . catarrhalis.

J Biol Chem, 2000 Jun 9, 275(23), 17693 - 9
The cysteine-rich protein A from Helicobacter pylori is a beta-lactamase; Mittl PR et al.; Among the large number of hypothetical proteins within the genomes of Helicobacter pylori, there is a family of unique and highly disulfide-bridged proteins, designated family 12, for which no function could originally be assigned . Sequence analysis revealed that members of this family possess a modular architecture of alpha/beta-units and a stringent pattern of cysteine residues . The H . pylori cysteine-rich protein A (HcpA), which is a member of this family, was expressed and refolded from inclusion bodies . Six pairs of cysteine residues, which are separated by exactly seven residues, form disulfide bridges . HcpA is a beta-lactamase . It slowly hydrolyzes 6-aminopenicillinic acid and 7-aminocephalosporanic acid (ACA) derivatives . The turnover for 6-aminopenicillinic acid derivatives is 2-3 times greater than for ACA derivatives . The enzyme is efficiently inhibited by cloxacillin and oxacillin but not by ACA derivatives or metal chelators . We suggest that all family 12 members possess similar activities and might be involved in the synthesis of the cell wall peptidoglycan . They might also be responsible for amoxicillin resistance of certain H . pylori strains.

J Biol Chem, 2000 Jun 9, 275(23), 17428 - 33
Functionally accepted insertions of proteins within protein domains; Collinet B et al.; Experiments were designed to explore the tolerance of protein structure and folding to very large insertions of folded protein within a structural domain . Dihydrofolate reductase and beta-lactamase have been inserted in four different positions of phosphoglycerate kinase . The resultant chimeric proteins are all overexpressed, and the host as well as the inserted partners are functional . Although not explicitly designed, functional coupling between the two fused partners was observed in some of the chimeras . These results show that the tolerance of protein structures to very large structured insertions is more general than previously expected and supports the idea that the natural sequence continuity of a structural domain is not required for the folding process . These results directly suggest a new experimental approach to screen, for example, for folded protein in randomized polypeptide sequences.

J Antimicrob Chemother, 2000 Apr, 45(4), 517 - 20
Combinatorial biochemistry and shuffling of TEM, SHV and Streptomyces albus omega loops in PSE-4 class A beta-lactamase; Sanschagrin F et al.; The class A PSE-4 beta-lactamase was used for studying the importance of amino acids in the omega (Omega) loop and its interactions for hydrolysis of beta-lactam antibiotics . By cassette mutagenesis, we replaced the amino acids 163-179 Omega loop in PSE-4 with TEM-1, SHV-1 and Streptomyces albus G beta-lactamase Omega loops . Phenotypic analysis of Escherichia coli recombinants expressing the Omega loop PSE-4 mutant enzymes gave MICs and kinetic data similar to those of wild-type PSE-4.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3160 - 5
Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases; Atanasov BP et al.; The pH dependence of the pK(a) values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 beta-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates . By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction intermediate, we found that acid-catalyzed protonation of the beta-lactam nitrogen is energetically favored as the initiating event, followed by base-catalyzed nucleophilic attack on the carbonyl carbon of the beta-lactam group . N-protonation is catalyzed through a hydrogen-bonded cluster involving the 2-carboxylate group of the substrate, the side chains of S130 and K234, and a solvent molecule . Nucleophilic attack on the carbonyl carbon is carried out by the side chain of S70 with proton abstraction catalyzed by a water molecule hydrogen-bonded to the side chain of E166 . Stabilization of ion pairs in the active site through interactions with distant clusters of charged residues in the enzyme was concluded to be an important driving force of the catalytic mechanism.

FEMS Microbiol Lett, 2000 Mar 15, 184(2), 303 - 6
Expression of beta-lactamase in Coxiella burnetii transformants; Suhan ML et al.; Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding beta-lactamase . However, non-plasmid emergence of resistance to ampicillin also develops . To validate the usefulness of the bla gene marker for selection and detection, transformed C . burnetii were examined for beta-lactamase expression by use of immunoblotting after SDS-PAGE . The 29-kDa mature form of the beta-lactamase protein was detected in C . burnetii lysates . Quantitation of these immunoblot signals showed that C . burnetii surprisingly expressed low levels of beta-lactamase . The results validate the use of plasmid-encoded ampicillin resistance as a means for selecting C . burnetii transformants; they also suggest that levels of ampicillin used for selection pressure should be empirically determined and that detection of beta-lactamase by antibody blotting done to confirm transformants.

Curr Microbiol, 2000 May, 40(5), 302 - 5
Development of a plasmid vector for easy selection of strong promoters; Pineiro SA et al.; A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed . The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site . Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E . coli . The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E . coli, and a chloramphenicol resistance gene for S . aureus . The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of beta-lactamase activity in liquid or solid media.

Biotechnol Bioeng, 2000 Apr 5, 68(1), 121 - 5
Rapid in vivo evolution of a beta-lactamase using phagemids; Long-McGie J et al.; RNA viruses are capable of undergoing extremely rapid evolution due to their high rates of reproduction, small genome size, and a high frequency of spontaneous mutagenesis . Here we demonstrate that a virus-like, evolutionary state can be created by propagating a phagemid population in a hypermutator strain of Escherichia coli in the presence of a helper phage . This enables one to subject individual phagemid-encoded genes to rapid in vivo evolution . We applied this approach to TEM-1 beta-lactamase which confers resistance to 0.05 mg/L of the antibiotic cefotaxime . After 3 weeks of in vivo evolution we were able to isolate a double mutant, E104K/G238S, of the enzyme which confers a 500-fold increased level of resistance to cefotaxime compared to the starting enzyme . In two independent experiments we obtained a triple mutant, E104K/G238S/T263M, which confers a 1000-fold increase in resistance compared to the wild type enzyme . The same three mutations have been previously observed in TEM-4 beta-lactamase which was discovered in a highly cefotaxime-resistant clinical isolate . The probability of randomly obtaining a beta-lactamase carrying three identical point mutations is less than 10(-10) . This indicates that phagemid evolution can rapidly reproduce evolution occurring in nature .

FEMS Microbiol Lett, 2000 Mar 1, 184(1), 85 - 9
Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP); Chanawong A et al.; Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone . Eight bacteria, each producing a different SHV beta-lactamase, were used in this study . These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12) . All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion . By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other . In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants . We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.

Bioorg Med Chem, 1999 Dec, 7(12), 2899 - 904
Synthesis and beta-lactamase inhibitory activity of new 6beta-cysteinesulfonamidopenicillanic acids; Changov LS et al.; New 6beta-cysteinesulfonamidopenicillanic acids and their sulfoxides were synthesized by sulfonylation of 6beta-aminopenicillanic acid or its (S)-sulfoxide with (R)-N-benzyloxycarbonylcysteinesulfonyl chloride ethyl ester (2a, 1b) and (R)-N-benzyloxycarbonylcysteinesulfonyl chloride benzyl ester (2a, 2b) . The corresponding 6beta-cysteinesulfonamidopenicillanic acids sulfones 1c and 2c were prepared by oxidation of the sulfoxides 1b and 2b with potassium permanganate in aqueous medium . When combined with ampicillin some of the compounds reduced the minimal inhibitory concentrations of ampicillin against beta-lactamase producing strains.

Biotechnol Bioeng, 2000 Mar 5, 67(5), 505 - 12
Enhanced protein renaturation by temperature-responsive polymers; Lin SC et al.; The application of temperature-sensitive polymer (PNIPAAm) for the renaturation of beta-lactamase from inclusion bodies was investigated . It was observed that PNIPAAm was more effective than PEG in enhancing protein renaturation . At a concentration of 0.1%, PNIPAAm improved the yield of beta-lactamase activity by 41% from 46 . 5 to 65.4 IU/mL, compared to 26% with PEG from 46.5 to 58.7 IU/mL . Kinetic study indicated that PNIPAAm did not significantly affect the initial rate of protein renaturation but did increase final activity yield . In the presence of PEG and PNIPAAm, the activity yields increased with temperature, indicating that hydrophobic interactions between denatured protein and polymer molecules contributed to the enhanced protein renaturation with polymers . The sequential addition approach, aiming at enhancing protein renaturation by reducing local protein concentration during renaturation, was also shown effective in enhancing protein renaturation, especially in the presence of polymers . With the sequential addition approach, the activity yield was increased by 60 . 5% from 46.5 to 74.6 IU/mL with PNIPAAm . Similar behavior was also observed with PEG . PNIPAAm exhibited similar behavior as PEG on the renaturation of beta-lactamase in terms of temperature effect and concentration effect, indicating that the mechanism for enhanced protein renaturation for the two polymers might be similar . PNIPAAm exhibits a lower critical solution temperature (LCST) of 32 degrees C and can be effectively separated from aqueous solution and recycled . A protein renaturation process employing PNIPAAm, which offers the advantages of enhanced renaturation efficiency, minimum loss of protein aggregates, and ease of polymers recycling, was proposed .

Antimicrob Agents Chemother, 2000 Feb, 44(2), 444 - 6
Molecular characterization of the beta-lactamases from clinical isolates of Moraxella (Branhamella) catarrhalis obtained from 24 U.S . medical centers during 1994-1995 and 1997-1998; Richter SS et al.; The beta-lactamases from 403 Moraxella (Branhamella) catarrhalis clinical isolates obtained during 1994-1995 and 1997-1998 U.S . multicenter surveillance studies were characterized by isoelectric focusing . The overall prevalences of the BRO-1 and BRO-2 enzymes among beta-lactamase-positive isolates were estimated to be 97.5 and 2.5%, respectively . The minimum inhibitory concentrations (MICs) of ampicillin for all BRO-2-producing isolates were </=1 microg/ml; however, numerous beta-lactamase-positive isolates for which the ampicillin MICs were </=1 microg/ml produced the BRO-1 enzyme (88 . 1%).

J Antimicrob Chemother, 2000 Jan, 45(1), 101 - 4
Detrimental effect of the combination of R164S with G238S in TEM-1 beta-lactamase on the extended-spectrum activity conferred by each single mutation; Giakkoupi P et al.; The non-naturally occurring TEM-1 beta-lactamase mutant R164S:G238S, as well as the R164S (TEM-12) and G238S (TEM-19) beta-lactamases, were constructed and expressed in Escherichia coli under isogenic conditions . Comparison of susceptibilities to beta-lactam antibiotics and substrate profiles showed that the combination of R164S with G238S drastically reduced the extended-spectrum activity of the respective single mutants.

Antimicrob Agents Chemother, 1999 Dec, 43(12), 3014 - 7
Distribution of beta-lactamases in actinomycetes; Ogawara H et al.; The distribution of beta-lactamase activities in a collection of actinomycete strains was surveyed . Six of 127 strains were found to produce beta-lactamase . This low frequency was in contrast to the case with Streptomyces species . The producing strains were not related phylogenetically . MICs of benzylpenicillin did not correlate with beta-lactamase production.

Enferm Infecc Microbiol Clin, 1999 Oct, 17(8), 401 - 4
{Incidence of extended spectrum beta lactamase in Escherichia coli in a university hospital from 1994-1996}; Sabate M et al.; BACKGROUND: The aim of this study was to evaluate the incidence of TEM- and SHV-type extended spectrum betalactamases (ESBLs) in Escherichia coli in a 700-bed teaching hospital between 1994 and 1996 . MATERIAL AND METHODS: Strains that presented reduced diameters to third-generation cephalosporins, as identified by disc diffusion techniques, were studied . The betalactamases involved were characterized by determination of the isoelectric point, hydrolysis profile, gene detection by polymerase chain reaction (PCR), and sequencing of the amplified products . RESULTS: 96 strains (1.4%) out of 7,054 strains of E . coli isolated between 1994 and 1996 showed decreased susceptibility to third-generation cephalosporins and only 4 strains (0.06%) produced ESBLs . Two strains produced SHV-2 and two produced TEM-12 . CONCLUSIONS: The contribution of ESBL production to resistance to third-generation cephalosporins was low: 0.06% of all the E . coli strains isolated between 1994 and 1996.

J Bacteriol, 1999 Nov, 181(22), 6922 - 8
Deletion of the pyc gene blocks clavulanic acid biosynthesis except in glycerol-containing medium: evidence for two different genes in formation of the C3 unit; Perez-Redondo R et al.; The beta-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine . A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases . Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid . Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant . The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid . However, the beta-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing D-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography . The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein . The pyc replacement mutant overproduces cephamycin.

Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13029 - 33
High pressure fosters protein refolding from aggregates at high concentrations; St John RJ et al.; High hydrostatic pressures (1-2 kbar), combined with low, nondenaturing concentrations of guanidine hydrochloride (GdmHCl) foster disaggregation and refolding of denatured and aggregated human growth hormone and lysozyme, and beta-lactamase inclusion bodies . One hundred percent recovery of properly folded protein can be obtained by applying pressures of 2 kbar to suspensions containing aggregates of recombinant human growth hormone (up to 8.7 mg/ml) and 0.75 M GdmHCl . Covalently crosslinked, insoluble aggregates of lysozyme could be refolded to native, functional protein at a 70% yield, independent of protein concentration up to 2 mg/ml . Inclusion bodies containing beta-lactamase could be refolded at high yields of active protein, even without added GdmHCl.

J Cell Sci, 1999 Nov, 112 ( Pt 22), 3889 - 98
The sorting determinant guiding Hsp150 to the COPI-independent transport pathway in yeast; Suntio T et al.; The COPI coatomer is thought to be required in yeast directly for retrograde transport from the Golgi to the endoplasmic reticulum (ER), and directly or indirectly for ER-to-Golgi transport . Unexpectedly, the secretory glycoproteins Hsp150 and invertase have been found not to require COPI for ER exit . The features according to which cargo proteins are selected for the COPI-independent pathway are not known . The ER form of Hsp150 has three distinct domains: an N-terminal fragment of 54 amino acids (subunit I) is followed by 11 repeats of a 19 amino acid peptide plus a unique C-terminal fragment of 114 amino acids (subunit II) . By fusing heterologous proteins to different Hsp150 domains and expressing them in sec21-1 and sec21-3 mutants with temperature-sensitive mutations in the gamma-COPI subunit, we show here that the repeats of subunit II function as sorting determinants for COPI-independent ER exit . The C-terminal fragment of Hsp150 could be replaced by E . coli beta-lactamase or rat nerve growth factor receptor ectodomain (NGFRe), and subunit I could be deleted, without inhibiting COPI-independent transport . However, when the repetitive region was omitted and beta-lactamase was fused directly to the C terminus of subunit I, COPI was required for efficient ER exit . Mass spectroscopic analysis demonstrated that both subunit I and II of Hsp150 were extensively O-glycosylated, suggesting that the O-glycosylation pattern was not decisive for cargo selection.

FEBS Lett, 1999 Oct 8, 459(2), 166 - 72
Circular beta-lactamase: stability enhancement by cyclizing the backbone; Iwai H et al.; We have cyclized the polypeptide backbone of beta-lactamase with a short peptide loop as a novel method for protein stabilization, using intein-mediated protein ligation . Successful cyclization was proven by mass spectrometry and subsequent re-linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase . Under the conditions of the experiment, no disulfide bond is present . The circular form of beta-lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form . The circular form could be purified from the linear one either by this heat treatment or by a his-tag which became exopeptidase-resistant by cyclization . The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states . While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N- and C-termini in close proximity.

Pharmacol Ther, 1999 Aug, 83(2), 141 - 51
Class A beta-lactamases--enzyme-inhibitor interactions and resistance; Yang Y et al.; Beta-Lactamases of Ambler's Class A are the most commonly encountered mechanism of bacterial resistance to beta-lactam antibiotics . In the face of selective pressure arising from use of either newer cephalosporins or beta-lactam/beta-lactamase inhibitor combinations, mutations arose among Class A beta-lactamase genes, leading to resistance . Clavulanic acid, a naturally occurring clavam, and the penicillanic acid sulfones sulbactam and tazobactam are the inhibitors in clinical use . This review focuses on the mechanism of inhibition by these currently marketed beta-lactamase inhibitors and on the mechanism by which specific amino acid substitutions might lead to resistance . The key amino acid positions important for inhibitor-resistance include Met69, Ser130, Arg244, Arg275, and Asn276 . Ser130 is vital to the chemical mechanism of inhibition . Arg244 appears to be coordinated to Arg275 and Asp276 by hydrogen bonds . Arg244 is involved in positioning beta-lactams, especially penicillins and beta-lactamase inhibitors, via their carboxyl groups . Site-directed mutagenesis studies confirm the role of Arg244 and its coordinating partners in beta-lactam turnover and in the reactions leading to enzyme inactivation . This mechanism is dependent on the donation of a proton via a water coordinated to Arg244 and Val216 to clavulanic acid to allow formation of a favorable leaving group . This proton donation is probably not required for formation of a favorable leaving group for the sulfone inhibitors sulbactam and tazobactam . Therefore, some amino acid substitutions have differing effects on inhibition by clavulanic acid compared with the penicillanic acid sulfones . Met69 may play a more structural role in beta-lactam positioning within the oxyanion hole.

Protein Eng, 1999 Sep, 12(9), 761 - 9
Susceptibility of beta-lactamase to core amino acid substitutions; Petrosino JF et al.; To determine which amino acids in TEM-1 beta-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme . A comprehensive screening of these libraries identified several TEM-1 beta-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity . An examination of positions 66 and 76 in the class A beta-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other beta-lactamases . It is possible that position 76 varies in the gene family because beta-lactamase mutants with non-conservative substitutions at position 76 retain partial function . In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function . By determining the beta-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions . An analysis of the Protein Data Bank files for three class A beta-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.

J Mol Biol, 1999 Sep 10, 292(1), 19 - 37
Activities of constitutive promoters in Escherichia coli; Liang S et al.; The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+ spoT+ strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient DeltarelADeltaspoT derivatives . The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the beta-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage lambda; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon . Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of a lac deletion strain . The amount of 5'-terminal lacZ mRNA and of beta-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively . In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates . By combining lacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined . In ppGpp-proficient (relA+ spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour . The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour . In contrast, PRNAIIactivity decreased threefold over this range of growth rates . In ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant . To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates . In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp . This caused a reduced activity of rrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+ spoT+) relative to ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains .

J Immunoassay, 1999 Aug, 20(3), 151 - 83
A comparison of performances of four enzymes used in ELISA with special reference to beta-lactamase; Khatkhatay MI et al.; Horse radish peroxidase, alkaline phaosphatase and beta-D-galactosidase are widely used as labels in the development of enzyme immunoassays (EIAs) . Enzyme beta-lactamase, though introduced as a label in late seventies has not yet become very popular inspite of having the necessary features of an enzyme to be used in EIAs . The present article reviews assays developed with this enzyme, highlights its salient features and brings out an argument in favour of its wide spread use in EIAs.

Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 153 - 8
Role of ser-237 in the substrate specificity of the carbapenem-hydrolyzing class A beta-lactamase Sme-1; Sougakoff W et al.; The role of the serine residue found at position 237 in the carbapenemase Sme-1 has been investigated by constructing a mutant in which Ser-237 was replaced by an alanine . The S237A mutant showed a catalytic behavior against penicillins and aztreonam very similar to that of Sme-1 . By contrast, S237A was characterized by a reduced catalytic efficiency against cephems, such as cephalothin and cephaloridine . In addition, the weak activity of Sme-1 against the cephamycin cefoxitin was hardly detectable with the mutant enzyme . Finally, the Ser-237-->Ala mutation resulted in a marked decrease in catalytic activity against imipenem, showing that Ser-237 contributes to the carbapenemase activity of the class A beta-lactamase Sme-1.

J Mol Biol, 1999 Aug 13, 291(2), 481 - 90
Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants; Dragani B et al.; Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding . Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule . Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase . Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates . The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II . At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained . The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions . The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding . The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis .

J Biol Chem, 1999 Aug 13, 274(33), 23052 - 60
Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEM(pUC19) beta-lactamase from Escherichia coli; Vakulenko SB et al.; We investigated the effects of mutations at positions 164 and 179 of the TEM(pUC19) beta-lactamase on turnover of substrates . The direct consequence of some mutations at these sites is that clinically important expanded-spectrum beta-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A beta-lactamases, bind the active site of these mutant enzymes more favorably . We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum beta-lactams by their enhanced ability to turn over these antibiotic substrates . Four of these mutant variants, Arg(164) --> Asn, Arg(164) --> Ser, Asp(179) --> Asn, and Asp(179) --> Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments . The effects on the kinetic parameters were such that either activity with the expanded-spectrum beta-lactams remained unchanged or, in some cases, the activity was enhanced . The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K(s)) invariably increased . Computation of the microscopic rate constants (k(2) and k(3)) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k(3)) or that neither the acylation nor deacylation became the sole rate-limiting step . In a few instances, the rate constants for both the acylation (k(2)) and deacylation (k(3)) of the extended-spectrum beta-lactamase were enhanced . These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM(pUC19) beta-lactamase . Our results indicated that severe steric interactions between the large 7beta functionalities of the expanded-spectrum beta-lactams and the Omega-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates . These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.

Antimicrob Agents Chemother, 1999 Aug, 43(8), 1881 - 7
Construction and characterization of mutants of the TEM-1 beta-lactamase containing amino acid substitutions associated with both extended-spectrum resistance and resistance to beta-lactamase inhibitors; Stapleton PD et al.; Extended-spectrum TEM beta-lactamases (ESBLs) do not usually confer resistance to beta-lactamase inhibitors such as clavulanate or tazobactam . To investigate the compatibility of the two phenotypes we used site-directed mutagenesis of the bla(TEM-1) gene to introduce into the TEM-1 beta-lactamase amino acid substitutions that confer the ESBL phenotype: TEM-12 (Arg164-->Ser), TEM-26 (Arg164-->Ser plus Glu104-->Lys), TEM-19 (Gly238-->Ser), and TEM-15 (Gly238-->Ser plus Glu104-->Lys) . These were combined with three sets of substitutions that confer inhibitor resistance: TEM-31 (Arg244-->Cys), TEM-33 (Met69-->Leu), and TEM-35 (Met69-->Leu and Asn276-->Asp) . Introduction of the Arg244-->Cys substitution gave rise to inhibitor-resistant hybrid enzymes that either lost ESBL activity (TEM-12, TEM-15, and TEM-19) or had reduced activity (TEM-26) against ceftazidime . In contrast, the introduction of Met69-->Leu or Met69-->Leu plus Asn276-->Asp substitutions did not significantly affect the abilities of the enzymes to confer resistance to ceftazidime, although increased susceptibility to cefotaxime was observed with Escherichia coli strains that expressed the TEM-19 and TEM-26 beta-lactamases . With the exception of the TEM-12 beta-lactamase, introduction of the Met69-->Leu substitution did not give rise to enzymes with increased resistance to clavulanate compared to that of the TEM-1 beta-lactamase . However, introduction of the double substitution Met69-->Leu plus Asn276-->Asp in the ESBLs did give rise to low-level (TEM-19, TEM-15, and TEM-26) or moderate-level (TEM-12) clavulanate resistance . None of the hybrid enzymes were as resistant to clavulanate as the corresponding inhibitor-resistant TEM beta-lactamase mutant, suggesting that active-site configuration in the ESBLs limits the degree of clavulanate resistance conferred.

Antonie Van Leeuwenhoek, 1999 Jan-Feb, 75(1-2), 125 - 33
Biosynthesis and molecular genetics of clavulanic acid; Jensen SE et al.; The biosynthesis of clavulanic acid and related clavam metabolites is only now being elucidated . Understanding of this pathway has resulted from a combination of both biochemical studies of purified biosynthetic enzymes, and molecular genetic studies of the genes encoding these enzymes . Clavulanic acid biosynthesis has been most thoroughly investigated in Streptomyces clavuligerus where the biosynthetic gene cluster resides immediately adjacent to the cluster of cephamycin biosynthetic genes . A minimum of eight structural genes have been implicated in clavulanic acid biosynthesis, although more are probably involved . While details of the early and late steps of the pathway remain unclear, synthesis proceeds from arginine and pyruvate, as the most likely primary metabolic precursors, through the monocyclic beta-lactam intermediate, proclavaminic acid, to the bicyclic intermediate, clavaminic acid, which is a branch point leading either to clavulanic acid or the other clavams . Conversion of clavaminic acid to clavulanic acid requires side chain modification as well as inversion of ring stereochemistry . This stereochemical change occurs coincident with acquisition of the beta-lactamase inhibitory activity which gives clavulanic acid its therapeutic and commercial importance . In contrast, the other clavam metabolites all arise from clavaminic acid with retention of configuration and lack beta-lactamase inhibitory activity.

Chem Biol, 1999 Aug, 6(8), 541 - 51
Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and antitumor drug potential of platinum complexes; Sandman KE et al.; BACKGROUND: The need for new platinum antitumor drugs is underscored by the usefulness of cisplatin and carboplatin in chemotherapy and the resistance of many tumors to these compounds . Combinatorial chemistry could aid in the search for cisplatin analogs if fast, high-throughput assays were available . Our goal was to develop rapid cell-based assays suitable for high-throughput screening that accurately predict the cytotoxicity of platinum complexes . We examined the effects of platinum complexes and other agents on reporter-gene expression in cancer cells . RESULTS: HeLa Tet-On cells with inducible enhanced green fluorescent protein (EGFP) were prepared . Cisplatin and other cis-disubstituted platinum complexes inhibited EGFP expression, with a strong positive correlation between EGFP inhibition and cytotoxicity . By contrast, trans-{Pt(NH(3))(2)Cl(2)}, other trans-platinum complexes, methyl methanesulfonate or heat shock stimulated EGFP expression . Northern and nuclear run-on analyses revealed that the changes in EGFP expression were at the level of transcription . In another reporter-gene assay in Jurkat cells, cisplatin, but not trans-{Pt(NH(3))(2)Cl(2)} or K(2){PtCl(4)}, inhibited beta-lactamase expression, as measured by hydrolysis of the fluorescent substrate CCF2 . CONCLUSIONS: The EGFP results indicate that cytotoxic stress enhances transcription from the inducible promoter, whereas compounds able to form the 1,2-intrastrand platinum-DNA cross-links repress transcription . Both fluorescence-based reporter-gene assays afford promising new approaches to platinum anticancer drug discovery.

Can J Microbiol, 1999 Apr, 45(4), 299 - 303
Construction of a genomic map of Moraxella (Branhamella) catarrhalis ATCC 25238 and physical mapping of virulence-associated genes; Nguyen KT et al.; A physical genome map of the Moraxella catarrhalis type strain (ATCC 25238) has been constructed using pulsed field gel electrophoresis . Macrorestriction analyses of the genome of M . catarrhalis were performed by digestion with the restriction enzymes SmaI, NotI, and RsrII, which cleave the single circular chromosome into 9, 10, and 6 fragments, respectively . The chromosomal fragments generated by pulsed field gel electrophoresis were converted to a linkage map utilizing a combination of partial digestions, and cross-hybridizations . Moraxella catarrhalis, like a number of other respiratory pathogens, has a relatively small genome estimated at 1750 kilobase pairs or about 40% of the size of the Escherichia coli genome . The locations of the four ribosomal RNA operons (rrnLS) were determined by Southern hybridization and by digestion with I-CeuI endonuclease . A number of genes involved in virulence have been placed onto the physical map by Southern hybridization including those encoding the predominant outer-membrane proteins and the chromosomal gene encoding beta-lactamase.

FEBS Lett, 1999 Jun 25, 453(3), 305 - 7
Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution; Doi N et al.; Protein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function . The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site . Here we describe a general method of creating entirely new molecular-recognition sites on GFPs . At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP . Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library . We have tested this methodology by using TEM1 beta-lactamase and its inhibitory protein as our model protein-ligand system . 'Allosteric GFP biosensors' constructed by this method may be used in a wide range of applications including biochemistry and cell biology.

Int J Antimicrob Agents, 1999 Jun, 12(1), 27 - 31
Beta-lactamases and outer membrane investigations in beta-lactam-resistant Comamonas acidovorans strains; Ravaoarinoro M et al.; Imipenem-induced beta-lactamase (level of expression, specific activity and kinetic parameters (Vmax and Km) in response to nitrocefin) and outer membrane proteins (OMPs) (hydrophobicity, permeability and electrophoretic pattern) were characterized in, one beta-lactam sensitive (PAC-9), one resistant (PAC-1) and two resistant laboratory mutants (PAC-9M, PAC-9M2) of Comamonas acidovorans strains . Beta-lactamases from both mutant strains showed different Vmax values compared to the parental strains . Beta-lactam resistance was found to be associated in PAC-1 with inducible beta-lactamase production and OMP alteration by the appearance of a 102-KDa protein . Moreover, PAC-1 was less permeable to nitrocefin than PAC-9 . These data indicate that C . acidovorans resistance to beta-lactam resulted from synergy between beta-lactamase and OMP alterations.

Clin Lab Sci, 1999 Mar-Apr, 12(2), 115 - 8
Drug-induced hemolytic anemias: increasing complications to therapeutic interventions; Wright MS; There are recent reports of severe drug-induced immune hemolysis caused by several different classes of drugs . Second and third generation cephalosporins, diclofenac, fludarabine, carboplatin, and beta-lactamase inhibitors are among the drugs associated with severe or fatal hemolysis . Studies on patients who exhibit hemolysis after ingesting these drugs indicate that the four classical mechanisms of drug-induced hemolytic anemia may overlap . These studies appear to support the unified theory for induction of drug-induced immune hemolytic anemia.

Biochim Biophys Acta, 1999 Jun 15, 1432(1), 125 - 36
OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors; Lin S et al.; 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.

J Antimicrob Chemother, 1999 Apr, 43(4), 447 - 58
Inhibitor-resistant TEM beta-lactamases: phenotypic, genetic and biochemical characteristics; Chaibi EB et al.; Beta-lactamases represent the main mechanism of bacterial resistance to beta-lactam antibiotics . The recent emergence of bacterial strains producing inhibitor-resistant TEM (IRT) enzymes could be related to the frequent use of beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam in hospitals and in general practice . The IRT beta-lactamases differ from the parental enzymes TEM-1 or TEM-2 by one, two or three amino acid substitutions at different locations . This paper reviews the phenotypic, genetic and biochemical characteristics of IRT beta-lactamases in an attempt to shed light on the pressures that have contributed to their emergence.

Protein Eng, 1999 Apr, 12(4), 313 - 8
Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242 in PER-1 beta-lactamase hydrolysing expanded-spectrum cephalosporins; Bouthors AT et al.; The class A beta-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum beta-lactamases (ESBLs), is characterized by a substrate profile similar to that conferred by these latter enzymes . The role of residues Ala164, His170, Ala171, Asn179, Arg220, Thr237 and Lys242, found in PER-1, was assessed by site-directed mutagenesis . Replacement of Ala164 by Arg yielded an enzyme with no detectable beta-lactamase activity . Two other mutants, N179D and A164R+N179D, were also inactive . Conversely, a mutant with the A171E substitution displayed a substrate profile very similar to that of the wild-type enzyme . Moreover, the replacement of Ala171 by Glu in the A164R enzyme yielded a double mutant which was active, suggesting that Glu171 could compensate for the deleterious effect of Arg164 in the A164R+A171E enzyme . A specific increase in kcat for cefotaxime was observed with H170N, whereas R220L and T237A displayed a specific decrease in activity towards the same drug and a general increase in affinity towards cephalosporins . Finally, the K242E mutant displayed a kinetic behaviour very similar to that of PER-1 . Based on three-dimensional models generated by homology modelling and molecular dynamics, these results suggest novel structure-activity relationships in PER-1, when compared with those previously described for the TEM-type ESBLs.

Biochemistry, 1999 May 4, 38(18), 5720 - 7
Structure of the SHV-1 beta-lactamase; Kuzin AP et al.; The X-ray crystallographic structure of the SHV-1 beta-lactamase has been established . The enzyme crystallizes from poly(ethylene glycol) at pH 7 in space group P212121 with cell dimensions a = 49.6 A, b = 55.6 A, and c = 87.0 A . The structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.18 for all data in the range 8.0-1.98 A resolution . Deviations of model bonds and angles from ideal values are 0.018 A and 1.8 degrees, respectively . Overlay of all 263 alpha-carbon atoms in the SHV-1 and TEM-1 beta-lactamases results in an rms deviation of 1.4 A . Largest deviations occur in the H10 helix (residues 218-224) and in the loops between strands in the beta-sheet . All atoms in residues 70, 73, 130, 132, 166, and 234 in the catalytic site of SHV-1 deviate only 0.23 A (rms) from atoms in TEM-1 . However, the width of the substrate binding cavity in SHV-1, as measured from the 104-105 and 130-132 loops on one side to the 235-238 beta-strand on the other side, is 0.7-1.2 A wider than in TEM-1 . A structural analysis of the highly different affinity of SHV-1 and TEM-1 for the beta-lactamase inhibitory protein BLIP focuses on interactions involving Asp/Glu104.

FEMS Microbiol Lett, 1999 Apr 15, 173(2), 459 - 65
Mutations in the ampC promoter of Escherichia coli isolates resistant to oxyiminocephalosporins without extended spectrum beta-lactamase production; Caroff N et al.; Escherichia coli strains showing an increased resistance to oxyiminocephalosporins without an extended spectrum beta-lactamase production have been screened for mutations in their ampC beta-lactamase gene promoter . Mutations were found by direct sequencing of seven promoters at positions -42, -32 (box -35), -18, -1, +5, +24 (attenuator), +31 (attenuator) and +58 . By using rapid and simple methods, three of these mutations (-42, -32 and +24), which could enhance transcription, were searched in 37 resistant and 25 sensitive isolates . The -42 mutation was present in 33 of the 37 promoters from the resistant isolates . The -32 and +24 mutations were present only in three and two promoters, respectively, and they were combined in the most resistant strain of the study . The +24 mutation was detected in another strain associated with a 1-bp insertion between the -35 and -10 conserved sequences . None of these mutations was detected in the ampC beta-lactamase from the sensitive isolates.

Antimicrob Agents Chemother, 1999 May, 43(5), 1206 - 10
Carbapenem resistance in Escherichia coli associated with plasmid-determined CMY-4 beta-lactamase production and loss of an outer membrane protein; Stapleton PD et al.; Three cefoxitin-resistant Escherichia coli isolates from stool specimens of a patient with leukemia were either resistant, intermediate, or sensitive to imipenem . Conjugation experiments showed that cefoxitin resistance, but not imipenem resistance, was transferable . All isolates were shown by isoelectric focusing to produce two beta-lactamases with isoelectric points of 5.4 (TEM-1, confirmed by sequencing of a PCR product) and >8.5 (consistent with a class C beta-lactamase) . The gene coding for the unknown beta-lactamase was cloned and sequenced and revealed an enzyme which had 99.9% sequence identity with the plasmid-determined class C beta-lactamase CMY-2 . The cloned beta-lactamase gene differed from blaCMY-2 at one nucleotide position that resulted in an amino acid change, tryptophan to arginine at position 221 . We propose that this enzyme be designated CMY-4 . Both the imipenem-resistant and -intermediate isolates lacked a 38-kDa outer membrane protein (OMP) that was present in the imipenem-sensitive isolate . The lack of an OMP alone did not explain the difference in carbapenem susceptibilities observed . However, measurement of beta-lactamase activities (including measurements under conditions where TEM-1 beta-lactamase was inhibited) indicated that the imipenem-intermediate isolate expressed six- to eightfold less beta-lactamase than did the other isolates . This study illustrates that carbapenem resistance in E . coli can arise from high-level expression of plasmid-mediated class C beta-lactamase combined with an OMP deficiency . Furthermore, in the presence of an OMP deficiency, the level of expression of a plasmid-mediated class C beta-lactamase is an important factor in determining whether E . coli isolates are fully resistant to carbapenems.

Biotechnol Prog, 1999 Mar-Apr, 15(2), 164 - 7
Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain; Park SJ et al.; Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase . HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases . HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation . The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272 . Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method . The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272.

Antimicrob Agents Chemother, 1999 Apr, 43(4), 862 - 7
Ampicillin-sulbactam and amoxicillin-clavulanate susceptibility testing of Escherichia coli isolates with different beta-lactam resistance phenotypes; Oliver A et al.; The activities of ampicillin-sulbactam and amoxicillin-clavulanate were studied with 100 selected clinical Escherichia coli isolates with different beta-lactam susceptibility phenotypes by standard agar dilution and disk diffusion techniques and with a commercial microdilution system (PASCO) . A fixed ratio (2:1) and a fixed concentration (clavulanate, 2 and 4 micrograms/ml; sulbactam, 8 micrograms/ml) were used in the agar dilution technique . The resistance frequencies for amoxicillin-clavulanate with different techniques were as follows: fixed ratio agar dilution, 12%; fixed concentration 4-micrograms/ml agar dilution, 17%; fixed ratio microdilution, 9%; and disk diffusion, 9% . Marked discrepancies were found when these results were compared with those obtained with ampicillin-sulbactam (26 to 52% resistance), showing that susceptibility to amoxicillin-clavulanic acid cannot be predicted by testing the isolate against ampicillin-sulbactam . Interestingly, the discrimination between susceptible and intermediate isolates was better achieved with 4 micrograms of clavulanate per ml than with the fixed ratio . In contrast, amoxicillin susceptibility was not sufficiently restored when 2 micrograms of clavulanate per ml was used, particularly in moderate (mean beta-lactamase activity, 50.8 mU/mg of protein) and high-level (215 mU/mg) TEM-1 beta-lactamase producer isolates . Four micrograms of clavulanate per milliliter could be a reasonable alternative to the 2:1 fixed ratio, because most high-level beta-lactamase-hyperproducing isolates would be categorized as nonsusceptible, and low- and moderate-level beta-lactamase-producing isolates would be categorized as nonresistant . This approach cannot be applied to sulbactam, either with the fixed 2:1 ratio or with the 8-micrograms/ml fixed concentration, because many low-level beta-lactamase-producing isolates would be classified in the resistant category . These findings call for a review of breakpoints for beta-lactam-beta-lactamase inhibitors combinations.

Biotechnol Bioeng, 1999 Jan 20, 62(2), 155 - 9
Site-protected fixation and immobilization of Escherichia coli cells displaying surface-anchored beta-lactamase; Freeman A et al.; Bacteria displaying heterologous receptors or enzymes on their surface hold great potential as whole-cell adsorbents and biocatalysts, respectively . For industrial applications, such surface-engineered cells need to be killed and chemically fixed to prevent disintegration and leakage of the displayed proteins under process conditions . It is also highly desirable to couple the chemically stabilized cells onto a solid support matrix for additional mechanical stability, flexibility in reactor choice, and easy separation from processed medium . Recently, we described the development of a readily scalable methodology for cell killing, fixation, and outer membrane stabilization via glutaraldehyde fixation followed by secondary crosslinking (Freeman, A., Abramov, S . and Georgiou, G . 1996 . Biotechnol . Bioeng . 52: 625-630) . Glutaraldehyde treatment was also found, however, to reduce the specific activity of a model enzyme, beta-lactamase displayed on the surface of E . coli . Here, we show that crosslinking carried out in the presence of beta-lactamase inhibitors, namely phenyl boronic acid or sodium borate, protects the active site from chemical modification resulting in up to threefold higher specific activities without affecting the cell-stabilizing effect of the glutaraldehyde treatment . To prepare an immobilized whole cell biocatalyst, residual unreacted surface aldehyde groups were employed to immobilize covalently the fixed bacteria onto chitosan-coated cellulose powder . The binding of the bacteria onto chitosan-coated cellulose was quantitative up to cell loading of 83 mg dry cell weight/g of support . Cell immobilization did not introduce mass transfer limitations and created only a modest reduction in Vmax . Thus, chemical crosslinking, affected in presence of reversible active-site inhibitors and coupled with cell immobilization on chitosan-coated cellulose represents a widely useful methodology for the process application of recombinant bacteria displaying surface-anchored heterologous proteins .

Biotechnol Bioeng, 1998 Sep 20, 59(6), 659 - 65
Construction and characterization of F plasmid-based expression vectors; Jones KL et al.; A low-copy expression vector has been constructed from a 9 Kbp region of the Escherichia coli F plasmid containing the oriV and oriS origins of replication . This plasmid carries the beta-lactamase gene (Apr) and the araBAD promoter/araC regulator for arabinose-inducible gene expression . A derivative which carries a lacZ reporter gene was found to be stably maintained for at least 150 generations . A related multi-copy plasmid was stably maintained in arabinose-free medium, but no plasmid-bearing segregants remained after 60 generations when lacZ expression was induced . Induced expression resulted in 27% (multi-copy) and 12% (low-copy) decreases in growth rate . The uninduced levels of beta-galactosidase were 200 units (multi-copy) and 15 units (low-copy) .

Anal Biochem, 1999 Apr 10, 269(1), 32 - 7
RNA-binding properties of in vitro expressed histidine-tagged RB69 RegA translational repressor protein; Allen SV et al.; To facilitate RNA-binding studies of the phage RB69 RegA translational repressor protein, regA was configured to add six histidines to the carboxyl end of the protein . In vitro transcription-translation from the T7 promoter on plasmid pSA1 yielded a RegA69-His6 protein that binds nickel-Sepharose and elutes with 0.5 M imidazole . The system was further modified to avoid cloning and the toxic effects of RegA on Escherichia coli by the polymerase chain reaction (PCR), producing linear templates with the configuration T7 promoter-TIR-regA-His6 . A translation initiation region was used that conforms to consensus E . coli and eukaryotic initiation sites and eliminates the target for RegA autogenous repression . RegA69-His6 synthesized in E . coli S30 or wheat germ extracts displayed RNA-binding properties similar to wild-type RB69 RegA . Specificity of RNA binding was demonstrated by in vitro repression of T4 gp44 and gp45 but not beta-lactamase, by differential binding to poly(U)- and poly(C)-agarose, and by site-specific binding to a 23-base gene 44 target RNA but not to mutant 44 RNA . Therefore, addition of the His6 tag to the C-terminus of RB69 RegA does not dramatically alter RNA binding, indicating that this region is not directly involved in site recognition . With access to several T4-like phage genomes and regA mutant sequences, in vitro synthesis of His-tagged proteins directly from linear PCR products provides a convenient and efficient system to study RegA and other interesting RNA-binding proteins .

Rev Mal Respir, 1999 Feb, 16(1), 65 - 70
{Evolution of the management of lung diseases in general medicine in Bordeaux (1992-1995)}; Vernejoux JM et al.; Lower respiratory tract infections (LRTI) are very often managed by General Practitioners (GPs) . In France, the 1991 Lille Consensus Conference set out guidelines for the management of respiratory tract infections; in 1994, the Ministry of Health published Official Medical Recommendations (OMR) to be applied to seasonal respiratory infections . The aim of the study is to evaluate the impact of these OMR in 1995 on GPs' attitude when confronted with a community-acquired pneumonia in a previously healthy 40-year-old adult, with no sign of complications . Sixty seven GPs took, part in the same study by questionnaire in 1992 and 1995; we observed an increase in the prescription of aminopenicillin without a beta-lactamase inhibitor (41% in 1992 vs 66% in 1995; p = 0.009), and a reduction in both the use of aminopenicillin with a beta-lactamase inhibitor (35% in 1992 vs 11% in 1995; p = 0.002) and the concomitant prescription of cortico-steroids (43% in 1992 vs 14% vs 14% in 1995; p = 0.0009) . Between 1992 and 1995, general practitioners in the Bordeaux region have changed their therapeutic choices in community-acquired pneumonia . In 1995, antibiotic prescriptions followed consensus guidelines more closely.

Int J Clin Pharmacol Ther, 1999 Feb, 37(2), 63 - 75
Pharmacokinetic properties of beta-lactamase inhibitors; de la Pena A et al.; OBJECTIVES: In recent years, the use of combinations of beta-lactam-antibiotics with beta-lactamase inhibitors has been proven to be a useful and effective strategy to improve upon the therapeutic value of beta-lactam antibiotics . The objective of this article is to evaluate the pharmacokinetic properties of three commercially available beta-lactamase inhibitors . MATERIALS AND METHODS: Based on published articles in the literature, the pharmacokinetic properties of the beta-lactamase inhibitors clavulanic acid, sulbactam and tazobactam are reviewed and compared . RESULTS: There are some considerable differences between these three compounds: Only clavulanic acid is orally bioavailable . Tazobactam and sulbactam are eliminated renally to a larger extent than clavulanic acid . Tazobactam's total body clearance is almost twice that of sulbactam, and the two drugs differ significantly in their degree of protein-binding . Furthermore, sulbactam has a larger volume of distribution than tazobactam . CONCLUSIONS: When choosing combinations of a beta-lactam antibiotic with a beta-lactamase inhibitor, it is important to make sure that the pharmacokinetic properties of drug and inhibitor are similar and remain similar under changing pathophysiological conditions . Hence, beta-lactam inhibitors should not be freely interchanged, but for each beta-lactam antibiotic the best partner needs to be identified . If this is done properly, fixed combinations of beta-lactam-antibiotic and beta-lactamase inhibitor are appropriate and convenient . This situation may represent one of the few cases in pharmacotherapy, where fixed combinations of two drugs are beneficial.

Arch Pharm Res, 1999 Feb, 22(1), 68 - 71
Synthesis of 6-exomethylenepenams as beta-lactamase inhibitors; Im C et al.; The 6,6-dibromopenam (6) was treated with CH3MgBr and carbaldehyde 5 to afford the hydroxy compound 7, which was reacted with acetic anhydride to give acetoxy compound 8 . The deacetobromination of 8 with zinc and acetic acid gave 6-exomethylenepenams, E-isomer 10 and Z-isomer 9, which was oxidized to sulfone 11 by m-CPBA . The p-methoxybenzyl compounds were deprotected by AlCl3 and neutralized to give the sodium salts 12, 13 and 14.

Protein Sci, 1999 Feb, 8(2), 404 - 9
pKa calculations for class A beta-lactamases: influence of substrate binding; Lamotte-Brasseur J et al.; Beta-Lactamases are responsible for bacterial resistance to beta-lactams and are thus of major clinical importance . However, the identity of the general base involved in their mechanism of action is still unclear . Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role . Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step . Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free beta-lactamases, showing that this residue is unlikely to act as a proton abstractor . On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base . To check this hypothesis, we performed continuum electrostatic calculations for five wild-type and three beta-lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri-Michaelis complex and in the tetrahedral intermediate . In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a beta-lactam substrate is bound in the enzyme active site . The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate . The influence of the beta-lactam carboxylate on the pKa values of the active-site lysines is also discussed.

J Mol Biol, 1999 Feb 5, 285(5), 2079 - 87
Crystal structure of the E166A mutant of extended-spectrum beta-lactamase Toho-1 at 1.8 A resolution; Ibuka A et al.; Bacterial resistance to beta-lactams is mainly due to the production of beta-lactamase . Especially through the production of extended-spectrum beta-lactamases (ESBLs), bacteria have acquired resistance not only to penicillins, but also to expanded-spectrum cephems . Here, we describe the crystal structure of the E166A mutant of class A beta-lactamase Toho-1 at 1.8 A resolution, the first reported tertiary structure of an ESBL . Instead of the wild-type enzyme, a mutant Toho-1, in which Glu166 was replaced with alanine, was used for this study, because of the strong tendency of the wild-type enzyme to form twinned crystals . The overall structure of Toho-1 is similar to the crystal structures of non-ESBLs, with no pronounced backbone rearrangement of the framework . However, there are some notable local changes . First, a difference in the disposition of an arginine residue, which is at position 244 in non-ESBLs but at position 276 in Toho-1 and other ESBLs, was revealed and the role of this arginine residue is discussed . Moreover, changes in the hydrogen-bonding pattern and in the formation of the hydrophobic core were also observed near the Omega loop . In particular, the lack of hydrogen bonds in the vicinity of the Omega loop could be a cause of the extended substrate specificity of Toho-1 . Through the generation of a model for the enzyme-substrate complex, a conformational change of Toho-1 occurring on complex formation is discussed based on the active-site cleft structure and the substrate profile .

Antimicrob Agents Chemother, 1999 Feb, 43(2), 393 - 6
Diversification of Escherichia coli expressing an SHV-type extended-spectrum beta-lactamase (ESBL) during a hospital outbreak: emergence of an ESBL-hyperproducing strain resistant to expanded-spectrum cephalosporins; Palucha A et al.; Twelve SHV-type extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli lac mutant isolates were recovered in October 1997 from 11 patients of the neonatal ward in a Warsaw hospital . The outbreak was clonal; however, some of the isolates expressed a much higher level of resistance to several beta-lactam antibiotics, including expanded-spectrum cephalosporins . This phenotype has been attributed to beta-lactamase hyperproduction correlating with the multiplication of ESBL gene copies, as was demonstrated for representative isolates.

Antimicrob Agents Chemother, 1999 Feb, 43(2), 367 - 70
Updated sequence information for TEM beta-lactamase genes; Goussard S et al.; The sequences of the promoter regions and of the structural genes for 13 penicillinase, extended-spectrum, and inhibitor-resistant TEM-type beta-lactamases have been determined, and an updated blaTEM gene nomenclature is proposed.

J Biol Chem, 1999 Jan 22, 274(4), 2424 - 31
Constitutive calcium-independent release of Toxoplasma gondii dense granules occurs through the NSF/SNAP/SNARE/Rab machinery; Chaturvedi S et al.; The signals and the molecular machinery mediating release of dense matrix granules from pathogenic protozoan parasites are unknown . We compared the secretion of the endogenous dense granule marker GRA3 in Toxoplasma gondii with the release of a stably transfected foreign reporter, beta-lactamase, that localizes to parasite dense granules . Both proteins were released constitutively in a calcium-independent fashion, as shown using both intact and streptolysin O-permeabilized parasites . N-Ethylmaleimide and recombinant bovine Rab-guanine dissociation inhibitor inhibited beta-lactamase secretion in permeabilized parasites, whereas recombinant hamster N-ethylmaleimide-sensitive fusion protein and bovine alpha-SNAP augmented release . Guanosine 5'-3-O-(thio)triphosphate, but not cAMP, augmented secretion in the presence but not in the absence of ATP . The T . gondii NSF/SNAP/SNARE/Rab machinery participates in dense granule release using parasite protein components that can interact functionally with their mammalian homologues.

Biochemistry, 1999 Jan 5, 38(1), 11 - 21
Biophysical characterization of the interaction of the beta-lactamase TEM-1 with its protein inhibitor BLIP; Albeck S et al.; BLIP is a secreted protein from Streptomyces clavuligerus that inhibits a wide range of beta-lactamases . Here we investigate the tight interaction of BLIP, expressed heterologousely in E . coli, with TEM-1 . Kinetic and thermodynamic constants were determined using methods with the proteins either in a homogeneous or in a heterogeneous phase . While values of Delta DeltaG(mut-wt) are similar whether measured by fluorescence quench, enzyme inhibition, or surface plasmon resonance, absolute values of DeltaG and kinetic constants vary . Association and dissociation rate constants of 10(5) M-1 s-1 and 10(-)4 s-1, respectively, and a nanomolar affinity were determined for the wild-type proteins . The highest affinity is measured at pH 7.5, with a decreasing association rate constant at higher pH values, and an increasing dissociation rate constant at lower pH values . The marginal effect of salt on the kinetics of binding, as well as the calculated surface potentials, suggests a limited role for electrostatic forces in guiding this reaction . Still, mutations of interfacial residues affect the rate of association significantly, so that an increase in the net negative charge on either protein reduces the association rate constant . We show that simple electrostatic rules can explain this behavior . BLIP inhibits the catalytic activity of TEM-1 by binding its active site . Yet, mutations of active site residues on TEM-1 only have a moderate though cooperative effect on the binding energy . This can be explained in light of the peripheral location of the active site in the interface between the two proteins.

Nat Biotechnol, 1998 Dec, 16(13), 1329 - 33
A genome-wide functional assay of signal transduction in living mammalian cells; Whitney M et al.; We describe a genome-wide functional assay for rapid isolation of cell clones and genetic elements responsive to specific stimuli . A promoterless beta-lactamase reporter gene was transfected into a human T-cell line to generate a living library of reporter-tagged clones . When loaded with a cell-permeable fluorogenic substrate, the cell library simultaneously reports the expression of a large number of endogenous genes . Flow cytometry was used to recover individual clones whose reporter-tagged genes were either induced or repressed following T-cell activation . Responsive clones were expanded and analyzed pharmacologically to identify patterns of regulation associated with specific genes . Although demonstrated using T cells, the genomic assay could be applied to map downstream transcriptional consequences for any propagating cell line in response to any stimulus of interest.

J Bacteriol, 1998 Dec, 180(24), 6625 - 34
Topological analysis of the aerobic membrane-bound formate dehydrogenase of Escherichia coli; Benoit S et al.; Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate, Escherichia coli synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis . FDH-O is a membrane-bound enzyme complex composed of three subunits, alpha (FdoG), beta (FdoH), and gamma (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N . The topology of these three subunits has been studied by using blaM (beta-lactamase) gene fusions . A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits . In contrast to previously reported predictions from sequence analysis, our data suggested that the alphabeta catalytic dimer is located in the cytoplasm, with a C-terminal anchor for beta protruding into the periplasm . As expected, the gamma subunit, which specifies cytochrome b, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm . Protease digestion studies of the 35S-labelled FDH-O heterotrimer in spheroplasts add further support to this model . Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.

Dakar Med, 1997, 42(1), 6 - 10
{Broad-spectrum beta-lactamases: profile and perspectives after 3 years of systematic detection at the Main Hospital of Dakar (1 February 1992 to 1 February 1995)}; Ndoye B et al.; After having undertaken an estimation upon infections from bacteria generating extended broad spectrum Beta-lactamase at "Hospital Principal" of Dakar between 1992 to 1993 and after having drawn the attention of authorities of the Hospital to the hazard caused by this sort of infections, authors come back in doing a second estimation during the two following years . The results of this estimation show that measures preciously taken, not with standing an certain impact, are clearly unsufficient and have got a limited effect in the future . They suggest practical solutions in the long run, solutions that have already been taken at "Hospital Principal" of Dakar and deserve to be kept on and encouraged.

Dev Biol, 1998 Nov 15, 203(2), 290 - 4
beta-lactamase as a marker for gene expression in live zebrafish embryos; Raz E et al.; In this report we describe the development of a sensitive assay for gene expression in zebrafish embryos using beta-lactamase as a reporter gene . We show that injection of a green fluorescent substrate for beta-lactamase allows the detection of reporter gene expression in live embryos . The beta-lactamase enzyme catalyzes the hydrolysis of the substrate, thereby disrupting fluorescence resonance energy transfer from the donor to the acceptor dye in the molecule . As a result, a blue fluorescent product is produced and retained specifically in cells within which the enzyme is expressed . beta-Lactamase is therefore suitable for monitoring spatially restricted patterns of gene expression in the early embryo . We suggest that this new reporter system provides a major advancement in sensitivity over the existing methods for monitoring gene expression in vivo during early embryogenesis .

J Med Chem, 1998 Nov 5, 41(23), 4577 - 86
Structure-based enhancement of boronic acid-based inhibitors of AmpC beta-lactamase; Weston GS et al.; The expression of beta-lactamases is the most common form of bacterial resistance to beta-lactam antibiotics . To combat these enzymes, agents that inhibit (e.g . clavulanic acid) or evade (e.g . aztreonam) beta-lactamases have been developed . Both the beta-lactamase inhibitors and the beta-lactamase-resistant antibiotics are themselves beta-lactams, and bacteria have responded to these compounds by expressing variant enzymes resistant to inhibition (e.g . IRT-3) or that inactivate the beta-lactamase-resistant antibiotic (e.g . TEM-10) . Moreover, these compounds have increased the frequency of bacteria with intrinsically resistant beta-lactamases (e.g . AmpC) . In an effort to identify non-beta-lactam-based beta-lactamase inhibitors, we used the crystallographic structure of the m-aminophenylboronic acid-Escherichia coli AmpC beta-lactamase complex to suggest modifications that might enhance the affinity of boronic acid-based inhibitors for class C beta-lactamases . Several types of compounds were modeled into the AmpC binding site, and a total of 37 boronic acids were ultimately tested for beta-lactamase inhibition . The most potent of these compounds, benzo{b}thiophene-2-boronic acid (36), has an affinity for E . coli AmpC of 27 nM . The wide range of functionality represented by these compounds allows for the steric and chemical "mapping" of the AmpC active site in the region of the catalytic Ser64 residue, which may be useful in subsequent inhibitor discovery efforts . Also, the new boronic acid-based inhibitors were found to potentiate the activity of beta-lactam antibiotics, such as amoxicillin and ceftazidime, against bacteria expressing class C beta-lactamases . This suggests that boronic acid-based compounds may serve as leads for the development of therapeutic agents for the treatment of beta-lactam-resistant infections.

J Med Chem, 1998 Oct 8, 41(21), 3972 - 5
Structure-based design of beta-lactamase inhibitors . 2 . Synthesis and evaluation of bridged sulfactams and oxamazins; Hubschwerlen C et al.; A series of bridged monocyclic beta-lactams activated by various groups on the beta-lactam nitrogen (X = OCH2CO2H, OSO3H) has been synthesized and evaluated . Among them, the bridged sulfactams (X = OSO3H) were found to be effective beta-lactamase inhibitors . They inhibit both class A and class C beta-lactamases.

Antimicrob Agents Chemother, 1998 Sep, 42(9), 2319 - 25
Characterization of a PSE-4 mutant with different properties in relation to penicillanic acid sulfones: importance of residues 216 to 218 in class A beta-lactamases; Sabbagh Y et al.; Class A beta-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and tazobactam . An examination of multiple alignments indicated that amino acids 216 to 218 differed among class A enzymes . By random replacement mutagenesis of codons 216 to 218 in PSE-4, a complete library consisting of 40,864 mutants was created . The library of mutants with mutations at positions 216 to 218 in PSE-4 was screened on carbenicillin and ampicillin with the inactivator sulbactam; a collection of 14 mutants was selected, and their bla genes were completely sequenced . Purified wild-type and mutant PSE-4 beta-lactamases were used to measure kinetic parameters . One enzyme, V216S:T217A:G218R, was examined for its peculiar pattern of inhibition . There was an increase in the Km from 68 microM for the wild type to 271 microM for the mutant for carbenicillin and 33 to 216 microM for ampicillin . Relative to the wild-type PSE-4 enzyme, 37- and 30-fold increases in Ki values were observed for the mutant enzyme for sulbactam and tazobactam, respectively . The results that were obtained suggested that positions 216 to 218 are important for interactions with penicillanic acid sulfone inhibitors . In contrast, V216 and A217 in the TEM-1 class A beta-lactamase do not tolerate amino acid residue substitutions . However, for the PSE-4 beta-lactamase, 11 of 14 mutants from the library of mutants with mutations at positions 216 to 218 whose sequences were determined had substitutions at position 216 (G, R, A, S) and position 217 (A, S) . The data showed the importance of residues 216 to 218 in their atomic interactions with inactivators in the PSE-4 beta-lactamase structure.

Biochem J, 1998 Jul 15, 333 ( Pt 2), 395 - 400
Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 beta-lactamase, an SHV-family class A enzyme; Lin S et al.; The Met69-->Ile mutant of the OHIO-1 beta-lactamase, an SHV-family enzyme, is resistant to inactivation by beta-lactamase inhibitors . Analysis of purified Met69-->Ile enzyme reveals that its isoelectric point (pI 7.0) and CD spectrum are identical with those of the OHIO-1 enzyme . Levels of beta-lactamase expression in Escherichia coli as determined by immunoblotting are similar for OHIO-1 and Met69-->Ile beta-lactamase . The kinetic constants of the Met69-->Ile enzyme compared with OHIO-1 are smaller for benzylpenicillin (Km = 6 microM compared with 17 microM; kcat = 234 s-1 compared with 345 s-1 respectively) and carbenicillin (Km = 3 microM compared with 17 microM; kcat = 131 s-1 compared with 320 s-1 respectively) . For the cephalosporins cephaloridine and 7-(thienyl- 2-acetamido)-3-{2-(4-N,N- dimethylaminophenylazo)pyridinium-methyl}-3-cephem-4-carboxylic acid (PADAC), a similar pattern is also seen (Km=38 microM compared with 96 microM and 6 microM compared with 75 microM respectively; kcat = 235 s-1 compared with 1023 s-1 and 9 s-1 compared with 50 s-1 respectively) . Consistent with minimum inhibitory concentrations that show resistance to beta-lactam beta-lactamase inhibitors, the apparent Ki values, turnover numbers and partition ratios (kcat/kinact) for the mechanism-based inactivators clavulanate, sulbactam and tazobactam are increased . The inactivation rate constants (kinact) are decreased . The difference in activation energy, a measurement of altered affinity for the wild-type and mutant enzymes leading to acylation of the active site, reveals small energy differences of less than 8.4 kJ/mol . In total, these results suggest that the Met-->Ile substitution at position 69 in the OHIO-1 beta-lactamase alters the active site, primarily affecting the interactions with beta-lactamase inhibitors.

J Bacteriol, 1998 Sep, 180(18), 4821 - 7
Topological analysis of DcuA, an anaerobic C4-dicarboxylate transporter of Escherichia coli; Golby P et al.; Escherichia coli possesses three independent anaerobic C4-dicarboxylate transport systems encoded by the dcuA, dcuB, and dcuC genes . The dcuA and dcuB genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity . A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S . Six, S . C . Andrews, G . Unden, and J . R . Guest, J . Bacteriol . 176:6470-6478, 1994) . These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a beta-lactamase protein lacking the N-terminal signal peptide . The resulting topological model differs from those previously predicted . It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6 . The N and C termini are located in the periplasm and the predicted orientation is consistent with the "positive-inside rule." Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region . The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB . Overproduction of DcuA to 15% of inner-membrane protein was obtained with the lacUV5-promoter-based plasmid, pYZ4.

Microbiology, 1998 Aug, 144 ( Pt 8), 2161 - 7
Cloning and heterologous expression of the gene for BLIP-II, a beta-lactamase-inhibitory protein from Streptomyces exfoliatus SMF19; Park HU et al.; A beta-lactamase-inhibitory protein (BLIP-II) was purified from the culture filtrate of Streptomyces exfoliatus SMF 19 and its N-terminal amino acid sequence was determined . A clone containing the gene encoding BLIP-II (bliB) was selected from a cosmid library by colony hybridization using an oligonucleotide probe based on the N-terminal amino acid sequence of BLIP-II . The bliB gene was isolated and sequenced . Analysis of the nucleotide sequence revealed that the gene consists of 1116 bp and encodes a mature protein of 332 amino acids preceded by a 40 amino acid signal sequence . bliB, expressed under the control of the T7 promoter in Escherichia coli, was accumulated in an inactive form in inclusion bodies, but beta-lactamase-inhibitory activity was recovered after refolding . In addition, bliB was heterologously expressed in Streptomyces lividans TK24 using the melC1 promoter . The BLIP-II protein produced in recombinant strains of S . lividans was secreted into the culture supernatant in a biologically active form.

Biochemistry, 1998 Aug 18, 37(33), 11660 - 9
Identification of the binding surface on beta-lactamase for GroEL by limited proteolysis and MALDI-mass spectrometry; Gervasoni P et al.; Escherichia coli beta-lactamase, alone or as a complex with GroEL at 48 degreesC, was partially digested with trypsin, endoproteinase Glu-C, or thermolysin . Peptides were analyzed by matrix-assisted laser desorption and ionization mass spectrometry and aligned with the known sequence . From the protease cleavage sites which become protected upon binding and those which become newly accessible, a model of the complex is proposed in which the carboxy-terminal helix has melted, two loops form the binding interface and the large beta-sheet become partially uncovered by the slight dislocation of other structural elements . This explains how hydrophobic surface on the substrate protein can become accessible while scarcely disrupting the hydrogen bond network of the native structure . An analysis of the GroEL-bound peptides bound after digestion of the beta-lactamase showed no obvious sequence motifs, indicating that binding is provided by hydrophobic patches in the three-dimensional structure.

FASEB J, 1998 Aug, 12(11), 1055 - 60
Functional mimicry: elicitation of a monoclonal anti-idiotypic antibody hydrolizing beta-lactams; Avalle B et al.; Antigen mimicry by anti-idiotypic antibodies is investigated as a reliable strategy to achieve molecular imprinting of an enzymatic activity . A monoclonal anti-idiotypic antibody (Ab2-9G4H9) was elicited by using a monoclonal antibody (Ab1-7AF9) specific for the beta-lactamase active site . Catalytic features of Ab2 were characterized with beta-lactamase substrates . The antibody combining site appeared to have retained a part of the catalytic specificity . The relevance of the idiotypic mimicry concept for the generation of catalytic antibodies was further demonstrated by eliciting a third generation antibody (Ab3), which was shown to recognize beta-lactamase: the complete internal image properties of Ab2 9G4H9, including binding and catalytic properties, were thus checked.

Chemotherapy, 1998 Jul-Aug, 44(4), 260 - 4
In vitro activity of loracarbef against TEM extended-spectrum beta-lactamase-producing Escherichia coli strains and its interaction with the enzymes; Prinarakis EE et al.; The in vitro activity of loracarbef was evaluated against Escherichia coli strains producing TEM extended-spectrum beta-lactamases (ESBLs) . The MICs of loracarbef were lower than those of cefaclor and cephalothin . The antibiotic was active (MICs < or = 2 mg/l) against strains producing TEM-6, TEM-8, TEM-10, TEM-16, TEM-26 and TEM-28 beta-lactamases . The MICs for TEM-3-, TEM-5- and TEM-24-producing strains were higher (> or = 16 mg/l) . Hydrolysis studies indicated that the latter beta-lactamases inactivated loracarbef more efficiently than the remaining TEM-type ESBLs examined.

Protein Expr Purif, 1998 Jul, 13(2), 184 - 90
Hyperexpression of rat spermatidal protein TP2 in Escherichia coli by codon optimization and engineering the vector-encoded 5' UTR; Meetei AR et al.; We have recently reported the cDNA cloning of rat spermatidal protein TP2 and its expression in Escherichia coli using pTrc 99A as the expression vector . However, the expression level was very low . We have now improved the expression of TP2 over fivefold by (1) optimizing the codons for lysine, arginine, proline, leucine, glycine, valine, threonine, alanine, and tyrosine and (2) by engineering the vector-encoded 5' UTR . The expressed protein was in the soluble phase and could be purified to homogeneity by successive chromatography on Zinc-NTA-agarose affinity matrix and heparin agarose . Serendipitously, we have also observed a concomitant hyperinduction of vector encoded beta-lactamase gene along with TP2 in the E . coli BL21 (DE3) cells .

Antimicrob Agents Chemother, 1998 Jul, 42(7), 1542 - 8
Selection and characterization of beta-lactam-beta-lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 beta-lactamase gene; Vakulenko SB et al.; Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics . This strategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation . We used PCR mutagenesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate . Eleven different mutant genes from these strains contained from 1 to 10 mutations . Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance . None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with beta-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes . Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors . Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance . Thus, despite PCR mutagenesis we found no new mutant TEM beta-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates . Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.

Anal Biochem, 1998 Jul 1, 260(2), 160 - 5
In vitro measurement of beta-lactamase-catalyzed ampicillin hydrolysis by recombinant Escherichia coli extracts using quantitative high-performance liquid chromatography; Urbain JL et al.; We report a rapid and simple protocol for measuring the beta-lactamase activity from recombinant Escherichia coli cells transformed with any of the common plasmid vectors that provide ampicillin resistance through constitutive expression and periplasmic localization of the beta-lactamase TEM-1 . The hydrolytic enzyme was extracted from the E . coli periplasm and the beta-lactamase activity determined by measuring conversion of ampicillin to aminobenzyl-penicilloic acid using quantitative high-performance liquid chromatography . Under saturating conditions the in vitro assay was linear as a function of both incubation time and enzyme concentration . Application of this assay to investigate TEM-1 expression, from two different protein expression vector systems, demonstrated the potential importance of this assay in studies of recombinant protein expression and translocation .

J Clin Microbiol, 1998 Jul, 36(7), 1977 - 83
Genetic diversity among strains of Moraxella catarrhalis: analysis using multiple DNA probes and a single-locus PCR-restriction fragment length polymorphism method; Walker ES et al.; Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce beta-lactamase and can be nosocomially transmitted . The typing methods used in epidemiological analyses of M . catarrhalis are not optimal for genetic analyses . Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M . catarrhalis . Nine randomly cloned M . catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains . For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative . A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification . DNAs from the 54 strains were subjected to PCR-RFLP . SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54) . Epidemiologically linked strains appeared genetically identical by both methods . PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains . PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness . Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations . The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches . High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones . Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites.

FEBS Lett, 1998 Jun 12, 429(2), 162 - 6
Different degradation pathways for heterologous glycoproteins in yeast; Holkeri H et al.; Rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli beta-lactamase were translocated into the yeast endoplasmic reticulum (ER), glycosylated, misfolded and rapidly degraded . NGFRe underwent ATP-dependent thermosensitive degradation independently of vesicular transport . Since no evidence for degradation by the cytoplasmic 26S proteosome complex could be obtained, NGFRe appeared to be degraded in the ER . Beta-lactamase exited the ER by vesicular traffic and was transported from the Golgi via the Vps10 receptor pathway to the vacuole for degradation . Machineries in the ER and the Golgi appear to recognize distinct structural features on misfolded heterologous proteins and guide them to different degradation pathways.

East Afr Med J, 1998 Mar, 75(3), 175 - 9
Human antibody response to Moraxella catarrhalis antigens; Gabre-Selasie S; Moraxella catarrhalis was isolated from 68 of 200 (34%) sputum and 56 (28%) nasopharyngeal swab samples of patients with community-acquired pneumonia . Of the 68 pneumonia patients, 42 (61.8%) were males and 26 (38.2%) females . Fifty one of the 68 patients (75%) had chronic underlying diseases . beta-lactamase was produced by 37 (54.4%) of the 68 sputum samples and 32(57.1%) of the 56 nasopharyngeal isolates . In an ELISA using outer membrane protein antigens of M . catarrhalis against patient sera showed 40 of 68 (58.8%), and 43 of 68 (63.2%) significant increase in convalescent to acute sera when IgA, IgM and IgG3 were used respectively . In control sera only of 30(3.3%) and none showed significant antibody rise when IgA, IgM and IgG3 conjugates were used respectively (P < 0.05).

J Cell Biol, 1998 Jun 15, 141(6), 1323 - 33
The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms; Karsten V et al.; All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules . To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T . gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E . coli beta-lactamase . Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space . Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface . Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules . The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail . Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network . Targeting of secreted proteins to T . gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.

Biophys J, 1998 Jun, 74(6), 3256 - 63
Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins; Vinckier A et al.; The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid . High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top . Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions . The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones . It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight . By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.

J Biol Chem, 1998 May 15, 273(20), 12109 - 15
Characterization of the C-terminal propeptide involved in bacterial wall spanning of alpha-amylase from the psychrophile Alteromonas haloplanctis; Feller G et al.; The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase . The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa) . In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide . The purified C-terminal propeptide displays several features common to beta-pleated transmembrane proteins . It has no intramolecular chaperone function because active alpha-amylase is expressed by Escherichia coli in the absence of the propeptide coding region . In E . coli, the 70-kDa precursor is directed toward the supernatant . When the alpha-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning . It can also accept a foreign passenger, as shown by the extracellular routing of a beta-lactamase-propeptide fusion protein.

Cell Mol Life Sci, 1998 Apr, 54(4), 341 - 6
The diversity, structure and regulation of beta-lactamases; Philippon A et al.; beta-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to beta-lactam antibiotics . A large number of enzymes have been described with very diverse primary structures and catalytic profiles . Nevertheless, all known three-dimensional structures of active-site serine beta-lactamases exhibit a high degree of similarity with apparently equivalent chemical functionalities in the same strategic positions . These groups might not, however, play identical roles in the various classes of enzymes . Structural data have also been recently obtained for the zinc metallo-beta-lactamases, but the detailed catalytic mechanisms might also differ widely, depending on the enzyme studied . Similarly, the induction of the synthesis of beta-lactamases is now better understood, but many questions remain to be answered.

Protein Eng, 1998 Feb, 11(2), 87 - 93
Recurrence quantification analysis in structure-function relationships of proteins: an overview of a general methodology applied to the case of TEM-1 beta-lactamase; Zbilut JP et al.; Protein structure-function relationships have been increasingly scrutinized by a variety of correlational and information theoretic measures . In an effort to extend this methodology, a technique originally developed in non-linear science, recurrence quantification analysis, was combined with traditional principal components analysis to study a large number (56) of TEM-1 beta-lactamase mutants . The hydrophobicity profiles corresponding to the primary structure of 13 naturally occurring mutations partially impairing function, together with 43 artificial non-tolerated mutations were subjected to discriminant analysis, derived from the results of recurrence quantification analysis, coupled to a principal exponents extraction . Eleven (85%) of the naturally occurring mutants and 36 (84%) of the artificial mutants were correctly classified (p < 0.0001) . We conclude that this technique may be useful in protein engineering and, in general, in structure-function studies of biopolymers.

Protein Sci, 1998 May, 7(5), 1164 - 71
Reactivation of thermally inactivated pre-beta-lactamase by DnaK, DnaJ, and GrpE; McCarthy D et al.; To understand the role of the 23-amino acid signal sequence in the folding and stability of beta-lactamase, the precursor and a mutant beta-lactamase with a 19-amino acid signal sequence deletion were synthesized in vitro using an Escherichia coli cell-free coupled transcription/translation system . Approximately 30% of the newly synthesized full-length precursor and 60% of the deletion mutant polypeptides were terminated and released from the ribosomes as active enzyme . Activity of the pre-beta-lactamase, but not the mutant, was unstable at 37 degrees C, suggesting that the signal sequence causes the enzyme to unfold . This inactivation was independent of ATP . Pre-beta-lactamase activity was stabilized by lowering the temperature to 30 degrees C . Furthermore, addition of the molecular chaperones DnaK/J and GrpE, in the presence of ATP and Mg2+, restored the activity of the temperature-inactivated precursor . The precursor formed a stable complex with DnaK and GrpE . Both ATP and DnaJ were required for recovery of enzymatic activity, indicating that DnaJ may bind transiently to the complex . These results suggest that the signal sequence of the pre-beta-lactamase causes a temperature-dependent unfolding of the synthesized enzyme and that DnaK/J and GrpE interact with unfolded pre-beta-lactamase to promote refolding of the protein into its native, enzymatically active conformation.

Biochem J, 1998 Mar 15, 330 ( Pt 3), 1443 - 9
Role of residues 104, 164, 166, 238 and 240 in the substrate profile of PER-1 beta-lactamase hydrolysing third-generation cephalosporins; Bouthors AT et al.; The class A beta-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum beta-lactamases (ESBLs), catalyses the hydrolysis of oxyimino-beta-lactams such as cefotaxime (CTX), ceftazidime (CAZ) and aztreonam (AZT) . Molecular modelling was used to identify in PER-1 the amino acid residues corresponding to those found at positions 104, 164, 238 and 240 in the TEM-type ESBLs, which are critical for hydrolysis of oxyimino-beta-lactams . The function of these residues in PER-1 was assessed by site-directed mutagenesis . In this enzyme, residue 104 could be either a glutamine, an asparagine or a threonine . The Gln-->Gly mutation did not significantly affect the catalytic efficiency, while Asn-->Gly and Thr-->Glu resulted in a marked decrease in catalytic activity, probably due to the alteration of a hydrogen bond network connecting the putative Asn-104 residue to Asn-132 and Glu-166 . Replacement of Ala-164 by Arg in PER-1 resulted in a mutant with no detectable activity, thus suggesting that Ala-164 is important for catalysis and stability of PER-1 . Conversely, Ser-238-->Gly and Gly-240-->Glu had little effect on kcat and Km values . Finally, the replacement of the catalytic residue Glu-166 by an alanine resulted in a complete loss of activity for CTX and a marked decrease of kcat for CAZ and AZT . These results suggest that Glu-166 is an important residue in PER-1 . However, residues other than Glu-166 could contribute in maintaining residual activity towards oxyimino-beta-lactams in the Ala-166 mutant.

Antimicrob Agents Chemother, 1998 May, 42(5), 1245 - 8
Molecular heterogeneity of the L-1 metallo-beta-lactamase family from Stenotrophomonas maltophilia; Sanschagrin F et al.; We have determined the nucleotide sequence of the blaS gene encoding the carbapenem-hydrolyzing L-1 beta-lactamase from Stenotrophomonas maltophilia GN12873 . Analysis of the DNA and deduced amino acid sequences identified a product of 290 amino acids . Comparisons of the L-1 amino acid sequence with those of other zinc beta-lactamases showed 88.6% identity with the L-1 enzyme from S . maltophilia IID1275 and less than 20% identity with other class B metalloenzymes.

Mol Biol Cell, 1998 Apr, 9(4), 817 - 27
Folding of active beta-lactamase in the yeast cytoplasm before translocation into the endoplasmic reticulum; Paunola E et al.; Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation . We show here that Escherichia coli beta-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells . beta-Lactamase is a globular trypsin-resistant molecule in authentic form . For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150delta, which conferred posttranslational translocation and provided sites for O-glycosylation . We devised conditions to retard translocation of Hsp150delta-beta-lactamase . This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles . Both proteins were trypsin resistant and had similar beta-lactamase activity and Km values for nitrocefin . The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium . Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls . This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation.

J Cell Sci, 1998 Jun, 111 ( Pt 11), 1575 - 82
Transient ER retention as stress response: conformational repair of heat-damaged proteins to secretion-competent structures; Saris N et al.; Mechanisms to acquire tolerance against heat, an important environmental stress condition, have evolved in all organisms, but are largely unknown . When Saccharomyces cerevisiae cells are pre-conditioned at 37 degrees C, they survive an otherwise lethal exposure to 48-50 degrees C, and form colonies at 24 degrees C . We show here that incubation of yeast cells at 48-50 degrees C, after pre-conditioning at 37 degrees C, resulted in inactivation of exocytosis, and in conformational damage and loss of transport competence of proteins residing in the endoplasmic reticulum (ER) . Soon after return of the cells to 24 degrees C, membrane traffic was resumed, but cell wall invertase, vacuolar carboxypeptidase Y and a secretory beta-lactamase fusion protein remained in the ER for different times . Thereafter their transport competence was resumed very slowly with widely varying kinetics . While the proteins were undergoing conformational repair in the ER, their native counterparts, synthesized after shift of the cells to 24 degrees C, folded normally, by-passed the heat-affected copies and exited rapidly the ER . The Hsp70 homolog Lhs1p was required for acquisition of secretion competence of heat-damaged proteins . ER retention and refolding of heat-denatured glycoproteins appear to be part of the cellular stress response.

Oral Microbiol Immunol, 1998 Feb, 13(1), 36 - 40
beta-Lactamase-producing strains in the species Prevotella intermedia and Prevotella nigrescens; Bernal LA et al.; A total of 96 strains were collected that included laboratory strains and clinical isolates classified Prevotella intermedia sensu lato and the type strains of the species P . intermedia sensu stricto and Prevotella nigrescens . Susceptibility to amoxicillin and amoxicillin-clavulanic acid was determined by the Etest . PCR-DNA probe assays were used to speciate each strain as P . intermedia sensu stricto or P . nigrescens . By Etest, 71 strains (74%) were susceptible to both amoxicillin and amoxicillin-clavulanic acid with minimum inhibitory concentrations in the 0.016-0.064 microgram/ml range . In contrast, amoxicillin minimum inhibitory concentrations of 25 strains (26%) were in the range of 1.5-96 micrograms/ml with concomitant amoxicillin-clavulanic acid minimum inhibitory concentrations in the low range 0.016-0.38 microgram/ml, indicating a production of beta-lactamase as confirmed by nitrocefin tests . Of these beta-lactamase-producing strains, 20% (5/25) were identified as P . intermedia sensu stricto by the PCR-DNA probe assay and 72% (18/25) as P . nigrescens . Our results provide support for the major role of P . nigrescens in the failure of therapy using beta-lactam antibiotics.

J Bacteriol, 1998 May, 180(9), 2387 - 94
Ferric citrate transport of Escherichia coli: functional regions of the FecR transmembrane regulatory protein; Welz D et al.; Transcription of the ferric citrate transport genes of Escherichia coli is induced by ferric citrate bound to the outer membrane receptor FecA . Additional ferric citrate-specific regulatory proteins are FecR in the cytoplasmic membrane and the FecI sigma factor in the cytoplasm . To further understand the assumed FecR-mediated signal transduction across the cytoplasmic membrane, the transmembrane topology of FecR (317 amino acids) was determined with hybrid proteins containing portions of FecR and mature BlaM beta-lactamase . BlaM fused to FecR regions extending from residues 107 to 149 and residues 230 to 259 conferred high ampicillin resistance to cells, while BlaM fused to sites between residues 159 and 210 and between residues 265 and 301 conferred low resistance . Cells that synthesized FecR'-BlaM with fusion joints between residues 8 and 81 of FecR were fully sensitive to ampicillin . The ampicillin resistance of the low-resistance FecR'-BlaM hybrids was increased 2- to 10-fold by cosynthesis of plasmid-encoded GroEL GroES and SecB chaperones and in degP and ompT protease mutants, which suggested that the decreased ampicillin resistance level of these hybrids was caused by the formation of inclusion bodies and proteolytic degradation . Replacement of glycine by aspartate residues in the only hydrophobic FecR sequence (residues 85 to 100) abolished the beta-lactamase activity of high-resistance FecR'-BlaM proteins, indicating that there are no other transmembrane regions in FecR that translocate BlaM into the periplasm independent of the hydrophobic sequence . All FecR'-BlaM proteins with at least 61 FecR residues complemented a fecR mutant such that it could grow on ferric citrate as the sole iron source and induced fecA-lacZ transcription independent of ferric citrate . The low resistance mediated by two FecR'-BlaM proteins in a fecA deletion mutant was increased 20-fold by transformation with a fecA-encoding plasmid . We propose that FecR spans the cytoplasmic membrane once, interacts in the periplasm with its C-terminal region with FecA occupied by ferric citrate, and transmits the information through the cytoplasmic membrane into the cytoplasm, where it converts FecI into an active sigma factor.

Anal Biochem, 1998 May 1, 258(2), 349 - 61
Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping; Opiteck GJ et al.; A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce . The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions . Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities . The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used . The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte . Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector . Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc . The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry . This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E . coli, as well as a number of native proteins comprising a small portion of the E . coli proteome.

Folia Microbiol (Praha), 1998, 43(1), 63 - 7
Effects of temperature and novobiocin on the expression of calf prochymosin gene and on plasmid copy number in recombinant Escherichia coli; Kapralek F et al.; Escherichia coli strain HB101 harboring an expression plasmid bearing calf prochymosin gene under the control of the tac promoter was grown in the presence of IPTG with or without novobiocin at 28 and 40 degrees C, respectively . The differential rates of synthesis of prochymosin inclusions, and, for comparison, of beta-lactamase and beta-galactosidase, as well as plasmid copy number, were determined during the first hours of steady state growth . At 28 degrees C the induced expression of prochymosin gene was almost blocked . Addition of novobiocin did not alleviate this effect . In fact, it strengthened it, and we conclude that both these additive inhibitory effects are a consequence of the decrease in negative superhelical tension of plasmid DNA to an insufficient level . At 40 degrees C the differential rate of prochymosin synthesis was markedly enhanced . Since the copy number of the expression plasmid increased approximately to the same extent, we conclude that an increase in gene dose is the cause . The stimulation of cloned heterologous gene expression at 40 degrees C and inhibition at 28 degrees C may be conveniently used in biotechnological-scale cultivations of some recombinant bacteria.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 581 - 98
Catalytic properties of class A beta-lactamases: efficiency and diversity; Matagne A et al.; beta-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related beta-lactam compounds . These enzymes inactivate the antibiotics by hydrolysing the amide bond of the beta-lactam ring . Class A beta-lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases . The introduction of new beta-lactam compounds, which are meant to be 'beta-lactamase-stable' or beta-lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne beta-lactamase genes or by the acquisition of new genes coding for beta-lactamases with different catalytic properties . On the basis of the X-ray crystallography structures of several class A beta-lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties . However, despite the many available kinetic, structural and mutagenesis data, the factors explaining the diversity of the specificity profiles of class A beta-lactamases and their amazing catalytic efficiency have not been thoroughly elucidated . The detailed understanding of these phenomena constitutes the cornerstone for the design of future generations of antibiotics.

Br J Haematol, 1998 Mar, 100(4), 777 - 83
Positive direct antiglobulin tests and haemolytic anaemia following therapy with beta-lactamase inhibitor containing drugs may be associated with nonimmunologic adsorption of protein onto red blood cells; Garratty G et al.; A high incidence (39%) of positive direct antiglobulin tests (DATs) has been reported in patients taking Unasyn {ampicillin sodium plus sulbactam sodium (a beta-lactamase inhibitor)} . Three of four patients, with positive DATs, receiving Unasyn or Timentin {ticarcillin disodium plus clavulanate potassium (also a beta-lactamase inhibitor)} developed a haemolytic anaemia (HA) associated with a positive DAT, which resolved when drug therapy was stopped . The patients' sera did not react with red blood cells (RBCs) in the presence of Unasyn or Timentin, but when drug-treated RBCs were tested, patients' sera and normal sera reacted equally by indirect antiglobulin test . Following incubation in normal sera, RBCs treated with Unasyn, Timentin, Augmentin (amoxicillin + clavulanate), sulbactam and clavulanate reacted with anti-human globulin and anti-human albumin (an index of non-specific adsorption); RBCs treated with ampicillin and amoxicillin were nonreactive . The beta-lactamase inhibitors sulbactam and clavulanate seem to cause nonimmunologic adsorption of protein onto RBCs in vitro . This may explain the high incidence of positive DATs detected in patients taking Unasyn, which contains sulbactam . It was not possible to prove that there was a direct association between the nonspecific uptake of protein onto drug-treated RBCs in vitro with the positive DATs or the HA.

J Clin Microbiol, 1998 Mar, 36(3), 827 - 9
Practical approach for detection and identification of OXA-10-derived ceftazidime-hydrolyzing extended-spectrum beta-lactamases; Vahaboglu H et al.; A practical approach to detect and identify ceftazidime-hydrolyzing extended-spectrum mutants of OXA-10 beta-lactamase is presented . Large numbers of bacteria were screened by colony hybridization, a 720-bp part of blaOXA was amplified by PCR from the hybridization-positive isolates, and the products were digested by PvuII and HaeIII.

Biochim Biophys Acta, 1998 Jan 15, 1382(1), 38 - 46
Clinical inhibitor-resistant mutants of the beta-lactamase TEM-1 at amino-acid position 69 . Kinetic analysis and molecular modelling; Chaibi EB et al.; The kinetic parameters of three IRT (Inhibitor-Resistant-TEM-derived-) beta-lactamases (IRT-5, IRT-6 and IRT-I69) were determined for substrates and the beta-lactamase inhibitors: clavulanic acid, sulbactam and tazobactam, and compared with those of TEM-1 beta-lactamase . The catalytic behaviour of the beta-lactamases towards substrates and inhibitors was correlated with the properties of the amino acid at position ABL69 . The three IRT beta-lactamases contain at that position a residue Ile, Leu and Val, amino acids whose side-chain are branched . Molecular modelling shows that the methyl groups of Ile-69 (C gamma 2) and Val-69 (C gamma 1) produced steric constraints with the side chain of Asn-170 as well as the main chain nitrogen of Ser-70, a residue contributing to the oxyanion hole . We suggest that hydrophobicity could be the main factor responsible for the kinetic properties of Met69Leu (IRT-5), as no steric effects could be detected by molecular modelling . Hydrophobicity and steric constraints are combined in Met69Ile and Met69Val, IRT-I69 and IRT-6, respectively.

Appl Microbiol Biotechnol, 1998 Jan, 49(1), 77 - 83
Enhanced biosynthesis of clavulanic acid in Streptomyces clavuligerus due to oxidative challenge by redox-cycling agents; Kwon HJ et al.; Streptomyces clavuligerus produces a clinically important beta-lactamase inhibitor, clavulanic acid . When several of the selected redox-cycling agents were treated, an increase in clavulanate production was observed . The stimulatory effect was seen when the reaction was fed with menadione, plumbagin and phenazine methosulfate (PMS), whereas feeding with methyl viologen had a negative effect . PMS exerted the strongest effect, enhancing the accumulation of clavulanic acid by 150% . Induction of superoxide dismutase upon the addition of PMS suggested an involvement of superoxide in the enhancing process . The stimulatory effect of PMS was offset by the addition of butylated hydroxyanisole, further supporting the involvement of the active oxygen . The enhanced production of clavulanic acid correlated well with the increased total activity of clavaminic acid synthase, a key enzyme in its biosynthesis, and the transcription of cas2, its coding gene . The results suggested that active oxygen species could enhance the biosynthesis of secondary metabolites through the transcriptional activation of the biosynthetic gene.

Biochemistry, 1998 Feb 17, 37(7), 1941 - 50
A collapsed intermediate with nonnative packing of hydrophobic residues in the folding of TEM-1 beta-lactamase; Vanhove M et al.; The kinetics of refolding of TEM-1 beta-lactamase from solution in guanidine hydrochloride have been investigated on the manual and stopped-flow mixing time scales . The kinetics of change of far-UV circular dichroism and of intrinsic and ANS fluorescence have been compared with changes in the quenching of fluorescence by acrylamide as a probe of the accessibility of solvent to tryptophan . The binding of ANS points to hydrophobic collapse in the very early stages of folding which take place in the burst phase . This is accompanied by regain of 60-65% of native ellipticity, indicating formation of a significant proportion of secondary structure . Also in the burst phase, the tryptophan residues, which are largely exposed to solvent in the native protein, become less accessible to acrylamide, and the intrinsic fluorescence increases markedly . An early intermediate is thus formed in which tryptophan is more buried than in the native protein . Further intermediates are formed over the next 20 s . Quenching by acrylamide increases during this period, as the transient nonnative state is disrupted and the tryptophan residue(s) become(s) reexposed to solvent . The two slowest phases are determined by the isomerization of incorrect prolyl isomers, but double jump tryptophan fluorescence and acrylamide quenching experiments show little, if any, effect of proline isomerization on the earlier phases . Hydrophobic collapse thus occurs to a folding intermediate in which there is a nonnative element of structure which has to rearrange in the later steps of folding, resulting in a nonhierarchical folding pathway . The C-terminal W290 is suggested as being involved in the nonnative intermediate . beta-Lactamase provides further evidence for the occurrence of nonnative intermediates in protein folding.

J Capillary Electrophor, 1997 May-Jun, 4(3), 123 - 30
Electrophoretic mobility of five polypeptides using nine different capillary chemistries over the pH range of 3.5-6.5; Treat-Clemons LG et al.; The performances of nine commercially available capillary zone electrophoresis (CZE) capillaries were tested and compared . Five model polypeptides, ranging in size from a tetrapeptide (604 D) to beta-lactamase 1 (approximately 29,000 D), were run at 32 degrees C on each of the nine capillaries using 10-mM ionic strength buffers at pH 3.5, 5.0, and 6.5 . These results were then used to evaluate each capillary's performance . Factors used to assess the performance included comparison of observed electrophoretic mobilities (mu) to predicted mu (Overbeek-Wiersema model, O-W model; see appendix); reproducibility and consistency of the electroosmotic (EO) flow marker or internal marker throughout testing; and peak shape, height, and signal-to-noise . Five of the nine capillaries were evaluated further for migration time (MT) reproducibility over 50 injections of a polypeptide . A direct correlation was observed between peak tailing and slower observed mu . Those capillaries that exhibited a greater amount of tailing also had observed mu that were slower than predicted by the O-W model . As expected for any model, other capillaries produced observed mu that were faster than those predicted.

Microbiol Res, 1997 Dec, 152(4), 385 - 94
Release of miniantibodies from E . coli cells into the supernatant at low and high cell densities; Morbe JL et al.; By chemical permeabilization of E . coli cells with detergents and membrane active peptides considerable amounts of the periplasmic anti-PC-miniantibodies can be released . Releases of active miniantibodies of 31%, respectively of 38%, were obtained by exposing low cell density suspensions to nonionic detergents like Triton X-100 and tetraethyleneglycolmonodecylether . At high cell densities releasing levels of 40% and 56% were observed for these detergents . The addition of cationic, membrane active peptides (PMBN, polylysine 115, magainin II and melittin) led to release of up to 20% of active miniantibodies at low cell densities . The excretion of miniantibodies at low cell densities was increased up to 35% by phospholipase A2 . Interestingly, the membrane associating properties of the anti-PC-miniantibodies influenced the permeability of the outer membrane and the excretion of beta-lactamase.

J Mol Biol, 1998 Jan 30, 275(4), 663 - 75
Two conformational states of beta-lactamase bound to GroEL: a biophysical characterization; Gervasoni P et al.; Escherichia coli RTEM beta-lactamase, in which both cysteine residues which form the single disulfide bond have been mutated to alanine residues, can form stable reversible complexes with GroEL under two different sets of conditions . Starting with the GdmCl-denatured enzyme, it is bound to GroEL in a state where no protons are protected against exchange with 2H2O, as determined by electrospray ionization mass spectrometry (ESI-MS) . In contrast, when native protein is destabilized at high temperature and added to GroEL, a conformation is bound with 18 protected protons after two hours of exchange . While the high-temperature complex can form both with the wild-type enzyme (with intact disulfide bond) and the Cys-Ala double mutant, only the latter protein can form a complex starting from GdmCl denatured states . Thus, two different sets of conformations of the same protein can be bound, depending both on the conditions used to form the complex and on the intrinsic stability of the intermediate recognized by GroEL, and we have characterized the properties of both complexes.

Appl Environ Microbiol, 1998 Feb, 64(2), 392 - 8
Optimization of bacteriocin release protein (BRP)-mediated protein release by Escherichia coli: random mutagenesis of the pCloDF13-derived BRP gene to uncouple lethality and quasi-lysis from protein release; van der Wal FJ et al.; Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli periplasm into the culture medium . However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality) . To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques . Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures . A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein beta-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium . These BRP derivatives can be more efficiently used for extracellular production of proteins by E . coli than can the original BRP.

J Antimicrob Chemother, 1997 Dec, 40(6), 823 - 31
Transcontinental importation into the UK of Escherichia coli expressing a plasmid-mediated AmpC-type beta-lactamase exposed during an outbreak of SHV-5 extended-spectrum beta-lactamase in a Leeds hospital; M'Zali FH et al.; Sixteen strains of Escherichia coli with high-level resistance to extended-spectrum cephalosporins and other classes of antibiotic have been isolated at St James' University Hospital, Leeds . They produce up to three separate beta-lactamases: TEM-1, SHV-5 and, in five isolates, a plasmid-mediated AmpC-type enzyme . With the exception of carbapenems, the isolates reported in this study were resistant to all beta-lactam antibiotics including extended-spectrum cephalosporins and the monobactam aztreonam . There was evidence of the spread of a plasmid encoding SHV-5, particularly amongst patients on the liver transplant unit . Sensitivity to beta-lactam antibiotics in five isolates expressing the AmpC-type beta-lactamase was not restored by the beta-lactamase inhibitor clavulanic acid . These bacteria also carried blaSHV-5 on a large plasmid . PCR-amplification of the structural gene and digestion with restriction endonucleases demonstrated that the plasmid-mediated blaAmpC probably identified as BIL-1 using the criteria available . Four of the five patients carrying isolates that carried the plasmid-located blaAmpC gene had recently visited the Indian subcontinent and we presume that they returned carrying these bacteria . Restriction fragment length polymorphism analysis using pulsed field gel electrophoresis (PFGE) suggests that at least four distinct strains existed amongst these five isolates . The two isolates that had very similar PFGE patterns had different plasmid profiles and were isolated from different locations in the hospital and at different times . This study demonstrates the ease with which highly resistant bacteria can be imported into the UK and spread within hospitals.

Science, 1998 Jan 2, 279(5347), 84 - 8
Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter; Zlokarnik G et al.; Gene expression was visualized in single living mammalian cells with beta-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester . Each enzyme molecule changed the fluorescence of many substrate molecules from green to blue by disrupting resonance energy transfer . This wavelength shift was detectable by eye or color film in individual cells containing less than 100 beta-lactamase molecules . The robust change in emission ratio reveals quantitative heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.

FEBS Lett, 1997 Dec 8, 419(1), 18 - 22
N-tail translocation of mature beta-lactamase across the Escherichia coli cytoplasmic membrane; Mitsopoulos C et al.; Mature beta-lactamase was attached to the N-terminus of human glycophorin C, an N-out membrane protein lacking a cleavable signal peptide (an N-tail membrane protein) . When synthesised in Escherichia coli more than 30% of the intact mature beta-lactamase-glycophorin C molecules assembled N-out, C-in into the cytoplasmic membrane . The N-tail translocated beta-lactamase folded into an enzymatically active form, but it was more susceptible to proteolysis than the equivalent portion of beta-lactamase-glycophorin C synthesised with an N-terminal signal peptide . Its translocation was virtually abolished when the N-out domain of glycophorin C was truncated or when the basic residues C-terminally flanking the glycophorin C membrane-spanning segment were replaced with neutral ones.

Infect Dis Clin North Am, 1997 Dec, 11(4), 875 - 87
Extended-spectrum beta-lactamases and other enzymes providing resistance to oxyimino-beta-lactams; Jacoby GA; Bacteria have once again demonstrated their remarkably versatility in meeting the introduction of new classes of beta-lactam antibiotics by modifying available plasmid mediated beta-lactamases to expand their spectrum of action and by incorporating chromosomal beta-lactamase genes onto plasmids that permit their spread to new hosts . Such resistance is more common than presently is appreciated because current NCCLS breakpoints for resistance underestimate its prevalence . A number of risk factors for acquisition of ESBL-producing K . pneumoniae have been defined, but most will be no easier to control than those for infection by MRSA or VRE . More clinical and animal model studies are needed to evaluate options for treatment . Most strains remain susceptible to imipenem and other carbapenems, but carbapenem resistance has appeared either by spread of metallo-beta-lactamase or by production of an AmpC enzyme combined with loss of an outer membrane porin channel . Attack on our adversaries' latest biological weapons is likely to require enhanced versatility on our part as well.

J Antimicrob Chemother, 1997 Nov, 40(5), 717 - 20
Susceptibility of 100 strains of Stenotrophomonas maltophilia to three beta-lactams and five beta-lactam-beta-lactamase inhibitor combinations; Lecso-Bornet M et al.; One hundred clinical strains of Stenotrophomonas maltophilia were investigated for their susceptibility to ticarcillin, piperacillin, ceftazidime and five combinations with beta-lactamase inhibitors (clavulanic acid, tazocillin or sulbactam) at three concentrations; 26% of the strains were susceptible to ticarcillin, 23% to ceftazidime and 12% to piperacillin . Eighty-two percent of the strains were susceptible to ticarcillin/clavulanic acid . In most cases, no synergy was observed with tazobactam or sulbactam . For eight ticarcillin-susceptible strains, antagonism was observed between ticarcillin and clavulanic acid.

Antimicrob Agents Chemother, 1997 Dec, 41(12), 2606 - 11
Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple Toho-1-like beta-lactamase genes; Yagi T et al.; Escherichia coli HKY56, which demonstrated resistance to various beta-lactams except carbapenems, was isolated from the throat swab of an inpatient in 1994 . Conjugal transfer of cephem resistance from HKY56 to E . coli CSH2 was not successful . Three cefotaxime-resistant E . coli clones harboring plasmid pMRE001, pMRE002, or pMRE003, each of which carried a 3.4-, 5.8-, or 6.2-kb EcoRI fragment insert, respectively, were obtained from HKY56 . Although restriction analysis suggested their different origins, these clones showed similar profiles of resistance to various beta-lactams . The sequence of 10 amino acid residues at the N terminus of beta-lactamase purified from E . coli HB101(pMRE001) was identical to that of Toho-1 . This Toho-1-like beta-lactamase-1 (TLB-1) was able to hydrolyze cefoperazone and cefotaxime efficiently, but it failed to hydrolyze cephamycins . A Toho-1-specific DNA probe was hybridized with three distinct EcoRI fragments derived from the chromosomal DNA of strain HKY56, and these fragments corresponded to DNA inserts carried by pMRE001, pMRE002, and pMRE003, respectively . PCR and Southern hybridization analysis suggested that all six cephem-resistant E . coli strains, strains HKY273, HKY285, HKY288, HKY305, HKY316, and HKY335, which were isolated in 1996 at the same hospital where strain HKY56 had been isolated, also possessed multiple Toho-1-like beta-lactamase (TLB) genes, and the hybridization patterns obtained with the Toho-1-specific probe were quite similar among these six isolates . The DNA fingerprinting patterns observed by pulsed-field gel electrophoresis revealed that among the E . coli isolates tested, all isolates except HKY56 possessed a similar genetic background . These findings suggested that E . coli strains that carry chromosomally multiplied TLB genes may have been proliferating and transmitted among patients in the same hospital.

J Biochem (Tokyo), 1997 Oct, 122(4), 696 - 702
Structure and function in Escherichia coli of plasmids containing pyrimidine/purine-biased stretch originated from the 5'-flanking region of the basidiomycete ras gene; Yamazaki T et al.; The Basidiomycete ras gene possesses a pyrimidine-rich stretch (CT-motif) with a short (7 bases) mirror repeat in which its major transcription start point is contained . To analyze the tertiary structure induced by the CT/AG-biased sequence and its effect on gene expression in supercoiled plasmids in Escherichia coli, the DNA fragment containing the ras CT/AG sequence was inserted into the EcoRI site on pBR322 in both orientations and the resulting pBR322 derivatives, named pBR-CT{ras} and pBR-invCT{ras} were introduced into E . coli strains DM800 (deltatopA gyrB225) and JM109 (topA+ gyrA96) . In pBR-CT {ras} the pyrimidine-rich sequence is on the pBR322 tetracycline-resistance gene (tet)coding strand and in pBR-invCT{ras} the complementary purine-rich sequence is on this strand . DNAs of pBR-CT{ras} and pBR-invCT{ras} isolated from DM800 were frequently cleaved with single-strand-specific S1 nuclease within the CT/AG sequence, showing the formation of extended open structure . Compared with those carrying pBR322, DM800 and JM109 carrying pBR-CT {ras} showed much higher levels of tetracycline resistance (Tcr), while both strains carrying pBR-invCT{ras} showed clearly lower levels of Tcr . pBR-CT {ras} and pBR-invCT {ras}, however, conferred reduced activity of beta-lactamase on DM800 and JM109 . pBR-CT {ras} derivatives lacking the counterpart of the mirror repeat did not form the S1-cleavable open structure within the CT/AG sequence and conferred pBR322-like Tcr and beta-lactamase activity . The tertiary structure formed in the CT/AG sequence via the mirror repeat was suggested to affect the expressions of pBR322-tet and -bla genes.

Antimicrob Agents Chemother, 1997 Nov, 41(11), 2547 - 9
Inhibitor-resistant TEM (IRT) beta-lactamases with different substitutions at position 244; Bret L et al.; A novel inhibitor-resistant TEM (IRT) beta-lactamase was detected in an Escherichia coli isolate resistant to amoxicillin-clavulanate and susceptible to cephalothin . The substrate and inhibitor profiles of this beta-lactamase were similar to those of IRT-1 and IRT-2 . The novel IRT's bla gene was sequenced, and the deduced amino acid sequence showed the amino acid replacement Arg for His-244 of the TEM-1 sequence . Substitutions for Arg-244 have been reported in three TEM-1 mutants: IRT-1 (which corresponds to TEM-31) (Cys), IRT-2/TEM-30 (Ser), and TEM-41 (Thr) . We designated this novel beta-lactamase, which corresponds to TEM-51, IRT-15.

Bioorg Med Chem, 1997 Sep, 5(9), 1783 - 8
Structure-activity studies of the inhibition of serine beta-lactamases by phosphonate monoesters; Li N et al.; A new series of phosphonyl derivatives has been prepared and tested for inhibition of serine (class A and C) beta-lactamases . Variations of the leaving group in a series of methyl phosphonates showed that leaving groups better than the previously employed p-nitrophenoxide could give more effective inhibitors . Inclusion of a negative charge in the leaving group did not, per se, lead to better inhibitors . Aryl phosphonates appeared more effective than those with electronically comparable but smaller leaving groups . The combination of a good leaving group, 2,4-dinitrophenoxide, with an amido side-chain, phenylmethylsulfonamido--the latter rather than phenylacetamido in order to increase the stability of the compound with respect to intramolecular nucleophilic catalysis of hydrolysis by the amide group--did not yield overall a better inhibitor than previously employed p-nitrophenyl phosphonates . These results give the first indication of specific interactions between a beta-lactamase and the leaving group of a phosphonate inhibitor . Only one enantiomer of a chiral thiophosphonate, presumably the Rp isomer, was an effective inhibitor . Addition of either a D- or a L-methyl group to the methylene group of a p-nitrophenyl amidomethylphosphonate did not enhance the inhibitory ability of the phosphonate . Class A beta-lactamases remain refractory to phosphonates.

J Bacteriol, 1997 Nov, 179(21), 6692 - 8
Topological analysis of the membrane-bound glucosyltransferase, MdoH, required for osmoregulated periplasmic glucan synthesis in Escherichia coli; Debarbieux L et al.; The MdoH protein is essential for synthesis of the osmoregulated periplasmic glucans, known as membrane-derived oligosaccharides (MDOs), in Escherichia coli . Mutants lacking MdoH are deficient in a glucosyltransferase activity assayed in vitro . The MdoH protein is the product of the second gene of an operon, and it has been shown to span the cytoplasmic membrane . The MdoH protein comprises 847 amino acids and is poorly expressed as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . We have experimentally measured the topological organization of MdoH within the membrane by construction of fusions to beta-lactamase as a reporter . Analysis of 51 different MdoH-beta-lactamase fusions suggested that the MdoH protein crosses the cytoplasmic membrane eight times, with the N and C termini in the cytoplasm . Moreover, a 310-amino-acid domain is present in the cytoplasm between the second and third transmembrane segments . It was deduced from the measurement of the MDO biosynthetic activity of truncated or fused MdoH proteins that almost all the C-terminal residues are necessary for this activity . The model of the MdoH protein in the membrane suggests that this protein could be directly involved in the translocation of nascent polyglucose chains to the periplasmic space.

Biochem Mol Biol Int, 1997 Oct, 43(3), 557 - 62
Characterization of a beta-lactamase from Mycobacterium smegmatis SN2; Basu D et al.; Beta-lactamases have been reported to be largely responsible for beta-lactam resistance in Mycobacteria . We report the characterization of a cell-associated beta-lactamase from Mycobacterium smegmatis . The enzyme hydrolyzed the "beta-lactamase-stable" oximinocephalosporins . Nitrocefin was the best substrate . 6-Beta-iodopenicillanate, clavulanate and sulbactam were effective inhibitors, whereas the Ki value for aztreonam was high . From its substrate and inhibitor profile, the enzyme appeared to be a cephalosporinase of group 2e.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11857 - 62
Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells; Kobayashi T et al.; DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif . The Cys-30-Cys-33 disulfide is donated to a pair of cysteines on the target proteins . Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo . DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning . It is not known, however, what might provide the overall oxidizing power to the DsbA-DsbB disulfide bond formation system . We now report that E . coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation . Disulfide bond formation of beta-lactamase was impaired under these conditions . Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA . This is followed by reduction of the bulk of DsbA molecules . These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB . It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

J Bacteriol, 1997 Oct, 179(19), 6035 - 40
The bla gene of the cephamycin cluster of Streptomyces clavuligerus encodes a class A beta-lactamase of low enzymatic activity; Perez-Llarena F et al.; A gene (bla) encoding a beta-lactamase is present in the cephamycin gene cluster of Streptomyces clavuligerus, the strain producing clavulanic acid and a beta-lactamase inhibitory protein . The bla gene is located 5.1 kb downstream from and in the opposite orientation to cefE, encoding the deacetoxycephalosporin C synthase . The bla gene encodes a 332-residue protein (Mr, 35,218), similar to other class A beta-lactamases produced by actinomycetes . Modification (to SDG) of the SDN conserved motif of class A beta-lactamases as well as of amino acids in otherwise conserved regions in the molecule may explain the low penicillinase and cephalosporinase activities of the protein . The beta-lactamase has been purified to homogeneity and found to bind {3H}benzylpenicillin, a result reflecting a rate-limiting deacylation step . Nucleotide sequences homologous to bla were found in all tested cephamycin producers, but several other Streptomyces species which produce a beta-lactamase do not contain genes for beta-lactam antibiotic biosynthesis.

Antimicrob Agents Chemother, 1997 Sep, 41(9), 1940 - 3
Construction and characterization of an OHIO-1 beta-lactamase bearing Met69Ile and Gly238Ser mutations; Bonomo RA et al.; Amino acid changes that influence activity and resistance to beta-lactams and beta-lactamase inhibitors were explored by constructing the Gly238Ser and Met69Ile-Gly238Ser mutants of the OHIO-1 beta-lactamase, a class A enzyme of the SHV family . The Km values of cefotaxime and ceftazidime for OHIO-1 and Met69Ile beta-lactamases were > or = 500 microM . The Km of cefotaxime for the Gly238Ser beta-lactamase was 26 microM, and that of ceftazidime was 105 microM . The Km of cefotaxime for the Met69Ile-Gly238Ser beta-lactamase was 292 microM, and that of ceftazidime was 392 microM . For the beta-lactamase inhibitors clavulanate and sulbactam, the apparent Ki values for the Met69Ile-Gly238Ser enzyme were 0.03 and 0.15 microM, respectively . Relative Vmax values indicate that the Met69Ile-Gly238Ser mutant of the OHIO-1 beta-lactamase possesses cephalosporinase activity similar to that of the Gly238Ser mutant but diminished penicillinase activity . In an Escherichia coli DH5alpha strain that possesses a Met69Ile beta-lactamase of the OHIO-1 family, the added Gly238Ser mutation resulted in a phenotype with qualities that confer resistance to expanded-spectrum cephalosporins and, to a lesser extent, beta-lactamase inhibitors.

Antimicrob Agents Chemother, 1997 Aug, 41(8), 1636 - 40
Assessment of biliary excretion of piperacillin-tazobactam in humans; Westphal JF et al.; Piperacillin-tazobactam concentrations in serum and bile were measured intraoperatively in 10 patients undergoing cholecystectomy (group 1) and 5 cholecystectomized patients provided with external bile duct drainage (group 2) . Each patient received a single intravenous dose of piperacillin at 4 g plus tazobactam at 0.5 g over 30 min . Drug concentrations in both serum and bile were measured by high-performance liquid chromatography . In group 1 patients, serum and bile specimens and gallbladder wall fragments were collected at mean times of 70 and 83 min postinfusion, respectively . The mean concentrations of piperacillin and tazobactam were, respectively, 69.1 +/- 41.5 (standard deviation) and 9.9 +/- 5.1 microg/ml in serum, 630.4 microg/ml (range, 24.8 to 1,194 microg/ml) and 11.8 microg/ml (range, 3.6 to 22 microg/ml) in choledochal bile, 342.3 microg/ml (range, 1.1 to 1,149 microg/ml) and 7.7 microg/ml, (range, 0.2 to 23.1 microg/ml) in gallbladder bile, and 49.3 microg/g (range, 9.7 to 223 microg/g) and 2.9 microg/g (range, 0.1 to 5.9 microg/g) in the gallbladder wall . In group 2 patients, the amounts of drugs recovered in bile drainage obtained over 12 h were 28.4 +/- 18.0 and 1.0 +/- 0.5 mg for piperacillin and tazobactam, respectively . Peak piperacillin and tazobactam concentrations in bile reached 358 +/- 242 and 10.8 +/- 4.2 microg/ml, respectively . Comparison of drug levels in serum and bile suggests an underlying active secretion process for piperacillin elimination into the bile, unlike that of tazobactam . From a therapeutic viewpoint, given the concentrations of tazobactam recorded in bile fluid and tissue, the addition of this beta-lactamase inhibitor to piperacillin therapy might be of interest in the management of biliary tract infections, mostly in patients at risk of mixed aerobic-anaerobic infections due to beta-lactamase-producing organisms.

Appl Environ Microbiol, 1997 Aug, 63(8), 2977 - 82
Amy as a reporter gene for promoter activity in Nocardia lactamdurans: comparison of promoters of the cephamycin cluster; Chary VK et al.; Promoter probe vectors containing the pA origin of replication and the Streptomyces griseus promoterless amy gene (encoding alpha-amylase) as reporter have been constructed to study transcription initiation regions in Nocardia lactamdurans . In some of the promoter probe vectors the phage fd terminator has been introduced to avoid readthrough expression from upstream sequences . By using these vectors, four different transcription initiation regions of the cephamycin gene cluster have been studied in N . lactamdurans . The bla gene encoding a beta-lactamase has a relatively strong promoter . Two other separate promoters corresponding to the lat and cefD genes (encoding, respectively, lysine-6-aminotransferase and isopenicillin N-epimerase) showed weak transcription initiation ability . These two promoters are arranged in a bidirectional transcription initiation region located in the center of the cephamycin gene cluster . The cmcH gene (encoding 3-hydroxymethylcephem carbamoyltransferase) upstream region did not contain a functional promoter, suggesting that cmcH is transcribed as a part of a polycistronic mRNA . The native amy promoter is used very efficiently in N . lactamdurans, resulting in secretion of high levels of extracellular alpha-amylase.

FEBS Lett, 1997 Jul 21, 412(1), 39 - 42
ATR-FTIR spectroscopic investigation of E . coli transconjugants beta-lactams-resistance phenotype; Bouhedja W et al.; Hyphenation of attenuated total reflection Fourier transform infrared spectroscopy and cluster analysis has been used to characterise a susceptible Escherichia coli K12 strain and the transconjugants TEM-1, TEM-2, TEM-3, SHV-2, SHV-3, SHV-4 . A good discrimination of the susceptible strain from the transconjugants was obtained . Although a limited success was achieved in the differentiation of SHV and TEM phenotypes in general, results obtained with TEM-2 and SHV-3 were convincing . Spectral differences observed are ascribed to the global effects of the conjugation process, particularly their repercussions in the nucleic acids and carbohydrate absorbing regions, rather than to beta-lactamase point-mutations.

FEMS Microbiol Lett, 1997 Jul 15, 152(2), 275 - 8
OHIO-1 beta-lactamase mutants: Asp179Gly mutation confers resistance to ceftazidime; Bonomo RA et al.; The Asp179Gly mutant of the OHIO-1 beta-lactamase, an SHV enzyme, was constructed to investigate the effect of disruption of the omega loop on beta-lactamase activity in this class A enzyme . In Escherichia coli DH5 alpha the strain possessing the Asp179Gly mutation of the OHIO-1 beta-lactamase demonstrated increased susceptibility to all beta-lactams except ceftazidime and ceftriaxone . The minimum inhibitory concentrations for ceftazidime and ceftriaxone increased from 0.25 and 0.015 microgram/ml to 4.0 and .25 micrograms/ ml respectively . For ceftazidime, a substrate not hydrolyzed by the wild-type enzyme (K(m) > or = 500 microM), the K(m) of the Asp179Gly mutant beta-lactamase was measured to be 7 microM and the Vmax was 0.13 microM/min . The minimum inhibitory concentrations, K(m), and Vmax for all other beta-lactams decreased . Our analysis of this OHIO-1 beta-lactamase mutant suggests that disruption of the salt bridge in the omega loop by substitution of a glycine at position 179 markedly decreases the catalytic efficiency of the enzyme.

J Biotechnol, 1997 Jun 13, 55(2), 69 - 83
Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI::SPA; Rhee JI et al.; Plasmid-free and plasmid-harbouring E . coli JM109 strains were investigated in shaken flasks, stirred tanks in batch and continuous operation . The shaken flask cultivations were performed in M9 minimal medium and in media with various protein supplements . The host hardly grows on M9 minimal medium as opposed to the plasmid-harbouring cells, which grow well on this medium . All of the investigated cells propagate well on protein-containing media . The influence of the combinations of repressor plasmid pRK248cI, the protection plasmid EcoR4 and the production plasmid pMTC48 were determined on the initial specific growth rate of the E . coli JM109 without gene expression, on the yield coefficient of cell growth, acetate concentration and acetate yield coefficient in the yeast extract-containing (HM) medium . The influence of various media on the induction of the gene expression were evaluated . In cultivation media with protein supplement, the growth rate and yield coefficient increased . The variation of the volumetric and specific beta-lactamase activities with the cultivation time were determined in a stirred tank reactor in HM medium . With increasing dilution rate the process performance decreased . Simple relationships exist between the substrate uptake rate and the specific growth rate of the continuous cultivated cells in M9 and HM media . The influence of the dilution rate on the cell mass concentration, colony forming units, acetate formation, yield coefficients of growth and acetate formation, substrate uptake rate, CO2 production rate, ammonium formation rate and beta-lactamase activity in M9 and HM media were determined as well . Carbon balances of the batch and continuous cultivations indicated high carbon recoveries . On account of the higher growth rate of plasmid-harbouring cells than than of the plasmid-free cells, the behaviour of the investigated plasmid-free and plasmid-harbouring E . coli JM109 cells deviates from the published properties of other plasmid-free and plasmid-harbouring E . coli cells.

Vaccine, 1997 Jun, 15(9), 968 - 75
Expression of a synthetic pertussis toxin operon in Escherichia coli; Pozza TD et al.; Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated of paroxysmal coughing . Pertussis toxin (PT) is a major virulence factor of B . pertussis and is a typical A/B bacterial toxin consisting of five subunits S1-S5 in a ratio of 1:1:1:2:1 . The PT subunit genes are organized into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production . We have expressed the five PT subunits individually in E . coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E . coli beta-lactamase leader sequence . We have developed a stepwise cloning method to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E . coli . Western blot analysis indicated that in E . coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed . Periplasmic extracts contained soluble S1 and S3; however, the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form . This work should allow an investigation of the potential of E . coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use.

Mol Gen Genet, 1997 Jun, 255(2), 187 - 93
The two beta-lactamase genes of Streptomyces cacaoi, blaL and blaU, are under the control of the same regulatory system; Magdalena J et al.; The production of beta-lactamase in Streptomyces cacaoi, which contains two beta-lactamase-encoding genes, blaL and blaU, is inducible by beta-lactam compounds . The two genes have been cloned independently in S . lividans TK24, a beta-lactamase-negative species . The blaU clone did not respond to the presence of beta-lactams, whereas the blaL clone appeared to be inducible in S . lividans . The latter clone contains two open reading frames, blaA and blaB, located just upstream of but transcribed divergently from blaL, which were shown to be required for the production as well as the induction of BlaL . The deduced BlaA protein belongs to the LysR family of transcription regulators . In order to examine the role of BlaA in regulation, we here report on over-expression of a GST-BlaA fusion protein in Escherichia coli and its use for antibody preparation . The GST-BlaA fusion protein was partially purified and bandshift assays showed that it bound the 197-bp blaL-blaA intergenic region . The BlaA DNA binding-site was further restricted to a 30-bp sequence containing a T-N11-A motif, a characteristic of LysR-type promoters . Another T-N11-A motif upstream of the blaU gene was also shown to bind BlaA . The affinities of these two T-N11-A motifs in BlaA binding were comparable . A plasmid bearing the blaU structural gene and the blaA-blaB regulatory region was constructed and shown to confer on an S . lividans host the capacity to produce inducible beta-lactamase . It can thus be concluded that the S . cacaoi blaL and blaU genes are controlled by the same regulatory system.

J Antimicrob Chemother, 1997 Jun, 39(6), 833 - 5
Evaluation of the resistance induction in enteric flora in children caused by oral ampicillin plus sulbactam; Soylu A et al.; To evaluate the effect on bacterial resistance of a beta-lactamase inhibitor, resistance patterns of predominant bacteria in enteric flora were evaluated before and after a 7-day course of oral ampicillin (100 mg/kg/days, qid, in 16 patients) and ampicillin-sulbactam (50 mg/kg/day of ampicillin, bd, in 32 patients) therapy . Ampicillin and ampicillin-sulbactam MICs for Escherichia coli, the predominant bacteria in all cases, and resistance rates of E . coli species to both antibiotics were 51.20 +/- 13.80 mg/L, 87.5% and 4.84 +/- 2.11 mg/L, 21% before the treatment respectively . Post-treatment MICs and resistance rates were 106.51 +/- 14.05 mg/L, 100% and 15.89 +/- 5.76 mg/L, 37.5% respectively, indicating a significant increase in MICs of both antibiotics (P < 0.05), being more prominent in the case of ampicillin-sulbactam (about four-fold) . We concluded that oral ampicillin-sulbactam could also decrease the susceptibility of the enteric flora to ampicillin.

J Mol Biol, 1997 May 9, 268(3), 607 - 18
Selectively-infective phage (SIP): a mechanistic dissection of a novel in vivo selection for protein-ligand interactions; Krebber C et al.; Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs . It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an "adapter" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle . Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle . Phage propagation is thus strictly dependent on the protein-ligand interaction . We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles . Any phages lacking the first N-terminal domain were not infective at all . In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium . An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments . Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages . Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration . Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event . Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.

Anal Biochem, 1997 May 1, 247(2), 389 - 93
Enzymatic assay of beta-lactamase using circular dichroism spectropolarimetry; Long DM; A method for measuring the rates of enzymatic hydrolysis of beta-lactam antibiotics based on circular dichroism spectropolarimetry is described . Unhydrolyzed beta-lactam antibiotics have high molar ellipticities, but the hydrolyzed compounds are circular dichroism (CD) inactive in the case of penams or have significantly different CD spectra in the case of cephems . By measuring CD at constant wavelength as a function of time for reaction mixtures containing beta-lactamase and beta-lactam antibiotics, rates of hydrolysis and steady-state enzyme kinetic constants can be derived . The method was applied to measurement of a wide range of enzymatic reaction constants for wild-type and four mutant RTEM-1 beta-lactamases . Compared to the commonly employed assay based on ultraviolet spectroscopy, the new method offers several advantages . These include the ability to measure larger enzymatic Michaelis-Menten constants, less interference from high concentrations of beta-lactamase, higher sensitivity, and potentially less interference from other uv-absorbing components of complex reaction mixtures.

Anal Biochem, 1997 May 1, 247(2), 203 - 9
The development of beta-lactamase as a highly versatile genetic reporter for eukaryotic cells; Moore JT et al.; We describe in this report that TEM-1 beta-lactamase has several desirable characteristics as a genetic reporter . First, it has no endogenous counterpart in eukaryotic cells and therefore provides a background-free measure of gene expression . Second, because of the uniqueness of the substrate cleavage reaction, a wide variety of substrates which are efficiently cleaved can be synthesized for beta-lactamase . Third, since the assays involve no more than addition of substrate to media, it is possible to continuously monitor a culture without destruction of the cells . Fourth, the enzyme is extremely versatile in that it can be fused to other proteins and retain activity . To demonstrate the versatility of beta-lactamase, we created three forms of the enzyme including secreted, intracellular, and membrane-bound forms of the enzyme, each form having distinct advantages as a reporter system . We also showed that levels of secreted beta-lactamase were proportional to both the levels of transfected DNA, beta-lactamase mRNA, as well as activity of the chloramphenicol acetyl transferase gene controlled by the same promoter, validating the reliability of this reporter . beta-Lactamase thus represents a novel and highly versatile genetic reporter.

Nippon Rinsho, 1997 May, 55(5), 1225 - 30
{Mechanisms of bacterial resistance to beta-lactams by beta-lactamases}; Sawai T; The beta-lactamases are bacterial enzymes that inactivate beta-lactam antibiotics by catalyzing the hydrolysis of the amide group of the beta-lactam ring . They are the source of much bacterial resistance to those antibiotics . Structural studies on the enzymes, i.e., penicillinases and cephalosporinases, are now well-advanced and the up-to-date knowledge of this research field were explained together with the proposed mechanism of beta-lactam hydrolysis by a cephalosporinase . After the introduction of the beta-lactamase-stable beta-lactams such as oxyimino cephalosporins, R plasmid-mediated penicillinases and a chromosomal cephalosporinase have been found to be adapted to the new drugs by mutation . The characteristics of the mutant beta-lactamases and their importance in the bacterial resistance to the new beta-lactams were explained.

Antimicrob Agents Chemother, 1997 May, 41(5), 1182 - 5
Cloning and sequence analysis of a class A beta-lactamase from Mycobacterium tuberculosis H37Ra; Hackbarth CJ et al.; A cosmid library from Mycobacterium tuberculosis H37Ra was introduced into Mycobacterium smegmatis, and eight recombinant clones with increased resistance to cefoxitin were identified . Isoelectric focusing detected an M . tuberculosis-derived beta-lactamase in one of these recombinant clones . A sequence analysis identified it as a class A beta-lactamase whose expression correlated with the increased resistance phenotype.

Nucleic Acids Res, 1997 May 1, 25(9), 1866 - 7
Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein; Hallet B et al.; A new insertion method for probing protein functional organization was developed . The method relies on the random insertion of transposon Tn 4430 and subsequent in vitro deletion of the bulk of the transposon after which a 15 bp insertion remains within the target gene . This results in pentapeptide insertions randomly distributed in the target protein . Characterization of 23 pentapeptide insertions in TEM-1beta-lactamase demonstrated the utility of the method . The phenotypes associated with the mutated beta-lactamase proteins equated both with the sorts of local peptide structures in which the pentapeptide insertions occurred and their position in the three-dimensional structure of the enzyme.

J Biol Chem, 1997 Apr 25, 272(17), 11103 - 8
Membrane topology of the ATP-binding cassette transporter associated with antigen presentation (Tap1) expressed in Escherichia coli; Gileadi U et al.; The ATP-binding cassette transporters associated with antigen presentation (Tap1 and Tap2) mediate the transport of peptide fragments across the endoplasmic reticulum membrane of mammalian cells . Tap1 and Tap2 are closely related to one another and are believed to function as a heterodimer . Each protein possesses a hydrophobic domain predicted to span the membrane multiple times and a highly conserved nucleotide-binding domain . We have assessed the transmembrane topology of Tap1 by expressing a series of fusions to a reporter protein, the mature form of beta-lactamase in Escherichia coli . From these data a topological model can be derived in which Tap1 spans the membrane eight times, with several large loops exposed in the lumen of the endoplasmic reticulum and with both the N and C termini (including the nucelotide-binding domain) residing in the cytoplasm.

FEMS Microbiol Lett, 1997 Apr 1, 149(1), 11 - 5
Purification and properties of the Mycobacterium smegmatis mc(2)155 beta-lactamase; Quinting B et al.; The beta-lactamase of Mycobacterium smegmatis mc(2)155 has been purified to protein homogeneity . Its N-terminal sequence and catalytic properties are similar to those of the beta-lactamase produced by Mycobacterium fortuitum D316 and establish this new enzyme as a member of molecular class A.

Antimicrob Agents Chemother, 1997 Apr, 41(4), 838 - 40
Emergence of an inhibitor-resistant beta-lactamase (SHV-10) derived from an SHV-5 variant; Prinarakis EE et al.; An inhibitor-resistant beta-lactamase (SHV-10), derived from an SHV-5 variant (SHV-9), was found in an Escherichia coli clinical isolate . In SHV-10, Ser-130 was replaced by Gly . The enzyme partially retained its ability to hydrolyze penicillins, but its activity against cephalosporins was drastically reduced . A Ser-130-->Gly mutant of the prototype SHV-5, obtained by site-directed mutagenesis, had properties similar to those of SHV-10.

Clin Infect Dis, 1997 Mar, 24(3), 498 - 505
Sulbactam/cefoperazone versus cefotaxime for the treatment of moderate-to-severe bacterial infections: results of a randomized, controlled clinical trial; Li JT et al.; We conducted a randomized, open-label, controlled, multicenter study to compare sulbactam/cefoperazone with cefotaxime in terms of efficacy and safety for the treatment of hospitalized patients with moderate-to-severe bacterial infections . More than two-thirds of the pathogens recovered from these patients produced beta-lactamase . Two hundred-seven (88.1%) of the 235 patients enrolled completed the study and were included in the efficacy and safety evaluations . One hundred-three patients received sulbactam/cefoperazone (2-4 g/d) administered in evenly divided doses every 12 hours by a 30-minute intravenous drip; 104 patients received cefotaxime (6-12 g/d) administered in evenly divided doses every 6 or 8 hours by a 30-minute intravenous drip . The overall efficacy rates (i.e., cure or markedly improved) were 95% for the sulbactam/cefoperazone group and 90% for the cefotaxime group (P = .186), whereas the bacterial eradication rates were 85% for the sulbactam/cefoperazone group and 81% for the cefotaxime group (P = .467) . Both drug regimens were well tolerated . Sulbactam/cefoperazone is effective and safe for the treatment of moderate-to-severe bacterial infections caused mainly by beta-lactamase-producing organisms.

J Bacteriol, 1997 Mar, 179(6), 2053 - 9
A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds; Perez-Llarena FJ et al.; A regulatory gene (ccaR), located within the cephamycin gene cluster of Streptomyces clavuligerus, is linked to a gene (blp) encoding a protein similar to a beta-lactamase-inhibitory protein . Expression of ccaR is required for cephamycin and clavulanic acid biosynthesis in S . clavuligerus . The ccaR-encoded protein resembles the ActII-ORF4, RedD, AfsR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs . Disruption of ccaR by targeted double recombination resulted in the loss of the ability to synthesize cephamycin and clavulanic acid . Complementation of the disrupted mutant with ccaR restored production of both secondary metabolites . ccaR was expressed as a monocistronic transcript at 24 and 48 h in S . clavuligerus cultures (preceding the phase of antibiotic accumulation), but no transcript hybridization signals were observed at 72 or 96 h . This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis . Amplification of ccaR in S . clavuligerus resulted in a two- to threefold increase in the production of cephamycin and clavulanic acid.

Antimicrob Agents Chemother, 1997 Mar, 41(3), 715 - 6
Novel extended-spectrum TEM-type beta-lactamase from an Escherichia coli isolate resistant to ceftazidime and susceptible to cephalothin; Chanal-Claris C et al.; A novel extended-spectrum TEM-type beta-lactamase was detected in an Escherichia coli isolate which was resistant to ceftazidime and susceptible to cephalothin . The corresponding bla gene was sequenced . The deduced amino acid sequence showed the following three amino acid replacements with respect to the TEM-2 sequence: Glu-->Lys-104, Arg-->Ser-164, and Glu-->Lys-240 . Since it confers a ceftazidimase-type resistance phenotype, we propose for this novel enzyme the designation CAZ-9, corresponding to TEM-46 in the sequential numbering scheme of TEM beta-lactamases.

Antimicrob Agents Chemother, 1997 Feb, 41(2), 374 - 8
Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type beta-lactamases; Canica MM et al.; IRT-14 (TEM-45) is a new mutant TEM-type beta-lactamase that was isolated from clinical Escherichia coli P37 and that confers resistance to broad-spectrum penicillins with reduced sensitivity to beta-lactamase inhibitors . The MICs of amoxicillin alone and of amoxicillin combined with 2 micrograms of clavulanic acid or 2 micrograms of tazobactam per ml were 4,096, 2,048, and 1,024 micrograms/ml, respectively . The strain was susceptible to cephalosporins, aztreonam, moxalactam, and imipenem . The enzyme was purified to homogeneity, and values of the kinetic parameters Kcat, Km, and Kcat/Km were determined for different substrates . This enzyme, with a pI of 5.2, was found to have reduced affinity for broad-spectrum penicillins and cephalosporins . The values of 50% inhibitory concentrations of clavulanic acid, sulbactam, tazobactam, and brobactam are correlated with the higher KmS for substrates . The resistance of E . coli P37 to mechanism-based inactivators results from a higher level of production of the TEM-derived enzyme due to the G-to-T substitution at position 162 (G-162-->T) in the promoter region of blaTEM and from the structural modifications resulting from the Met-69-->Leu and Arg-275-->Gln substitutions that characterize IRT-14 beta-lactamase.

FEBS Lett, 1997 Jan 20, 401(2-3), 138 - 42
Folding intermediates of beta-lactamase recognized by GroEL; Gervasoni P et al.; beta-Lactamase, from which the disulfide bond was removed by two Cys-->Ala mutations, forms stable complexes with GroEL only during the first 30 s of folding, while wild-type beta-lactamase forms no stable complex under these conditions . The 3-phasic kinetics of folding are very similar between wild-type and mutant . After 4 s, Trp-210 is already juxtaposed to the disulfide bond, but proline cis-trans isomerization has not yet taken place and almost no enzymatic activity is observed . This shows that GroEL is unable to bind late folding intermediates and also discriminates between the degree of unfolding possible in wild-type disulfide-containing beta-lactamase and the Cys-Ala mutant.

Biochem J, 1997 Jan 15, 321 ( Pt 2), 413 - 7
Kinetic and thermodynamic consequences of the removal of the Cys-77-Cys-123 disulphide bond for the folding of TEM-1 beta-lactamase; Vanhove M et al.; Class A beta-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123 . To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 beta-lactamase, this bond was removed by introducing a Cys-77-->Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation . The unfolding and refolding rates were monitored using tryptophan fluorescence . At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses . The characteristics of the two phases were not significantly affected by the mutation . Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions . Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N<-->H<--U) . Both the folded mutant protein (N) and, to a lesser extent the thermodynamically stable intermediate, H . were destabilized relative to the fully unfolded state, U . Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization . Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.

FEBS Lett, 1997 Jan 3, 400(2), 155 - 7
Site-specific introduction of an electroactive label into a non-electroactive enzyme (beta-lactamase I); Di Gleria K et al.; A cysteine residue was introduced close to the active site of beta-lactamase I by site-directed mutagenesis to replace tyrosine-105 and was subsequently modified with an electroactive SH-specific reagent, N-(2-ferrocene-ethyl)maleimide . The resulting modified enzyme became electroactive, showing good quasireversible electrochemistry which was characteristic of the attached ferrocene moiety while retaining its specific enzymatic activity . In the presence of a suicide substrate, 6beta-iodopenicillanic acid, the redox potential shifted +20 mV suggesting that the label was sensitive to changes in the active site of the enzyme.

Pathol Biol (Paris), 1997 Jan, 45(1), 77 - 81
{Enzyme determination in vitro of inhibitor activity of beta-lactamase from tazobactam . Comparison with high performance liquid chromatography}; Sotto A et al.; We developed an enzymatic method using nitrocefin to assay tazobactam in vitro . Tazobactam was incubated with TEM-1 beta-lactamase . Then, residual beta-lactamase activity was assayed by adding nitrocefin . This activity corresponded indirectly to the initial concentration of tazobactam . Within-assay, between-assay and accuracy coefficients of variation were below 15% . The correlation coefficients between enzymatic method and high performance liquid chromatography (the reference method)was 0.98 . The enzymatic method is rapid, easy to perform and should be applied to daily clinical practice.

J Mol Evol, 1997 Jan, 44(1), 57 - 65
Molecular diversity and evolution of blaTEM genes encoding beta-lactamases resistant to clavulanic acid in clinical E . coli; Canica MM et al.; The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing . The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and beta-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region . These mutations were found to lie on two different gene sequences, described here as "TEM-1B like" and "TEM-2 like" restriction linkage groups . Further analysis, of nucleotide sequences of promoter and coding regions of the beta-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework . These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of beta-lactam-beta-lactamase inhibitor combinations.

Microbiol Res, 1996 Dec, 151(4), 337 - 42
Influence of phospholipid composition on excretion of beta-lactamase from a stringent/relaxed Escherichia coli K 12 strain pair; Gitter B et al.; In comparison to stringent (relA+) controlled Escherichia coli cells, relaxed (relA) controlled E . coli cells excreted more recombinant beta-lactamase into the culture medium . The analysis of the composition of phospholipid fractions of the cells yielded increased levels of phosphatidylserine in relaxed controlled cells . We added various phospholipid vesicles to growing cells in order to influence the excretion rate via their incorporation in the membranes . The addition of vesicles to phosphatidic acid, phosphatidylglycerol and phosphatidylserine reduced the excretion of beta-lactamase whereas vesicles of phosphatidylethanolamine and phosphatidylcholine decreased or increased the excretion of beta-lactamase in dependence on the individual fatty acid residues of the added phospholipids . The lower the degree of saturation of the added phospholipids the more permeable was the cell envelope for beta-lactamase.

Protein Eng, 1996 Dec, 9(12), 1103 - 19
Detection of non-topological motifs in protein structures; Alesker V et al.; We present an efficient technique for the comparison of protein structures . The algorithm uses a vector representation of the secondary structure elements and searches for spatial configurations of secondary structure elements in proteins . In such recurring protein folds, the order of the secondary structure elements in the protein chains is disregarded . The method is based on the geometric hashing paradigm and implements approaches originating in computer vision . It represents and matches the secondary structure element vectors in a 3-D translation and rotation invariant manner . The matching of a pair of proteins takes on average under 3 s on a Silicon Graphics Indigo2 workstation, allowing extensive all-against-all comparisons of the data set of non-redundant protein structures . Here we have carried out such a comparison for a data set of over 500 protein molecules . The detection of recurring topological and non-topological, secondary structure element order-independent protein folds may provide further insight into evolution . Moreover, as these recurring folding units are likely to be conformationally favourable, the availability of a data set of such topological motifs can serve as a rich input for threading routines . Below, we describe this rapid technique and the results it has obtained . While some of the obtained matches conserve the order of the secondary structure elements, others are entirely order independent . As an example, we focus on the results obtained for Che Y, a signal transduction protein, and on the profilin-beta-actin complex . The Che Y molecule is composed of a five-stranded, parallel beta-sheet flanked by five helices . Here we show its similarity with the Escherichia coli elongation factor, with L-arabinose binding protein, with haloalkane dehalogenase and with adenylate kinase . The profilin-beta-actin contains an antiparallel beta-pleated sheet with alpha-helical termini . Its similarities to lipase, fructose disphosphatase and beta-lactamase are displayed.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 113 - 6
Construction of pRES18 and pRES19, Streptomyces-Escherichia coli shuttle vectors carrying multiple cloning sites; Ishikawa J et al.; We developed two Streptomyces-Escherichia coli shuttle vectors . The plasmid pRES102, consisting of the essential region of pRES1 and the thiostrepton resistance gene (tsr) fragment of pIJ702, was combined with the E . coli plasmid vector pUC18 or pUC19 . The resulting shuttle vectors, designated pRES18 and pRES19, respectively, have relatively compact size (6.25 kb), low copy number, multiple cloning sites reciprocally arranged in opposite directions, and selection markers for both Streptomyces (tsr) and E . coli (beta-lactamase (bla) and beta-galactosidase (lacZ) . These shuttle vectors are capable of carrying DNA fragments as long as 10 kb, of being maintained in S . griseus, S . lavendulae and S . lividans, and are compatible with pIJ702.

Conn Med, 1996 Nov, 60(11), 643 - 7
Hemophilus influenzae septic arthritis and pneumonia in an adult as the first presentation of multiple myeloma; Bhatnagar SK et al.; We describe an apparently healthy woman who presented with monoarthritis of the right knee and a history of respiratory infection . The synovial fluid cultures grew beta lactamase-negative H . influenzae . Chest roentgenogram showed patchy densities throughout the right lung . Investigations for a predisposing factor for this H . influenzae infection revealed underlying multiple myeloma . As H . influenzae pyoarthrosis is extremely rare in adults, we suggest that an underlying systemic cause be sought in all such patients.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12189 - 94
beta-Lactamase binds to GroEL in a conformation highly protected against hydrogen/deuterium exchange; Gervasoni P et al.; Escherichia coli RTEM beta-lactamase reversibly forms a stable complex with GroEL, devoid of any enzymatic activity, at 48 degrees C . When beta-lactamase is diluted from this complex into denaturant solution, its unfolding rate is identical to that from the native state, while the unfolding rate from the molten globule state is too fast to be measured . Electrospray mass spectrometry shows that the rate of proton exchange in beta-lactamase in the complex at 48 degrees C is slower than in the absence of GroEL at the same temperature, and resembles the exchange of the native state at 25 degrees C . Similarly, the final number of protected deuterons is higher in the presence of GroEL than in its absence . We conclude that, for beta-lactamase, a state with significant native structure is bound to GroEL . Thus, different proteins are recognized by GroEL in very different states, ranging from totally unfolded to native-like, and this recognition may depend on which state can provide sufficient accessible hydrophobic amino acids in a suitably clustered arrangement . Reversible binding of native-like states with hydrophobic patches may be an important property of GroEL to protect the cell from aggregating protein after heat-shock.

J Biol Chem, 1996 Oct 4, 271(40), 24625 - 33
The role of charged residues in determining transmembrane protein insertion orientation in yeast; Harley CA et al.; The first 79 residues of the yeast Ste2p G protein-coupled pheromone receptor, including the negatively charged N-terminal domain, the first transmembrane segment, and the following positively charged cytoplasmic loop, has been fused to a Kex2p-cleavable beta-lactamase reporter . Insertion orientation was determined by analysis of cell-associated and secreted beta-lactamase activities and independently corroborated by analysis of membrane association and glycosylation patterns . This fusion inserts with exclusively N terminus exofacial (Nexo) topology, serving as a model type III membrane protein . Orientation is unaffected by removal of all three positively charged residues in the cytoplasmic loop or by deletion of all but 12 residues from the N-terminal domain . The residual -2 N-terminal charge apparently provides a signal sufficient to determine Nexo topology . This is entirely consistent with the statistically derived rule in which the charge difference, Delta(C-N), counted for the 15 immediately flanking residues, is the primary topology determinant . Mutations altering Delta(C-N) to zero favors Nexo insertion by 3 to 1, whereas increasingly negative values cause increasing inversion of orientation . All results are consistent with the charge difference rule and indicate that whereas positive charges promote cytoplasmic retention, negative charges promote translocation.

Antimicrob Agents Chemother, 1996 Oct, 40(10), 2434 - 6
Implication of Ile-69 and Thr-182 residues in kinetic characteristics of IRT-3 (TEM-32) beta-lactamase; Farzaneh S et al.; The substitution of a methionine for an isoleucine at position 69 (Met69Ile), which causes inhibitor resistance to TEM-type beta-lactamases (IRT-3 and IRT-I69), altered the positions of the Asn-170 and Glu-166 side chains as well as the position of the catalytic water molecule . A novel hydrogen bond between the hydroxyl of Thr-182 and the carbonyl of Glu-64 was expected to be responsible for the increase in the catalytic activity of the IST-T182 and IRT-3 enzymes compared with those of TEM-1 and IRT-169, respectively.

Biochemistry, 1996 Sep 24, 35(38), 12421 - 32
Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry; Brown RP et al.; Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism . Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme . These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da . Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate . The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme . Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme . Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70 . A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130 . A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde . Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.

FEMS Microbiol Lett, 1996 Sep 15, 143(1), 29 - 33
A kinetic study of NMC-A beta-lactamase, an Ambler class A carbapenemase also hydrolyzing cephamycins; Mariotte-Boyer S et al.; In order to analyze its kinetic parameters, an Ambler class A carbapenemase NMC-A was purified . NMC-A demonstrated unusually strong hydrolytic activity towards imipenem and meropenem . Moreover, it hydrolyzed cephamycins with kcat values uncommonly high for this class of beta-lactamases . Clavulanic acid and tazobactam had comparable inhibitory activity against NMC-A, whereas sulbactam was the least active inhibitor . Noticeably, NMC-A was more readily inhibited by brobactam . All these catalytic properties suggest that NMC-A possesses an original structure of its active site allowing hydrolysis of beta-lactams usually stable to the hydrolytic activity of class A beta-lactamases.

Biochemistry, 1996 Sep 3, 35(35), 11386 - 95
Competition between DsbA-mediated oxidation and conformational folding of RTEM1 beta-lactamase; Frech C et al.; Similar to other proteins of the periplasm of Escherichia coli, TEM 1 beta-lactamase contains only a single disulfide bond . It can fold to its native conformation in both the presence and the absence of this disulfide bond . The GdmC1-dependent equilibrium unfolding of beta-lactamase in vitro is well described by a N reversible I reversible U three-state model in which the native protein (N) first reacts to an intermediate of the molten globule type (I) and then to the unfolded state (U) . We find that the disulfide bond of beta-lactamase stabilizes I relative to U, but does not change the stability of N relative to I . The I reversible U transition is an extremely rapid reaction for both reduced and oxidized beta-lactamase, but the N reversible I folding kinetics are slow and identical in the presence and the absence of the disulfide bond . This insensitivity of the N reversible I equilibrium and kinetics suggests that the region around the disulfide bond is already native-like folded and is presumably buried in the intermediate I, prior to the slow and rate-limiting events of folding . This was confirmed by measuring the stability of the disulfide bond, which, to a first approximation, is identical in N and I . In native, reduced beta-lactamase, the thiol groups are inaccessible for oxidation by DsbA, but at the stage of the molten globule intermediate I oxidation is still possible, because I is in fast exchange with the unfolded protein U . The introduction of the disulfide bond into beta-lactamase by DsbA competes with conformational folding at the stage of the final slow steps in the folding of the reduced protein . The major problem in the oxidation of proteins with one or two disulfide bonds (such as beta-lactamase) is not the formation of incorrect disulfide bonds, but the premature burial of the thiol groups by the rapid conformational folding of the reduced protein . DsbA, the major thiol/ disulfide isomerase of the bacterial periplasm, meets this problem . It is a very strong oxidant, and its reaction with cysteine residues in unfolded proteins is extremely fast.

Neurochem Res, 1996 Sep, 21(9), 1117 - 24
The other kind of biological NMR--studies of enzyme-substrate interactions; Roberts GC; NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates . The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism . Cytochromes P450 play a crucial role in metabolism of a wide range of exogenous chemicals . NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes . The other two enzymes, chloramphenicol acetyltransferase and beta-lactamase, are responsible for bacterial resistance to specific antibiotics . In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of beta-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism.

J Biol Chem, 1996 Aug 30, 271(35), 21579 - 82
The amino-terminal charge and core region hydrophobicity interdependently contribute to the function of signal sequences; Izard JW et al.; We have constructed a series of signal sequence mutants that contain negatively charged amino termini and simplified core regions of varying hydrophobicity levels . This series provides a means of exploring the relative roles of the amino terminus and the hydrophobic core region during transport . The signal peptides with highly hydrophobic core regions support a rapid rate of transport in the presence of a negatively charged amino terminus . We have found that these negatively charged mutants are secreted in a manner similar to the wild-type signal sequence; sodium azide and carbonyl cyanide 3-chlorophenylhydrazone treatments indicate that the negatively charged mutants depend on SecA and the protonmotive force, respectively . These same mutants also demonstrate reduced competition with coexpressed beta-lactamase, reflecting the lower overall affinity for the transport pathway due to the net negative charge at the amino terminus . In addition, the pronounced effects of introducing three negative charges support the conclusion that the two regions function in a concerted manner.

Biochim Biophys Acta, 1996 Aug 15, 1296(1), 85 - 94
Characterization of a molten globule intermediate during GdnHCl-induced unfolding of RTEM beta-lactamase from Escherichia coli; Sarkar D et al.; GdnHCl-induced unfolding and reversible folding of beta-lactamase from E . coli have been investigated by measuring enzymatic activity, fluorescence emission and far-UV circular dichroism as indices of the extent of denaturation . The non-coincidence of far-UV CD and fluorescence data and existence of an inflection point clearly suggest the presence of an equilibrium intermediate . The existence of the equilibrium intermediate at around 1 M is corroborated by its enhanced binding of fluorophobic probe 1,8-ANS . The intermediate was found to have a compact shape as measured by its Stokes radius by size-exclusion chromatography . Furthermore, near-UV CD analysis of this enzymatically inactive intermediate showed a significantly disrupted tertiary structure with only a minor change in the secondary structure, which is a characteristic of typical molten globule states . Estimation of the activation energy from the kinetics of unfolding of the protein monitored by fluorescence and CD suggests that the intermediate may be separated from the native and the unfolded state by a high activation-energy barrier.

J Clin Microbiol, 1996 Aug, 34(8), 1995 - 2000
Characterization of two unusual clinically significant Francisella strains; Clarridge JE 3rd et al.; We have isolated two phenotypically distinct nonfastidious Francisella strains (Fx1 and Fx2) from the blood of compromised patients with pneumonia and compared them with eight other Francisella strains, including Francisella tularensis biovar tularensis, F . tularensis biovar novicida, and F . philomiragia . Our isolates grew well on sheep blood agar, chocolate agar, modified Thayer-Martin agar, and Trypticase soy agar . Fx1 and Fx2 were determined to be within the Francisella genus by cellular fatty acid analysis and by the utilization of glucose, production of H2S and catalase, and lack of motility, oxidase, nitrate reductase, and gelatinase . They were additionally shown to belong to the species F . tularensis by sequencing of two variable regions comprising approximately 500 nucleotides of the 16S rRNA gene . Also, RNA probe hybridization confirmed their belonging to the species F . tularensis . However, the new strains, which are not identical, are distinguished from other F . tularensis strains by growth characteristics, repetitive extragenic palindromic PCR fragment pattern, and some biochemical tests . Key biochemical differences included the findings that Fx1 was positive for beta-galactosidase and arabinose hydrolysis and that both strains were citrulline ureidase positive and glycerol negative . Commercial F . tularensis antiserum agglutinated stock F . tularensis strains but not Fx1, Fx2, F . tularensis biovar novicida, or F . philomiragia; serum from either patient failed to agglutinate or only weakly agglutinated commercial antigen but showed agglutination when tested against each patient's respective isolate . Fx1 and Fx2 produced beta-lactamase . Because of their good growth, negative serology, and biochemical profile, the organisms could be misidentified in the clinical laboratory if standard strategies or commercial identification systems are used.

J Bacteriol, 1996 Jul, 178(13), 3748 - 54
Role of the propilin leader peptide in the maturation of F pilin; Majdalani N et al.; F-pilin maturation and translocation result in the cleavage of a 51-amino-acid leader sequence from propilin and require LepB and TraQ but not the SecA-SecY secretion pathway . The unusual propilin leader peptide and the dependence of its cleavage on TraQ suggested that TraQ recognition may be specific for the leader peptide . An in vitro propilin cleavage assay yielded propilin (13 kDa), the pilin polypeptide (7 kDa), and a 5.5-kDa protein as the traA products . The 5.5-kDa protein comigrates with the full-length 51-amino-acid leader peptide, and {14C}proline labeling confirmed its identity since the only proline residues of propilin are found within the leader peptide . The in vitro and in vivo propilin-processing reactions proceed similarly in a single polypeptide cleavage step . Furthermore, TraQ dependence is a property of F-pilin maturation specifically rather than a property of the leader peptide . A propilin derivative with an amino-terminal signal sequence generated by deleting codons 2 to 28 required TraQ for processing in vivo . On the other hand, a chimeric protein with the propilin wild-type leader peptide fused to the mature portion of beta-lactamase was processed in a TraQ-independent manner . Thus, despite its unusual length, the propilin leader peptide seems to perform a function similar to that of the typical amino-terminal signal sequence . This work suggests that TraQ is not necessary for the proteolysis of propilin and therefore is likely to act as a chaperone-like protein that promotes the translocation of propilin.

EMBO J, 1996 Jun 17, 15(12), 2901 - 13
The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene; Rehling P et al.; The import of peroxisomal matrix proteins is dependent on one of two targeting signals, PTS1 and PTS2 . We demonstrate in vivo that not only the import of thiolase but also that of a chimeric protein consisting of the thiolase PTS2 (amino acids 1-18) fused to the bacterial protein beta-lactamase is Pas7p dependent . In addition, using a combination of several independent approaches (two-hybrid system, co-immunoprecipitation, affinity chromatography and high copy suppression), we show that Pas7p specifically interacts with thiolase in vivo and in vitro . For this interaction, the N-terminal PTS2 of thiolase is both necessary and sufficient . The specific binding of Pas7p to thiolase does not require peroxisomes . Pas7p recognizes the PTS2 of thiolase even when this otherwise N-terminal targeting signal is fused to the C-terminus of other proteins, i.e . the activation domain of Gal4p or GST . These results demonstrate that Pas7p is the targeting signal-specific receptor of thiolase in Saccharomyces cerevisiae and, moreover, are consistent with the view that Pas7p is the general receptor of the PTS2 . Our observation that Pas7p also interacts with the human peroxisomal thiolase suggests that in the human peroxisomal disorders characterized by an import defect for PTS2 proteins (classical rhizomelic chondrodysplasia punctata), a functional homologue of Pas7p may be impaired.

Eur J Biochem, 1996 Jun 15, 238(3), 760 - 8
Substitution of fifty four homologue (Ffh) in Escherichia coli with the mammalian 54-kDa protein of signal-recognition particle; Patel S et al.; The fifty four homologue (Ffh) of Escherichia coli promotes the translocation of a subset of periplasmic, membrane and secreted proteins across the cytoplasmic membrane . The ffh gene product is essential for cell viability and efficient protein export . Here we show that the mammalian homologue signal-recognition particle (SRP) 54 kDa is not able to suppress the translocation defect in an Ffh conditional mutant Wam 113 {Phillips, G.J . & Silhavy, T.J . (1992) Nature 359, 744-746} . The expression of SRP 54kDa, which is increased when Ffh is suppressed in the Wam 113 strain, causes a pleiotropic defect characterised by cell elongation, and increased accumulation of precursor proteins . The accumulation of precursors of outer membrane protein A (Omp A) and maltose-binding protein (MBP), See-B dependent pre-proteins, was less than the Ffh-dependent proteins ribose-binding protein (RBP) and beta-lactamase . Sec B expression was suppressed by Ffh expression . The recombinant SRP 54 kDa, which forms a ribonucleoprotein complex in E coli, was shown to bind to precursor proteins, but is unable to interact with the filamentous temperature-sensitive Y (Fts Y) membrane receptor of the translocation machinery.

J Bacteriol, 1996 Jun, 178(12), 3608 - 13
Involvement of the DnaK-DnaJ-GrpE chaperone team in protein secretion in Escherichia coli; Wild J et al.; We used depletion studies designed to further investigate the role of the DnaK, DnaJ, and GrpE heat shock proteins in the SecB-dependent and SecB-independent secretion pathways . Our previous finding that SecB-deficient strains containing the grpE280 mutation were still secretion proficient raised the possibility that GrpE was not involved in this secretory pathway . Using depletion studies, we now demonstrate a requirement for GrpE in this pathway . In addition, depletion studies demonstrate that while DnaK, DnaJ, and GrpE are involved in the secretion of the SecB-independent proteins (alkaline phosphatase, ribose-binding protein, and beta-lactamase), they are not the primary chaperones in this process.

J Mol Biol, 1996 May 17, 258(4), 688 - 703
Amino acid sequence determinants of beta-lactamase structure and activity; Huang W et al.; TEM-1 beta-lactamase catalyzes the hydrolysis of beta-lactam antibiotics such as the penicillins and cephalosporins, thus providing for bacterial resistance to these compounds . To determine the amino acid residues critical for the structure and function of TEM-1 beta-lactamase, the codons for each of the 263 amino acid residues that constitute the mature form of the enzyme were randomized using a site-directed mutagenesis procedure . Functional random mutants were selected based on their ability to confer ampicillin resistance to Escherichia coli . The DNA sequence of several functional mutants was determined for each set of random mutants . It was found that 43 out of the 263 amino acid residues do not tolerate substitutions and therefore are critical for the structure and activity of the enzyme . In addition, a comparison of conserved residue positions among functional beta-lactamase mutants with conserved residues in the beta-lactamase gene family identified many positions which did not tolerate substitutions in the mutagenesis studies but are freely substituted among members of the gene family . This observation may be due to the accumulation of compensating mutations among members of the gene family . Finally, the sequence variability at residue positions among functional mutants was quantitated by calculating the effective number of substitutions at each position using information-theoretical entropy . These values were used to obtain a quantitative estimate of the correlation between the sequence variability at a position and the fractional accessible surface area of the residue . The correlation is found to be statistically significant in that buried residues tend to exhibit low variability and invariant residues tend to exhibit low solvent exposure . However, the correlation is weak because most residues are neither completely buried nor invariant.

Zh Mikrobiol Epidemiol Immunobiol, 1996 May-Jun, (3), 34 - 9
{A genetic and molecular biological study of the pathogenicity factors in Brucella}; Kulakov IuK et al.; The use of gene engineering techniques made it possible to obtain strain GSE830, capable of a higher level of expression of the gene of 38-kD protein in immunoblotting with sheep and rabbit antibrucellar sera in comparison with the expression of this gene of other Escherichia coli strains, containing recombinant plasmids with this gene . Due to the presence of the gene of 38-kD protein, recombinant E.coli strains were capable of survival in macrophage-like cell line U937 3.6-6.3 times more effectively . The model of interaction of Brucella pathogenic and nonpathogenic species with HeLa cells was studied . The bank of insertion mutants of B.suis virulent strain 1330 was studied with the use of transposon TnblaM . Out of 380 insertion mutants, 7 clones expressing beta-lactamase and having decreased capacity for multiplication in HeLa cells 48 hours after inoculation were selected . Detailed analysis revealed that 3 of them had lower adhesive capacity, 1 of them had lower invasive capacity and 3 other mutants were less capable of intracellular multiplication in HeLa cells than the initial B.suis strain 1330 . All these 7 mutants had different sites of TnblaM insertion into the chromosome of B.suis strain 1330.

Proteins, 1996 May, 25(1), 104 - 11
The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 beta-lactamase is the trans to cis isomerization of a non-proline peptide bond; Vanhove M et al.; The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 beta-lactamase have been investigated as a function of guanidine hydrochloride concentration . The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166-Thr167 peptide bond, like the Glu166-Pro167, is cis . The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein . In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme . The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding . The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans-->cis isomerization of the Glu166-Thr167 peptide bond.

J Bacteriol, 1996 May, 178(9), 2701 - 8
Transformation of Coxiella burnetii to ampicillin resistance; Suhan ML et al.; A 5.8-kb chromosomal fragment isolated from Coxiella burnetii initiates plasmid replication in Escherichia coli and was characterized as an autonomous replication sequence, ars (M . Suhan, S.-Y . Chen, H.A . Thompson, T.A . Hoover, A . Hill, and J.C . Williams, J . Bacteriol . 176:5233-5243, 1994) . In the present study, an ars replicon was used to transform C . burnetii to ampicillin resistance . Plasmid pSKO(+)1000 contained the C . burnetii ars sequence cloned into a ColE1-type replicon encoding beta-lactamase . pSKO(+)1000 was introduced into C . burnetii by electroporation . Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization . Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination . The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C . burnetii chromosome . The bla gene was also located in an episome . However, an ampicillin resistance plasmid lacking the C . burnetii ars sequence did not stably transform C . burnetii . A biological assay analyzing beta-lactamase activity of C . burnetii transformants during acid activation in vitro provided evidence for expression of the bla (beta-lactamase) gene.

Gene, 1996 Apr 17, 170(1), 143 - 4
Construction of beta-lactamase-encoding ApR gene cassettes for rapid identification of cloned genes; Yokochi T et al.; We have constructed two Escherichia coli plasmids, pYK18 and pYK19, from which the BamHI, SmaI or EcoRI DNA fragments containing the ApR gene, conferring resistance to ampicillin, can be excised . The ApR cassettes have an annealing site for the sequencing primer of pUC plasmids at each end . Therefore, when the cassette is inserted into a gene, we can determine the nucleotide sequence of the gene from the insertion site using the sequencing primers of the pUC plasmids . This method is useful for identifying a cloned gene.

Microb Drug Resist, 1996 Spring, 2(1), 105 - 12
The convergence of murein recycling research with beta-lactamase research; Park JT; The story of how investigation of Escherichia coli cell wall elongation evolved into a study of murein recycling and how this led to the discovery that ampG and ampD were required for both murein recycling and beta-lactamase regulation is chronicled . Preliminary information on two other genes believed to be involved in recycling, nagZ, the structural gene for beta-N-acetylglucosaminidase, and tpl, the presumed structural gene for the hypothetical tripeptide-adding enzyme, is presented . The possibility that recycling of murein fragments serves a signaling function is discussed.

Antimicrob Agents Chemother, 1996 Apr, 40(4), 966 - 72
Molecular characterization of the BRO beta-lactamase of Moraxella (Branhamella) catarrhalis; Bootsma HJ et al.; A rapid increase in the prevalence of beta-lactamase-producing Moraxella (Branhamella) catarrhalis strains has been noticed during the last decades . Today, more than 80% of strains isolated worldwide produce beta-lactamase . To investigate beta-lactamase(s) of M . catarrhalis at the molecular level, the BRO-1 beta-lactamase gene (bla) was isolated as part of a 4,223-bp HindIII fragment . Sequence analysis indicated that bla encodes a polypeptide of 314 amino acid residues . Insertional inactivation of bla in M . catarrhalis resulted in complete abrogation of beta-lactamase production and ampicillin resistance, demonstrating that bla is solely responsible for beta-lactam resistance . Comparison with other beta-lactamases suggested that M . catarrhalis beta-lactamase is a unique enzyme with conserved residues at the active sites . The presence of a signal sequence for lipoproteins suggested that it is lipid modified at its N terminus . In keeping with this assumption was the observation that 10% of beta-lactamase activity was found in the membrane compartment of M . catarrhalis . M . catarrhalis strains produce two types of beta-lactamase, BRO-1 and BRO-2, which differ in their isoelectric points . The BRO-1 and BRO-2 genes from two ATCC strains of M . catarrhalis were sequenced, and only one amino acid difference was found between the predicted products . However, there was a 21-bp deletion in the promoter region of the BRO-2 gene, possibly explaining the lower level of production of BRO-2 . The G + C content of bla (31%) was significantly lower than those of the flanking genes (47 and 50%), and the overall G + C content of the M . catarrhalis genome (41%) . These results indicate that bla was acquired by horizontal gene transfer from another, still unknown species.

Mol Microbiol, 1996 Apr, 20(2), 361 - 73
Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence; Odenbreit S et al.; Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans . In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H . pylori . The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H . pylori gene library established in Escherichia coli . A total of 192 E . coli clones expressing active beta-lactamase fusion proteins (BlaM+) were obtained, indicating that the corresponding target plasmids carry H . pylori genes encoding putative extracytoplasmic proteins . Natural transformation of H . pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H . pylori mutant strains (70%) . Screening of the H . pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines . Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III) . Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci . In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line KatoIII . Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.

J Antimicrob Chemother, 1996 Apr, 37(4), 797 - 802
Detection of mutations conferring extended-spectrum activity on SHV beta-lactamases using polymerase chain reaction single strand conformational polymorphism (PCR-SSCP); M'Zali FH et al.; Single strand conformational polymorphism (SSCP) is a recently developed technique used to detect single base mutations in short PCR-generated amplimers . The method has been adapted and applied to differentiation of beta-lactamase genes . Each of the five standard SHV strains used produced a unique SSCP pattern, allowing the possibility of rapid identification of the SHV genes of other isolates . A clinical isolate that phenotypically produced SHV-5 yielded a pattern of major bands indistinguishable from that of the SHV-5 standard strain, illustrating the applicability of this technique . We therefore report a reliable and reproducible technique that can be applied to the characterisation of the SHV beta-lactamases.

J Antimicrob Chemother, 1996 Apr, 37(4), 697 - 701
In-vitro evaluation of beta-lactamase inhibition by latamoxef and imipenem; Sotto A et al.; The in-vitro 50% inhibitory concentrations (IC50s) of latamoxef and imipenem against a set of plasmid-mediated beta-lactamases including TEM-1, TEM-3, TEM-5 and TEM-10 were determined by an enzymatic method using nitrocefin as substrate . The IC50s of both antibiotics against extended-spectrum beta-lactamases were below 0.2 mg/L . The conventional spectrum beta-lactamase TEM-1 was not inhibited by either antibiotic at the highest concentration tested . Except for TEM-10 for which the IC50s of the two antibiotics were the same, imipenem showed significantly greater activity than latamoxef against TEM-3 and TEM-5 . Clavulanic acid taken as a control demonstrated greater and wider inhibitory activity, but on the other hand it has no significant antibiotic activity.

Cell Biol Int, 1996 Apr, 20(4), 293 - 9
Novel markers for constitutive secretion used to show that tissue plasminogen activator is sorted to the regulated pathway in transfected PC12 cells; Harrison TM et al.; The rat pheochromocytoma cell line PC12 contains two distinct pathways of protein secretion . Proteins secreted via the regulated pathway are stored in secretory vesicles and exocytosed only in response to a specific signal, whereas proteins secreted via the constitutive pathway are exported continuously . Analysis of regulated secretion of a heterologous protein in this system often relies on comparison of secretion rates with those of endogenous proteins known to be secreted via the constitutive route . In order to improve these controls, we have evaluated a number of secreted enzymes, selected for the sensitivity and convenience of their assays, as transgenic markers for the constitutive pathway . We show that both human-secreted placental alkaline phosphatase (SEAP) and bacterial beta-lactamase operate in this way in transfected PC12 cells . In contrast, transfected human tissue plasminogen activator (tPA) is shown to be sorted to the regulated pathway.

Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 112 - 9
Characterization of Escherichia coli expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the outer membrane; Stathopoulos C et al.; The Lpp'OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E . coli surface, thus providing a system for several possible biotechnology applications . Here we show that fusion between Lpp'OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane . However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane . Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid . Furthermore, E . coli expressing the Lpp'OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability . Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors . Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane . The large size and quaternary structure of PhoA may define a limitation of the Lpp'OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface . Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (beta-lactamase, alkaline phosphatase, Cex cellulase Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp'OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.

Biotechnol Prog, 1996 Mar-Apr, 12(2), 205 - 8
Effects of glycerol of beta-lactamase production during high cell density cultivation of recombinant Escherichia coli; Kwon S et al.; Recombinant Escherichia coli was cultured up to OD600 90 with glycerol fed by the DO-STAT method to produce recombinant beta-lactamase . Optimum feeding and induction conditions were determined . When glycerol was maintained at a high concentration (40-50 g/L) after isopropyl thiogalactoside (IPTG) induction at OD600 75, a 2-fold increase in the amount of soluble beta-lactamase was obtained . Similarly, a 3-fold increase was obtained by the addition of NaCl (0.4 M) at low glycerol concentration (10 g/L).

Nat Struct Biol, 1996 Mar, 3(3), 233 - 9
Molecular docking programs successfully predict the binding of a beta-lactamase inhibitory protein to TEM-1 beta-lactamase; Strynadka NC et al.; Crystallization of the 1:1 molecular complex between the beta-lactamase TEM-1 and the beta-lactamase inhibitory protein BLIP has provided an opportunity to put a stringent test on current protein-docking algorithms . Prior to the successful determination of the structure of the complex, nine laboratory groups were given the refined atomic coordinates of each of the native molecules . Other than the fact that BLIP is an effective inhibitor of a number of beta-lactamase enzymes (KI for TEM-1 approximately 100 pM) no other biochemical or structural data were available to assist the practitioners in their molecular docking . In addition, it was not known whether the molecules underwent conformational changes upon association or whether the inhibition was competitive or non-competitive . All six of the groups that accepted the challenge correctly predicted the general mode of association of BLIP and TEM-1.

J Biol Chem, 1996 Feb 2, 271(5), 2427 - 32
The chromosomal arsR gene of Escherichia coli encodes a trans-acting metalloregulatory protein; Xu C et al.; Plasmid-encoded arsenical resistance (ars) operons confer high level resistance to arsenicals and antimonials, while the chromosomally encoded ars operon of Escherichia coli bestows low level resistance . The transcriptional start site of the chromosomal ars mRNA was mapped by primer extension, and putative -10 and -35 promoter recognition sites were identified . The arsR gene, the first gene in this operon, was cloned using polymerase chain reaction . The arsR gene product, the ArsR repressor, was expressed and purified . The results of gel mobility shift assays indicated that the repressor is a DNA binding protein that binds to a fragment of DNA containing the chromosomal ars promoter . The specific binding site, as determined by DNase I footprint analysis, spans 33 nucleotides in the promoter region, including the putative -35 promoter element . By construction and expression of a series of in-frame fusions between truncated arsR genes and the coding region for the mature form of beta-lactamase (blaM'), it was shown that ArsR is a trans-acting repressor that regulates expression of the chromosomal ars operon . In addition, the chromosomally-encoded repressor can regulate expression of the ars operon of plasmid R773, and the R773 repressor can cross-regulate expression from the chromosomal operon.

Biol Res, 1996, 29(1), 127 - 40
Protein engineering as a powerful tool for the chemical modification of enzymes; Moreno-Hagelsieb G et al.; This article discusses the techniques of site-specific mutagenesis and protein engineering and their application in the study of enzyme active sites and the mechanism of enzyme action . Particular emphasis is given to beta-lactamase.

Antimicrob Agents Chemother, 1996 Jan, 40(1), 260 - 2
TEM-28 from an Escherichia coli clinical isolate is a member of the His-164 family of TEM-1 extended-spectrum beta-lactamases; Bradford PA et al.; TEM-28 (pI 6.1), expressed by an Escherichia coli clinical isolate, is a novel beta-lactamase which hydrolyzed ceftazidime, cefotaxime, and aztreonam with rates of 25, 1.1, and 5.6, respectively, relative to that for benzylpenicillin (100) . The nucleotide sequence of blaTEM-28 differed from that of blaTEM-1 by two base changes, resulting in amino acid substitutions of Arg-164 to His and Glu-240 to Lys.

Bioconjug Chem, 1996 Jan-Feb, 7(1), 150 - 8
Dextran modification of a Fab'--beta-lactamase conjugate modulated by variable pretreatment of Fab' with amine-blocking reagents; Mikolajczyk SD et al.; The physical and pharmacological properties of proteins can be altered by chemical modification with polymers . Preliminary studies showed that attachment of oxidized dextran to the bacterial protein, beta-lactamase (beta L) effectively reduced in vivo immunogenicity in mice with no loss of enzymatic activity . This report describes a general method for differentially dextran modifying the Fab' component of a Fab'--beta-lactamase conjugate by the use of amine-blocking reagents . Methyl acetimidate (MeAcm) and the N-succinimidyl derivative of (methylsulfonyl)ethyl carbonate (NHS-Msc), reagents which can reversibly block primary amines, were used in model studies to modulate the level of available reactive amines on the F(ab')2 fragments of both the anti-carcinoembryonic antigen antibody, ZCE025, and the antitumor-associated glycoprotein-72 antibody, CC49 . MeAcm had little or no effect on immunoreactivity and was maximally effective in modulating dextran attachment, while NHS-Msc was much less effective . A comparison of NHS-Msc and MeAcm is described . Treatment of F(ab')2 with 5-300 mM MeAcm prior to dextran treatment showed a proportional decline in the level of dextran attachment as well as intramolecular cross-linking of the protein by the dextran polymers (6 kDa or 33-mer) . A conjugate of beta L coupled to MeAcm-treated ZCE025 Fab' {reduced F(ab')2} was constructed under standard conditions using sulfosuccinimidyl N-{(4-carboxycyclohexyl)methyl}maleimide . After dextran modification, this conjugate maintained good immunoreactivity and enzymatic activity . Biodistribution studies in tumor-bearing nude mice of dextranated and nondextranated conjugate showed comparable overall distribution profiles except that the clearance of the dextranated conjugate from both blood and tumor was delayed about 48-72 h.

FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 227 - 32
Comparison of the phenotypes of the lpxA and lpxD mutants of Escherichia coli; Vuorio R et al.; We compared the phenotype of two thermosensitive Escherichia coli mutants defective in lipid A biosynthesis, i.e . SM101 (lpxA) and CDH23-213 (lpxD) . More than 40% of the periplasmic 27-kDa marker enzyme beta-lactamase was released from SM101 at 28 degrees C . At this temperature, the mutant still grew with a generation time (67 min), not much longer than that of the parent control strain (57 min) . CDH23-213 released beta-lactamase only at higher temperatures . SM101 and CDH23-213 were both unable to grow in hypo-osmotic conditions . Derivatives of SM101 and CDH23-213 with mdoA::Tn10 had identical phenotypes (including thermosensitivity and defective outer membrane permeability barrier to hydrophobic probes) to those of SM101 and CDH23-213, indicating that the potential loss of membrane-derived oligosaccharides (MDO) did not explain these phenotype properties . A method for the estimation of lipid A synthesis rate was developed.

J Mol Biol, 1995 Dec 8, 254(4), 566 - 78
Adjacent upstream superhelical writhe influences an Escherichia coli promoter as measured by in vivo strength and in vitro open complex formation; Hirota Y et al.; This work investigates the effect on transcription of superhelical writhe located in the region immediately upstream of the -35 consensus sequence of Escherichia coli promoters . A set of double-stranded oligonucleotides, each with an unique DNA configuration, were designed, synthesized and substituted into an area of naturally occurring right-handed superhelical curvature immediately upstream of the beta-lactamase promoter in plasmid pUC19 . All the mutants showed reduced promoter activities in E . coli cells . However, rightward superhelical writhe clearly facilitated transcription when compared with the effect produced by a straight DNA segment . Leftward writhe greatly repressed transcription . A plane curve showed an intermediate effect . This phenomenon was due not only to the difference in the ability of the segment to drive an open complex formation, but also to the difference in the binding affinity of RNA polymerase to the promoter . The positive effect of rightward writhe was also observed in vivo for the promoter of the tetracycline resistance gene of pBR322 . The sense and extent of superhelical writhe of a DNA curvature seem to determine its influence on promoter activity.

EMBO J, 1995 Dec 1, 14(23), 6028 - 33
In vivo reactivation of heat-denatured protein in the endoplasmic reticulum of yeast; Jamsa E et al.; Saccharomyces cerevisiae cells grown at 24 degrees C acquire thermotolerance and survive exposure to 50 degrees C, but only if they are first incubated at 30 degrees C, the temperature where heat shock genes are activated . We show here that the enzymatic activity of a secretory beta-lactamase fusion protein, pre-accumulated at 37 degrees C in the endoplasmic reticulum, was abolished by exposure of the cells to 50 degrees C . When the cells were returned to 24 degrees C, beta-lactamase activity was resumed . Reactivation occurred in the endoplasmic reticulum, but not in the Golgi apparatus . It was dependent on metabolic energy, but did not require de novo protein synthesis . According to co-immunoprecipitation experiments, immuno-globulin-binding protein (BiP/Kar2p) was associated with the fusion protein . We suggest that recovery from thermal insult involves, in addition to cytoplasmic and nuclear events, refolding of heat-damaged proteins in the endoplasmic reticulum by a heat-resistant machinery, which forms part of a fundamental survival mechanism.

J Antimicrob Chemother, 1995 Dec, 36(6), 927 - 39
Mechanisms of hyperproduction of TEM-1 beta-lactamase by clinical isolates of Escherichia coli; Wu PJ et al.; The mechanisms of hyperproduction (defined as > or = 200 nmoles nitrocefin hydrolysed per minute per mg of protein) of TEM-1 beta-lactamase by 38 isolates of Escherichia coli were investigated . The copy numbers of TEM-encoding plasmids were determined for the hyperproducing isolates and for 39 TEM-1-producing isolates that did not hyperproduce the enzyme . Allele-specific PCR was used to determine if the promoter region of the TEM-1 gene was of the TEM-1 or TEM-2 type . Twenty three of the 38 hyperproducers had the TEM-1-type promoter but 15 had the more efficient TEM-2-type promoter; in contrast, 37 of the 39 isolates with lower activities had the TEM-1-type promoter and only two had the TEM-2-type promoter . Many of the TEM-1-hyperproducing isolates possessed small plasmids (< or = 20 MDal) with high copy numbers, in some cases together with large, low copy number plasmids; the average total copy number of TEM-encoding plasmids was 14; if only isolates with the TEM-1-type promoter were included, average total copy number was 22 . Hyperproduction was attributed to high copy number (> or = 10) plasmids in 11 isolates; another seven had plasmids with moderately high copy numbers (4-9) . The average total copy number for isolates that produced relatively small amounts of TEM-1 beta-lactamase (< or = 100 nmoles/min/mg protein) was 2.2, and for the 12 isolates with TEM-1 activities of 101-200 nmoles/min/mg protein it was 6.8 . We conclude that both high copy number plasmids and a more efficient promoter are common causes of hyperproduction of TEM-1.

Appl Environ Microbiol, 1995 Dec, 61(12), 4474 - 6
The beta-lactamase secreted by the antarctic psychrophile Psychrobacter immobilis A8; Feller G et al.; A class C beta-lactamase has been purified from the culture supernatant of the antarctic psychrophile Psychrobacter immobilis A8 . This psychrophilic beta-lactamase displays a low level of thermal stability and a low optimal temperature of activity . In contrast to other cold-adapted enzymes, its level of specific activity is not higher than that of mesophilic class C beta-lactamases.

Antimicrob Agents Chemother, 1995 Nov, 39(11), 2478 - 83
Incidence and mechanisms of resistance to the combination of amoxicillin and clavulanic acid in Escherichia coli; Stapleton P et al.; Among Escherichia coli organisms isolated at St . Thomas's Hospital during the years 1990 to 1994, the frequency of resistance to amoxicillin-clavulanic acid (tested by disk diffusion in a ratio of 2:1) remained constant at about 5% of patient isolates (10 to 15% of the 41 to 45% that were amoxicillin resistant) . Mechanisms of increased resistance were determined for 72 consecutively collected such amoxicillin-clavulanic acid-resistant isolates . MICs of the combination were 16-8 micrograms/ml for 51 (71%) of these and > or = 32-16 micrograms/ml for the remainder . The predominant mechanism was hyperproduction of enzymes isoelectrically cofocusing with TEM-1 (beta-lactamase activities, > 200 nmol of nitrocefin hydrolyzed per min per mg of protein) which was found in 44 isolates (61%); two isolates produced smaller amounts (approximately 150 nmol/min/mg) of such enzymes, and two isolates hyperproduced enzymes cofocusing with TEM-2 . Eleven isolates produced enzymes cofocusing with OXA-1 beta-lactamase, which has previously been associated with resistance to amoxicillin-clavulanic acid . Ten isolates produced increased amounts of chromosomal beta-lactamase, and four of these additionally produced TEM-1 or TEM-2 . Three isolates produced inhibitor-resistant TEM-group enzymes . In one of the enzymes (pI, 5.4), the amino acid sequence change was Met-67-->Val, and thus the enzyme is identical to TEM-34 . Another (pI, 5.4) had the substitution Met-67-->Ile and is identical to IRT-I67, which we propose now be given the designation TEM-40 . The third (pI, 5.2) had the substitution Arg-241-->Thr; this enzyme has not been reported previously and should be called TEM-41 . The rarity and diversity of inhibitor-resistant TEM-group enzymes suggest that they are the result of spontaneous mutations that have not yet spread.

Res Microbiol, 1995 Nov-Dec, 146(9), 761 - 71
{beta-Lactamases of Branhamella catarrhalis and their phenotypic implications}; Chaibi EB et al.; Of the 50 strains of beta-lactamase-producing Branhamella catarrhalis isolated at Saint Joseph's Hospital (Paris) that were studied, 94% produced BRO-1 type beta-lactamase and 6% produced the BRO-2 type . We examined the transfer of BRO-1 and BRO-2 genes and found that, among 7 donor strains producing BRO-1, all were able to transfer the gene for BRO-1 production by conjugation . Of the 4 donor strains producing BRO-2, 2 were able to transfer the gene for BRO-2 production by conjugation . Three BRO-1 beta-lactamase-producing transformants were obtained from total DNA extracted from 3 strains producing BRO-1 . Plasmid bands were demonstrated in strains of B . catarrhalis, but no change in plasmid profiles was seen in beta-lactamase-positive recombinants, supporting previous studies that suggested the beta-lactamases are chromosomal . In vitro activity of oral beta-lactams was tested for 67 strains of B . catarrhalis (56 beta-lactamase-producing strains) . Cefixime, cefpodoxime and the combination ampicillin-clavulanic acid were very active against the beta-lactamase-producing strains . BRO-1 beta-lactamase appears to affect the activity of cefaclor, cefuroxime and loracarbef . BRO-2 beta-lactamases have no effect on the activity of these cephalosporins . Cefixime and cefpodoxime seemed the least affected by beta-lactamase production.

Biochemistry, 1995 Oct 17, 34(41), 13642 - 50
Oxidation of kinetically trapped thiols by protein disulfide isomerase; Walker KW et al.; The formation of a stabilized structure during oxidative protein folding can severely retard disulfide formation if the structure must be disrupted to gain access to buried cysteines . These kinetic traps can slow protein folding and disulfide bond formation to the extent that unassisted folding is too slow to be kinetically competent in the cell . Protein disulfide isomerase (PDI) facilitates the oxidation of a kinetically trapped state of RTEM-1 beta-lactamase in which two cysteines that form the single disulfide bond in the native protein are buried and approximately 500-fold less reactive than exposed cysteines . Under second-order conditions, PDI-dependent oxidation of reduced, folded beta-lactamase is 500-fold faster than GSSG-dependent oxidation . The rate difference observed between PDI and GSSG can be accounted for by the 520-fold higher kinetic reactivity of PDI as an oxidant . Noncovalent interactions between PDI (35 microM) and beta-lactamase increase the reactivity or unfolding of beta-lactamase in the steady-state by less than 3-fold . At high concentrations of PDI or alkylating agents, the reaction of beta-lactamase cysteines approaches a constant rate, limited by the spontaneous unfolding of the protein (kunfold = 0.024 +/- 0.005 min-1) . PDI does not substantially increase the rate of beta-lactamase unfolding; however, once beta-lactamase spontaneously unfolds, PDI at concentrations greater than 44 +/- 4 microM, oxidizes the unfolded substrate before it can refold (kfold = 1.5 +/- 0.2 min-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1995 Oct 16, 164(1), 49 - 53
Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides; Stemmer WP et al.; Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos) . The method, which is derived from DNA shuffling {Stemmer, Nature 370 (1994a) 389-391}, does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process . A 1.1-kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length . The synthetic gene was PCR amplified and cloned in a vector containing the tetracycline-resistance gene (TcR) as the sole selectable marker . Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene . We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alpha-fragment of the lacZ gene and the pUC origin of replication . Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid . Assembly PCR is well suited for several in vitro mutagenesis strategies.

FEMS Microbiol Lett, 1995 Oct 1, 132(1-2), 61 - 6
Bleomycin-induced beta-lactamase overexpression in Escherichia coli carrying a bleomycin-resistance gene from Streptomyces verticillus and its application to screen bleomycin analogues; Yuasa K et al.; A bleomycin-resistance gene, designated blmA, has been cloned from bleomycin-producing Streptomyces verticillus by Sugiyama et al . (Gene 151 (1994) 11-16) . The present study shows that Escherichia coli harboring the blmA-carrying pUC plasmid overproduced beta-lactamase, encoded by an ampicillin-resistance gene on the plasmid, when cultured in the presence of bleomycin, which suggests that bleomycin may act as an inducer (or an activator) for the expression of the specific gene in the presence of blmA . We constructed a vector, designated pMAB50, which senses bleomycin and produces a pigment, using blmA and a Streptomyces tyrosinase gene located under the control of beta-lactamase promoter: E . coli harboring pMAB50 produced the melanin pigment in the presence of bleomycin-type antibiotics, suggesting that the transformed E . coli can be employed as a reporter organism to screen bleomycin analogues.

Proteins, 1995 Sep, 23(1), 63 - 72
Stability of TEM beta-lactamase mutants hydrolyzing third generation cephalosporins; Raquet X et al.; The stability properties of six natural mutants of the TEM-1 beta-lactamase have been studied . The glutamate to lysine substitution at positions 104 and 240 stabilize the enzyme . Conversely, the G238S mutant's decreased stability might reflect an altered conformation of the active site and thus be related to the modified substrate profile . The relative stability of the R164S and R164H mutants is explained by the formation of a hydrogen bond between these residues and Asp-179 conferring a somewhat different structure to the omega loop and thus also explaining the extended substrate profile of these mutants . The loss of stability of the R164H mutant with increasing pH values can be explained by the titration of a hydrogen bond between the N delta of His-164 and the O delta of Asp-179 . The properties of the G238S + E104K double mutant which is the most active against third-generation cephalosporins result from a balance of destabilizing and stabilizing substitutions, and their effects seem to be additive . The behavior of the R164S + E240K mutant might be explained on the basis of a similar compensation phenomenon.

Biosci Biotechnol Biochem, 1995 Sep, 59(9), 1727 - 31
Production of biologically active mature brain-derived neurotrophic factor in Escherichia coli; Fukuzono S et al.; A gene coding for a brain-derived neurotrophic factor (BDNF) obtained from rat BDNF cDNA was used to construct an expression plasmid, pTRSBDNF . It contained a BDNF gene that was fused, in frame, to a region encoding the beta-lactamase signal peptide . The E . coli HB101 harboring pTRSBDNF produced 18 mg/liter of mature protein . The E . coli cells produced a larger amount of BDNF than that of human nerve growth factor (hNGF), which was produced using the same expression system . The E . coli cells harboring pTRSBDNF were sonicated and centrifuged to obtain a supernatant and precipitate . The BDNF purified from the supernatant containing 20% of the total BDNF had high biological activity (EC50 of 30 pg/ml) against neurons of chick dorsal root ganglia . On the other hand, the BDNF purified from the precipitate had low biological activity (EC50 of 2 ng/ml) and incorrect disulfide bonds.

J Biol Chem, 1995 Aug 4, 270(31), 18240 - 5
The asparagine to aspartic acid substitution at position 276 of TEM-35 and TEM-36 is involved in the beta-lactamase resistance to clavulanic acid; Saves I et al.; TEM-35 (inhibitor resistant TEM (IRT)-4) and TEM-36 (IRT-7) clavulanic acid-resistant beta-lactamases have evolved from TEM-1 beta-lactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276 . The substitutions at position 69 have previously been shown to be responsible for the resistance to clavulanic acid, and they are the only mutations encountered in TEM-33 (IRT-5) and TEM-34 (IRT-6) . However, the N276D substitution has never been found alone in inhibitor-resistant beta-lactamases, and its role in resistance to clavulanic acid was thus unclear . The N276D mutant was constructed, purified, and kinetically characterized . It was shown that the substitution has a direct effect on substrate affinities and leads to slightly decreased catalytic efficiencies and that clavulanic acid becomes a poor substrate of the enzyme . Electrospray mass spectrometry demonstrated the simultaneous presence of free and inhibited enzymes after incubation with clavulanic acid and showed that a cleaved moiety of clavulanic acid leads to the formation of the major inactive complex . The kinetic properties of the N276D mutant could be linked to a salt-bridge interaction of aspartic acid 276 with arginine 244 that alters the electrostatic properties in the substrate binding area.

Mol Microbiol, 1995 Aug, 17(3), 421 - 6
Why does Escherichia coli recycle its cell wall peptides?
Park JT.
This review discusses the phenomenon of recycling of cell wall peptides . It is a major, though non-essential, pathway of the cell and is required for induction of beta-lactamase . Consequently, the recycling pathway is viewed as a possible signalling vehicle, informing the cell of the condition of the murein sacculus, an essential structure existing outside the cell itself . As the study of this phenomenon is in its infancy, several speculations are offered for a possible regulatory function.

EMBO J, 1995 Aug 1, 14(15), 3609 - 16
Membrane insertion and assembly of ductin: a polytopic channel with dual orientations; Dunlop J et al.; Ductin is a highly conserved and polytopic transmembrane protein which is the subunit c component of the vacuolar H(+)-ATPase (V-ATPase) and a component of a connexon channel of gap junctions . Previous studies have suggested that ductin in the V-ATPase has the opposite orientation of ductin in a connexon . Using an in vitro translation system coupled to microsomes derived from the endoplasmic reticulum, we show that ductin is co-translationally inserted into the membrane bilayer, suggesting a dependency on the signal recognition particle for synthesis . By attaching a C-terminal polypeptide derived from beta-lactamase and by using cysteine replacement coupled to chemical labelling, we show that ductin is inserted into the microsomal membrane in both orientations in similar proportions . In contrast, squid rhodopsin appears to be inserted in a single orientation . Changing conserved charged residues at the N-terminus of ductin does not affect the ratio of the two orientations . Once in the microsomal membrane, ductin assembles into an oligomeric complex which contains a pore accessible to a water-soluble probe, reminiscent of the ductin complex found in the V-ATPase and a connexon.

Antimicrob Agents Chemother, 1995 Aug, 39(8), 1878 - 80
Effects of Asp-179 mutations in TEMpUC19 beta-lactamase on susceptibility to beta-lactams; Vakulenko SB et al.; To examine the effect of disruption of the salt bridge (between Arg-164 and Asp-179 {numbering of Ambler et al . (Biochem J . 267:269-272, 1991)}) that anchors the conserved omega-loop in class A beta-lactamases, we obtained mutant enzymes with each of the 19 other amino acid residues replacing Asp-179 in the TEM beta-lactamase encoded by pUC19 and studied the level of resistance to various beta-lactams conferred by each enzyme . All mutations of Asp-179 compromised the level of resistance to ampicillin, but most of them enhanced resistance to ceftazidime . In contrast, mutations of Asp-179 generally impaired the low levels of resistance to cefepime and aztreonam . One might expect to find clinical isolates with mutant TEM beta-lactamases with replacements of Asp-179 that express an expanded spectrum of resistance to beta-lactams including ceftazidime.

Antimicrob Agents Chemother, 1995 Aug, 39(8), 1856 - 8
Effect of pH on activities of novel beta-lactamases and beta-lactamase inhibitors against these beta-lactamases; Ohsuka S et al.; The effects of acidic conditions on activities of seven beta-lactamases--TEM-1 (class A), KOXY (class A), IMP-1 (class B), AmpC (class C), MOX-1 (class C), OXA-5 (class D), and PSE-2 (class D)--and their inhibitors were measured . The enzymatic activities of KOXY, IMP-1, and MOX-1 at pH 5.8 were slightly lower than those at pH 7.5 . However, the activities of PSE-2 and OXA-5 were greatly reduced at pH 5.8 . All of the beta-lactamase inhibitors tested had poorer inhibitory activities at pH 5.8 than at pH 7.5 except clavulanic acid for TEM-1.

J Bacteriol, 1995 Jul, 177(13), 3714 - 20
Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis; Wu TK et al.; Proclavaminate amidino hydrolase (PAH) catalyzes the reaction of guanidinoproclavaminic acid to proclavaminic acid and urea, a central step in the biosynthesis of the beta-lactamase inhibitor clavulanic acid . The gene encoding this enzyme (pah) was tentatively identified within the clavulanic acid biosynthetic cluster in Streptomyces clavuligerus by translation to a protein of the correct molecular mass (33 kDa) and appreciable sequence homology to agmatine ureohydrolase (M.B.W . Szumanski and S.M . Boyle, J . Bacteriol . 172:538-547, 1990) and several arginases, a correlation similarly recognized by Aidoo et al . (K . A . Aidoo, A . Wong, D . C . Alexander, R . A . R . Rittammer, and S . E . Jensen, Gene 147:41-46, 1994) . Overexpression of the putative open reading frame as a 76-kDa fusion to the maltose-binding protein gave a protein having the catalytic activity sought . Cleavage of this protein with factor Xa gave PAH whose N terminus was slightly modified by the addition of four amino acids but exhibited unchanged substrate specificity and kinetic properties . Directly downstream of pah lies the gene encoding clavaminate synthase 2, an enzyme that carries out three distinct oxidative transformations in the in vivo formation of clavulanic acid . After the first of these oxidations, however, no further reaction was found to occur in vitro without the intervention of PAH . We have demonstrated that concurrent use of recombinant clavaminate synthase 2 and PAH results in the successful conversion of deoxyguanidinoproclavaminic acid to clavaminic acid, a four-step transformation . PAH has a divalent metal requirement, pH activity profile, and kinetic properties similar to those of other proteins of the broader arginase class.

Arch Otolaryngol Head Neck Surg, 1995 Jul, 121(7), 729 - 36
Pilot study of outcomes in pediatric rhinosinusitis; Rosenfeld RM; OBJECTIVE: To determine outcomes achieved by treating refractory pediatric rhinosinusitis with a stepped protocol of additional antibiotics, adenoidectomy, and functional endoscopic sinus surgery (FESS) . DESIGN: Prospective cohort with follow-up at 2 to 3 months and at 10 to 12 months . SETTING: Hospital-based pediatric otolaryngology practice with community satellite offices . PARTICIPANTS: Consecutive 10-month sample of 41 children aged 2 to 13 years who met the following criteria: (1) roentgenographically documented sinusitis, (2) at least one 3-week course of a beta-lactamase stable antibiotic, (3) 3 months or more of clinical symptoms or three or more annual recurrences, (4) no obstructive adenoid hyperplasia, and (5) no cystic fibrosis . INTERVENTIONS: Fifteen children (37%) received additional antibiotics alone . Nonresponders underwent adenoidectomy (n = 10) when adenoids were present or FESS (n = 16) when adenoids were scant or if adenoidectomy did not provide relief (n = 2) . MAIN OUTCOME MEASURES: Unblinded, oral survey of symptomatic response, caregiver expectations, and quality-of-life issues . Responses from the 12 survey questions were combined to produce a sinusitis response score . RESULTS: Caregiver expectations were met in 88% of children 1 year after treatment but were more often exceeded in patients undergoing FESS (50%) compared with those receiving antibiotics with or without adenoidectomy (13%) . Functional endoscopic sinus surgery improved all major symptoms in 100% of children compared with 67% of those receiving antibiotics alone and 75% of those receiving antibiotics and undergoing adenoidectomy . In contrast, complete symptom resolution occurred in only 27% of patients, with no significant differences among groups . The median sinusitis response score of 84% was not associated with treatment level but was negatively associated with male gender . CONCLUSIONS: A stepped treatment approach to refractory sinusitis can improve quality of life for children and caregivers . Additional antibiotic therapy and adenoidectomy should be considered before FESS, even if the adenoids are nonobstructing.

Microbiology, 1995 Jul, 141 ( Pt 7), 1585 - 92
Recombinant BCG expressing the leishmania surface antigen Gp63 induces protective immunity against Leishmania major infection in BALB/c mice; Abdelhak S et al.; We have cloned and expressed the gp63 gene of Leishmania major in BCG to develop a recombinant vaccine against zoonotic cutaneous leishmaniasis . Two different expression systems were investigated . The first system consists of pAN, a Mycobacterium paratuberculosis promoter, which drives expression of ORF2, an open reading frame in IS900 . This system allows the production of heterologous polypeptides as hybrids with the ORF2 gene product . The second expression system relies on the production of antigenic fragments as fusion proteins with the N-terminal region of Mycobacterium fortuitum beta-lactamase . Both constructs resulted in the production of Gp63 in BCG . The ability of the two recombinant BCG strains to induce protective immunity against a challenge with L . major amastigotes was evaluated after vaccination of susceptible (BALB/c), and resistant (C57BL/6) mice . Recombinant BCG producing Gp63 as a hybrid protein with the N-terminal region of the beta-lactamase elicited significant protection against a challenge with L . major in BALB/c-immunized mice.

Pediatr Pulmonol, 1995 Jul, 20(1), 40 - 3
Sensitivity and specificity of oropharyngeal suction versus bronchoalveolar lavage in identifying respiratory tract pathogens in children with chronic pulmonary infection; Avital A et al.; The purpose of this study was to compare the pathogens obtained by oropharyngeal suction (OPS) and bronchoalveolar lavage (BAL) in children with chronic pulmonary infections . Forty-four children (mean age of 6.1 years; range 4 months to 15 years) were included in the study (27 children with recurrent localized lung infection, 5 with bronchiectasis, 5 with cystic fibrosis, 2 with foreign body aspiration, 2 with bronchiolitis obliterans, and 3 with recurrent episodes of cough and shortness of breath) . In 27 out of 44 BAL samples (61%) bacterial cultures were positive . The sensitivity of OPS in detecting the same BAL pathogen was 89% (24/27 samples), the specificity was 94% (16/17 samples), and the predictive value was 91% (40/44 samples) . Hemophilus influenzae beta-lactamase negative was the main organism recovered from BAL in non-cystic fibrosis patients with recurrent or persistent pneumonia . We conclude that OPS is a simple and efficient noninvasive procedure which may be helpful in the diagnosis and treatment of recurrent or chronic pulmonary infection.

Structure, 1995 Jun 15, 3(6), 603 - 13
Electrostatic analysis of TEM1 beta-lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations; Swaren P et al.; BACKGROUND: Escherichia coli TEM1 is a penicillinase and belongs to class A beta-lactamases . Its naturally occurring mutants are responsible for bacterial resistance to beta-lactamin-based antibiotics . X-ray structure determinations show that all class A beta-lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated . We address the questions of what the molecular contexts during the acylation and deacylation steps are and how they contribute to the efficiency of these penicillinases . RESULTS: Electrostatic analysis of the 1.8 A resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl-enzyme complexes, showed that substrate binding induces an upward shift in the pKa of the unprotonated Lys73 by 6.4 pH units . The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote acylation . In the acyl-enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the estercarbonyl carbon of the acyl-enzyme complex, in an electrostatic potential gradient amounting to 30 kTe-1 A-1 . Other residues, not directly involved in catalysis, also contribute to the formation of this gradient . The deacylation rate is related to the magnitude of the gradient . The kinetic behavior of site-directed mutants that affect the protonation state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism . CONCLUSIONS: In the wild-type enzyme, the very high rates of acylation and deacylation of class A beta-lactamases arise from an optimal chemical setup in which the acylation reaction seems triggered by substrate binding that changes the general base property of Lys73 . In site-directed mutants where Lys73 is protonated, acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.

J Med Microbiol, 1995 Jun, 42(6), 429 - 32
Back mutations to the TEM-1 beta-lactamase from TRC-1 lead to restored sensitivity to clavulanic acid; Thomson CJ et al.; Back mutations from the TRC-1 beta-lactamase to the TEM-1 enzyme were selected in vitro . The revertant beta-lactamase was obtained from Escherichia coli strain J62.2 carrying plasmid pUK901 which encodes the TRC-1 beta-lactamase . The revertant was obtained after repeated subculture of E . coli J62.2 (pUK901) in amoxycillin 512 mg/L for 5 days . The revertant beta-lactamase had the same pI as TEM-1 (5.4) and had restored inhibition by clavulanic acid (ID50 reduced from 4.2 microM to 0.15 microM) . The prevalence of these beta-lactamases in the clinical population may be the result of a two-way flux, with mutations in both forward and backward directions.

J Bacteriol, 1995 Jun, 177(12), 3596 - 600
Physiological and biochemical analysis of the effects of alkaline phosphatase overproduction in Escherichia coli; Kadokura H et al.; Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase (PhoA), on multicopy plasmids caused a severe defect in the precursor processing (secretion) of PhoA, beta-lactamase, and the outer membrane protein OmpA . This secretion defect continued even after the repression of phoA expression, indicating that protein secretion was irreversibly impaired in cells . Among the secretory proteins, only OmpA gradually secreted posttranslationally . The inverted inner membrane vesicles prepared from cells with the secretion defect showed appreciably reduced translocation activity in vitro . But the membrane vesicles retained the ability to generate a proton motive force which, together with ATP, is essential as an energy source for the efficient secretion of proteins in E . coli . An appreciable amount of incompletely translocated PhoA molecules was detected in the inner membranes of cells with the secretion defect.

J Virol Methods, 1995 Jun, 53(2-3), 263 - 72
Generation of an expression library in the baculovirus expression vector system; Ward VK et al.; The construction and screening of a small cDNA library consisting of 2 x 10(4) clones in the baculovirus expression vector system are described . This library consists of antibody heavy chain sequences isolated from the spleen of a mouse immunized with tetanus toxoid fragment C . A portion of this library was used to produce a pool of recombinant baculoviruses which were screened for production of antibody fragments reactive to tetanus toxoid without prior expression in Escherichia coli . The pool of 30 clones was found to contain at least 6 different populations of antibody indicating that diversity existed within the library . Positive clones were isolated from the baculovirus system and confirmed as being capable of producing a tetanus reactive antibody by expression as a beta-lactamase fusion protein in E . coli . One of these clones was returned to the baculovirus system using a different transfer vector, and tetanus binding reconfirmed . The results presented here show that the concept of the construction and screening of a baculovirus expression library is feasible even with 'difficult' proteins, such as antibody heavy chain fragments, and that the baculovirus expression vector system has the potential to produce cDNA expression libraries which can be screened directly for the desired protein.

Antimicrob Agents Chemother, 1995 Jun, 39(6), 1379 - 82
Molecular characterization of the OXA-7 beta-lactamase gene; Scoulica E et al.; The OXA-7 gene, which encodes an oxacillinase, was cloned from plasmid pMG202 of Escherichia coli isolate 7181 (A . A . Medeiros, M . Cohenford, and G . A . Jacoby, Antimicrob . Agents Chemother . 27:715-719, 1985) and sequenced . The nucleotide sequence of the OXA-7 gene was closely related to that of the OXA-10 (PSE-2) gene, with a derived amino acid sequence of the OXA-7 enzyme showing greater than 95% homology with those of OXA-10 and OXA-11.

Proteins, 1995 Jun, 22(2), 110 - 8
Investigation of the folding pathway of the TEM-1 beta-lactamase; Vanhove M et al.; The TEM-1 beta-lactamase is a globular protein containing 12 proline residues . The folding mechanism of this enzyme was investigated by kinetic and equilibrium experiments with the help of fluorescence spectroscopy and circular dichroism . The equilibrium denaturation of the protein induced by guanidine hydrochloride occurs in two discrete steps, indicating the existence of a thermodynamically stable intermediate state . This state is 5.2 +/- 0.4 kcal/mol less stable than the native conformation and 5.7 +/- 0.2 kcal/mol more stable than the fully denatured protein . This intermediate state exhibits a high content of native secondary structure elements but is devoid of specific tertiary organization; its relation to the "molten globule" is discussed . Refolding kinetic experiments revealed the existence of a transient intermediate conformation between the thermodynamically stable intermediate and the native protein . This transient intermediate appears rapidly during the folding reaction . It exhibits a secondary structure content very similar to that of the native protein and has also recovered a significant amount of tertiary organisation . The final refolding step of the TEM-1 beta-lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probably reflect a very complex process kinetically limited by proline cis/trans isomerization.

Antimicrob Agents Chemother, 1995 Apr, 39(4), 899 - 905
SHV-7, a novel cefotaxime-hydrolyzing beta-lactamase, identified in Escherichia coli isolates from hospitalized nursing home patients; Bradford PA et al.; Four ceftazidime-resistant Escherichia coli strains were isolated from elderly nursing home patients in a New York hospital during 1993 . Strains MCQ-2, MCQ-3, and MCQ-4 were determined to be identical by pulsed-field gel electrophoresis and plasmid profiles, whereas strain MCQ-1 was unique . Strain MCQ-1 was determined to produce a TEM-10 beta-lactamase . Strains MCQ-2, MCQ-3, and MCQ-4 were also noted to be resistant to cefotaxime . These three strains produced two beta-lactamases with pIs of 5.4 (TEM-1) and 7.6 . beta-Lactamase assays revealed that the pI 7.6 enzyme hydrolyzed cefotaxime faster (at a relative hydrolysis rate of 30% compared with that of benzylpenicillin) than either ceftazidime or aztreonam (relative hydrolysis rates of 13 and 3.3%, respectively) . Nucleotide sequencing of the gene encoding the pI 7.6 beta-lactamase from strain MCQ-3 revealed a blaSHV-type gene differing from the gene encoding SHV-1 at four nucleotides which resulted in amino acid substitutions: phenylalanine for isoleucine at position 8, serine for arginine at position 43, serine for glycine at position 238, and lysine for glutamate at position 240 . This novel SHV-type extended-spectrum beta-lactamase is designated SHV-7.

Appl Microbiol Biotechnol, 1995 Apr, 43(1), 89 - 92
Influence of stringent and relaxed response on excretion of recombinant proteins and fatty acid composition in Escherichia coli; Gitter B et al.; In contrast to stringent (relA+) cells of Escherichia coli, relaxed (relA) cells excreted recombinant proteins (beta-lactamase, interferon alpha 1) into the culture medium during amino acid limitation . Comparative analyses of overall fatty acid composition in relA+ cells and relA cells were performed and revealed that, in wild-type cells, drastic alterations occurred during the stringent response . The portion of saturated fatty acids (C16:0) and the fractions of cyclopropane fatty acids (C17cyc and C19cyc) increased whereas the portions of unsaturated fatty acids (C16:1 and C18:1) decreased . In cells of the relaxed mutant, no significant changes in the overall composition of the fatty acids were observed after the onset of amino acid limitation . These results indicate that a change in fatty acid composition of membrane lipids after starvation of cells may be responsible for the prevention of loss of cellular proteins into the culture medium in stringent controlled cells of Escherichia coli.

J Chemother, 1995 Apr, 7(2), 106 - 8
Beta-lactamase activity in mycobacteria other than M . tuberculosis; Coira A et al.; Beta-lactamase activity was studied in 142 non-tuberculosis mycobacterial strains . The distribution according to species was as follows: four M . avium, 14 M . chelonae, 10 M . fortuitum, 59 M . gordonae, 55 M . kansasii . The spectrum of hydrolysis of the beta-lactamases was screened using an acidimetric method and the characterization was performed by analytical isoelectric focusing.

Pathol Biol (Paris), 1995 Apr, 43(4), 306 - 9
{Detection of beta-lactamases resistant to inhibitors (IRT) by the disk diffusion method}; Romaszko JP et al.; 85 E . coli strains producing various beta-lactamase were studied: TEM low level: n = 11, TEM high level: n = 4, oxacillinase (OXA): n = 5, cephalosporinase (Case) +/- TEM: n = 5, inhibitor resistant TEM (IRT) n = 50, IRT+TEM: n = 3, IRT+Case: n = 7 . Their susceptibility to amoxicillin (AMX), amoxicillin+clavulanate (AMC), ticarcillin (TIC), ticarcillin+clavulanate (TCC), piperacillin (PIP), piperacillin+tazobactam (TAZ), mecillinam (MEC), cephalotin (CF), cefoxitin (FOX) et ceftazidime (CAZ) were evaluated by a disk diffusion method, in order to determine the resistance pattern which allows a reliable detection of IRT-producing strains . The phenotype AMX R, TIC R, AMCI/R, TCCI/R et CFS was observed in 46 out of 53 IRT +/- TEM strains, 4/5 of OXA strains and 1/11 of TEM low level strains . A better sensitivity could be obtained by a modification of breakpoints for AMC, TCC and CF, however OXA-producing strains remain indistinguishable from IRT-strains.

J Chromatogr B Biomed Appl, 1995 Mar 24, 665(2), 363 - 71
Rapid determination of sulbactam and tazobactam in human serum by high-performance liquid chromatography; Guillaume Y et al.; A simple and rapid HPLC method for the determination of tazobactam and sulbactam, two beta-lactamase inhibitors, in serum for the therapeutic follow-up of patients is described . The effect of the pH of the aqueous mobile phase and column temperature on column efficiency and retention were examined and equations for their dependences were derived . The use of a chromatographic response function showed that methanol-buffer (5:95, v/v) (pH 6.3) as the mobile phase and a 45 degrees C column temperature were optimum values for chromatographic separation . The analytical method was linear from 10 to 200 micrograms/ml . This assay limit range is sufficient for the analysis of human serum . The limit of detection was 10 micrograms/ml for sulbactam and 5 micrograms/ml for tazobactam . The coefficient of variation was less than 5% . The speed at which this assay can be performed makes it especially useful for estimating the levels of these drugs in human serum.

Am J Health Syst Pharm, 1995 Mar 15, 52(6 Suppl 2), S23 - 8
Combination beta-lactam and beta-lactamase-inhibitor therapy: pharmacokinetic and pharmacodynamic considerations; Dudley MN; Pharmacokinetic and pharmacodynamic considerations in in vitro susceptibility testing are described, and the integration of pharmacokinetic and pharmacodynamic concepts in dosage-regimen design is explored . Both the amount of beta-lactamase produced per unit of time and the amount of beta-lactamase inhibitor supplied greatly influence susceptibility to beta-lactam antibiotics . The goal should be to supply enough beta-lactamase to render the bacteria functionally beta-lactamase negative . In susceptibility testing, the amount of inhibitor may be more important than the ratio between the beta-lactam antibiotic and the inhibitor . Irreversible inactivation of beta-lactamases by inhibitors allows for a period of killing of bacteria by beta-lactams, even when concentrations of the inhibitor fall to concentrations below those tested in vitro . The integration of pharmacokinetic and pharmacodynamic concepts allows for comparisons between in vitro and in vivo drug exposure . With some antibiotic-inhibitor combinations, it may be possible to extend the dosage interval if an adequate amount of inhibitor is provided over the course of therapy.

J Biol Chem, 1995 Mar 3, 270(9), 4262 - 9
Expression and purification of two isozymes of clavaminate synthase and initial characterization of the iron binding site . General error analysis in polymerase chain reaction amplification; Busby RW et al.; Clavaminate synthase is an Fe(2+)-, O2-, and alpha-ketoglutarate-dependent oxygenase that catalyzes three transformations in the biosynthesis of the important beta-lactamase inhibitor clavulanic acid . The genes from Streptomyces clavuligerus encoding two isoenzymes of clavaminate synthase have been over-expressed in Escherichia coli to give soluble proteins whose reactions, kinetic properties, and molecular masses are in excellent agreement with the wild-type isozymes . Preliminary investigation of the active site of clavaminate synthase was undertaken using diethyl pyrocarbonate and N-ethylmaleimide . Each was inhibitory to catalytic activity . Protection from inactivation in the presence of these reagents by Fe2+, O2, and alpha-ketoglutaric acid was thwarted by the rapid self-inactivation of the enzyme in the absence of substrate . However, protection was achieved when Co2+, a potent competitive inhibitor of clavaminate synthase 2 with respect to Fe2+, was substituted . This is consistent with the presence of histidine and cysteine, respectively, at or near the active site and possibly involved in iron binding . In the course of constructing the expression vector, a simply applied general error analysis of the polymerase chain reaction was formulated to calculate the proportion of correctly replicated DNA and guide the design of experiments using this method.

J Bacteriol, 1995 Mar, 177(5), 1137 - 43
Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export; Rosenow C et al.; In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster . This gene cluster is composed of three regions . The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression . The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced . Region 1 contains the kpsE, -D, -U, -C, and -S genes . In this communication we describe the KpsE protein, the product of the kpsE gene . A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE') . This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits . Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside . It was purified by affinity chromatography with immobilized anti-KpsE antibodies . Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain . By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.

J Antimicrob Chemother, 1995 Mar, 35(3), 429 - 33
An enzymatic method for assaying sulbactam in human serum: comparison with high performance liquid chromatography; Sotto A et al.; An enzymatic method using nitrocefin as substrate was developed to assay sulbactam in human serum . Serum containing sulbactam was incubated with purified titrated TEM-1 beta-lactamase and nitrocefin was then added to the mixture to determine the remaining beta-lactamase activity and consequently the concentration of sulbactam . Assays were carried out on five patients with pulmonary infections receiving sulbactam plus amoxycillin iv . The values for serum sulbactam concentrations determined by the enzymatic method were compared with those determined by high performance liquid chromatography (HPLC) . The correlation coefficient was 0.990 for serum sulbactam concentrations below 15 mg/L.

Biochem J, 1995 Feb 15, 306 ( Pt 1), 123 - 8
Kinetic study in vitro of Escherichia coli promoter closure during transcription initiation; Schmitt B et al.; The rate of closure of two Escherichia coli promoters borne by plasmid pBR322, following transcription initiation from the open complex, was probed in vitro by the protection of unpaired thymines in the open complex against oxidation by KMnO4 . Run-off transcription kinetics were also studied under identical conditions . Closure of the open promoter appears to be by far the rate-limiting step of transcription initiation and elongation for the linearized beta-lactamase gene, and is strongly dependent on template topology for the RNAI gene . It is suggested that the corresponding signals are deposited 30 bases at least downstream of transcription initiation and that promoter closure, and its clearance by elongating RNA polymerase, may occur almost simultaneously.

FEMS Microbiol Lett, 1995 Feb 1, 126(1), 49 - 54
Genomic heterogenicity amongst phenotypically similar Legionella micdadei strains; Luck PC et al.; Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis . All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media . They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, beta-lactamase and gelatinase . None of the strains showed autofluorescence under long-wave ultraviolet light . A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L . micdadei strains . None of these monoclonal antibodies reacted with L . maceachernii and L . longbeachae serogroup 2, the only species that cross-react with polyclonal antisera . Each of the nine L . micdadei strains showed individual restriction patterns of the genomic DNA when using both SfiI and NotI restriction enzymes in the pulsed-field gel electrophoresis . Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L . micdadei.

Mol Microbiol, 1995 Feb, 15(4), 771 - 83
Topological analyses of the L-fucose-H+ symport protein, FucP, from Escherichia coli; Gunn FJ et al.; The transport of L-fucose into Escherichia coli is mediated by the L-fucose-H+ symport protein (FucP) . The fucP gene has been sequenced and encodes a hydrophobic protein that contains 438 amino acid residues, with a predicted M(r) of 47,773 . The hydropathic profile of FucP indicates 10 to 12 hydrophobic regions that could span the membrane as alpha-helices . A 12-helix model with the N- and C-termini located in the cytoplasm was derived from the hydropathic profile and from application of the 'positive inside' rule . This model was tested using beta-lactamase fusion technology . Analyses of 62 different FucP-beta-lactamase fusions suggested that the FucP protein crosses the cytoplasmic membrane of E . coli 12 times, with the N- and C-termini in the cytoplasm . From measurements of {14C}-L-fucose uptake, it was deduced that the last putative transmembrane region must be complete for transport activity to be retained and that the four C-terminal residues were unnecessary for transport activity . Fourier transform analyses show that all the predicted helices contain a periodicity that enables hydrophobic/hydrophilic faces to be identified; these were particularly evident in putative helices 1, 3, 4, 5, 6, 10 and 11.

J Chemother, 1995 Feb, 7(1), 3 - 7
DD-peptidases and beta-lactamases: catalytic mechanisms and specificities; Galleni M et al.; DD-peptidases and beta-lactamases share several common properties, including the formation of an acylenzyme intermediate in their catalytic pathways . In their interactions with beta-lactam antibiotics, the stability of this intermediate is much higher with the peptidases than with the beta-lactamases . The structural factors responsible for this difference have not been identified . The evolution of beta-lactamases is taking place before our eyes, since mutants are constantly selected which can hydrolyze the molecules newly introduced as "beta-lactamase resistant" in the chemotherapeutic arsenal.

J Biol Chem, 1995 Jan 13, 270(2), 781 - 7
A new TEM beta-lactamase double mutant with broadened specificity reveals substrate-dependent functional interactions; Viadiu H et al.; Using a random combinatorial mutagenesis of TEM beta-lactamase, directed against residues potentially involved in substrate discrimination, followed by selection on third generation cephalosporins, we obtained the double mutant E104M/G238S . Additionally, by using cloning strategies and site-directed mutagenesis we constructed the individual single mutants and also the single modification E104K and the double mutant E104K/G238S, which broaden the specificity of clinically isolated TEM beta-lactamase variants . The kinetic characterization of the purified double mutant E104M/G238S and its single counterparts E104M and G238S was carried out . The single mutant E104M exhibited increased kcat values against all substrates tested . Km values remained similar to the values shown by the wild-type enzyme . The mutation at E104M was responsible for the increased hydrolysis rate against cefuroxime shown by the double mutant E104/G238S . The effect of mutation G238S varied more pronouncedly, depending on the substrate . In general, a lower Km was observed, but also a decreased kcat . The double mutant E104M/G238S exhibited a higher hydrolytic rate against cefotaxime compared with the corresponding single mutations . We observed nearly a 1000-fold greater kcat/Km for the double mutant than for the wild type . This improvement in catalysis was the consequence of increased kcat and decreased Km values . Computed contact interactions from modeling substrate complexes show reliable results only for benzylpenicillin . The modeling results with this substrate confirmed the observed enzyme activities for the different single and double mutants . Analysis of the apparent coupling energies, as calculated from the kinetic parameters of the single and double mutants, showed that the quantitative effect of a second mutation on a single mutant was either absent, additive, partially additive, or synergistic with respect to the first mutation, depending on the substrate analyzed.

J Bacteriol, 1995 Jan, 177(1), 59 - 65
Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions; Lim EM et al.; The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane . A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria . Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M . smegmatis of protein fusions with PhoA activity . A genomic library from M . tuberculosis was constructed in pJEM11 and screened in M . smegmatis for clones with PhoA activity . Sequences of the M . tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined . They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M . leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences . This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M . tuberculosis.

J Antimicrob Chemother, 1995 Jan, 35(1), 7 - 22
TEM- and SHV-derived extended-spectrum beta-lactamases: relationship between selection, structure and function; Du Bois SK et al.; The later-generation cephalosporins were developed to overcome beta-lactamases which conferred resistance to earlier beta-lactam drugs . Within two years of their clinical introduction, the ubiquitous TEM and SHV plasmid-encoded beta-lactamase genes underwent simple point mutations that changed key amino acids around the active site of the protein and enabled the enzyme to bind and hydrolyse these new drugs . Successive mutations interacted in concert, radically increasing the enzymes' abilities to bind and confer resistance to later-generation cephalosporins . These modified enzymes have been classified in three groups, based on activity, and altered functions have been correlated with changes in the enzyme structure.

Antimicrob Agents Chemother, 1995 Jan, 39(1), 227 - 31
Kinetic study of interaction between BRL 42715, beta-lactamases, and D-alanyl-D-alanine peptidases; Matagne A et al.; A detailed kinetic study of the interactions between BRL 42715, a beta-lactamase-inhibiting penem, and various beta-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4) is presented . The compound was a very efficient inactivator of all active-site serine beta-lactamases but was hydrolyzed by the class B, Zn(2+)-containing enzymes, with very different kcat values . Inactivation of the Streptomyces sp . strain R61 extracellular DD-peptidase was not observed, and the Actinomadura sp . strain R39 DD-peptidase exhibited a low level of sensitivity to the compound.

Antimicrob Agents Chemother, 1995 Jan, 39(1), 260 - 3
Cloning, sequencing, and site-directed mutagenesis of beta-lactamase gene from Streptomyces fradiae Y59; Kurai S et al.; The beta-lactamase gene from Streptomyces fradiae Y59 was cloned and sequenced . To determine which amino acid residues are critical in binding activity to blue dextran, chimera beta-lactamases were constructed and their binding abilities were determined . The results suggested that blue dextran binding may depend more on overall conformation of about two-thirds of the beta-lactamase molecule from the N terminus than on the primary structure.

Gene, 1994 Dec 30, 151(1-2), 131 - 5
Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli; Skerra A; A generic vector, pASK75, was developed for the synthesis of foreign proteins in Escherichia coli under transcriptional control of the tetA promoter/operator . Tight regulation was achieved by placing the structural gene for the tet repressor, as a transcriptional fusion, downstream from the beta-lactamase-encoding gene (bla) on the same plasmid . Strong expression of the foreign gene was conveniently induced by adding anhydrotetracycline at a low concentration . Using the production of a recombinant murine immunoglobulin F(ab) fragment as an example, the system was shown to function independently of the host-strain background and to be extremely well repressed in the absence of the inducer . Thus, it represents an economic and independent alternative to IPTG-inducible promoter constructs . Additional features of pASK75 include a signal sequence and a multiple cloning site followed by a region encoding the Strep tag affinity peptide to facilitate purification of a bacterially produced protein.

J Mol Biol, 1994 Dec 16, 244(5), 625 - 39
TEM beta-lactamase mutants hydrolysing third-generation cephalosporins . A kinetic and molecular modelling analysis; Raquet X et al.; The catalytic properties of six "natural" mutants of the TEM-1 beta-lactamase have been studied in detail, with special emphasis on their activity versus third-generation cephalosporins . On the basis of the recently determined high-resolution structure of the wild-type enzyme, and of the substrates' structures optimized by the AMI quantum chemistry method, we have attempted to explain the influences of the mutations on the substrate profiles of the enzymes . Some of the kinetic results have thus received a satisfactory, semi-quantitative interpretation, especially in the case of single mutations . Analysis of the double mutants proved more hazardous . Extending the comparison to some other class A beta-lactamases showed that similar properties could result from different sequences, supplying an interesting example of convergent evolution within a generally diverging family.

Gene, 1994 Dec 2, 150(1), 193 - 4
The construction of a beta-lactamase-encoding ApR gene cassette flanked by recognition sites for NotI, SacII, MluI, SplI, BssHII and NarI; Hoglund M et al.; An ApR gene cassette was constructed using the gene present in the PBR322 derivative pTZ18 . The cassette is maintained in the plasmid pcLINK-1 and may be excised with any of the six rare cutting enzymes NotI, SacII, MluI, SplI, BssHII or NarI . By using a double-digestion procedure, the ApR gene may be excised with two different protruding ends . In the process of constructing the pcLINK-1 plasmid the multiplecloning site of pTZ18 was extended with recognition sites for the enzymes NotI, SacII, MluI, SplI, BssHII and NarI.

Appl Microbiol Biotechnol, 1994 Dec, 42(4), 595 - 603
Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli; Somerville JE Jr et al.; The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined . Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen . However, immediately after induction at either 30 degrees C or 37 degrees C there was a significant loss in bacterial viability, which was followed by a loss in absorbance . The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant . The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic beta-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis . Bacteria incubated at 30 degrees C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A660 at the lower incubation temperature . More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded . Osmotic-shock procedures did not release L6 sFv . However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location . French press disruption resulted in the release of correctly but not incorrectly folded material . Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane . These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis . It is also suggested that membrane association in the periplasmic space may impede proper folding.

Mol Microbiol, 1994 Dec, 14(5), 871 - 81
Topological and mutational analysis of KpsM, the hydrophobic component of the ABC-transporter involved in the export of polysialic acid in Escherichia coli K1; Pigeon RP et al.; The 17 kb kps gene cluster of Escherichia coli K1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule of E . coli K1, is divided into three functional regions . Region 3 contains two genes, kpsM and kpsT, essential for the transport of capsule polymer across the cytoplasmic membrane . The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP-binding site . KpsM and KpsT belong to the ATP-binding cassette (ABC) superfamily of membrane transporters . In this study, we investigate the topology of KpsM within the cytoplasmic membrane using beta-lactamase fusions and alkaline phosphatase sandwich fusions . Our analysis provides evidence for a model of KpsM having six membrane-spanning regions, with the N- and C-terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV-SVI linker localizing in the membrane . Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space . In vivo cross-linking studies provide support for dimerization of KpsM within the cytoplasmic membrane . Linker-insertion and site-directed mutagenesis define the N-terminus, the first cytoplasmic loop, and the SV-SVI linker as regions that are important for the function of KpsM in K1 polymer transport.

Hum Gene Ther, 1994 Dec, 5(12), 1445 - 55
Delivery of a secretable adenosine deaminase through microcapsules--a novel approach to somatic gene therapy; Hughes M et al.; Many current gene therapy protocols require genetic modification of autologous cells . An alternate approach is to use universal recombinant cell lines engineered to secrete in vivo the desired gene products . Enclosing these cells within immunoprotective devices before implantation would prevent rejection of the nonautologous donor cells . To overcome the limitation that not all therapeutic gene products are secreted, we now propose to fuse a signal sequence to the amino terminus of a nonsecreted protein such as human adenosine deaminase (ADA), thus directing the product into a secretory pathway for release from the cells . A fusion gene constructed between the cDNA of the beta-lactamase signal sequence and human ADA expressed a product after in vitro transcription and translation that was immunologically similar to the human protein . Mouse fibroblasts transfected with the fusion gene demonstrated secreted ADA activity that resembled the human cytosolic enzyme in its heat stability, pH optimum, KM, electrophoretic mobility, and immunologic reactivity . Hence, the secreted enzyme expressed from the fusion gene is antigenically and enzymatically similar to the authentic human form . When transfected mouse fibroblasts or myoblasts were enclosed in permselective alginate-poly-L-lysine alginate microcapsules, ADA activity was secreted from the microcapsules and the cells remained viable for over 5 months . Hence, a secretable and functional human ADA has been constructed that can be delivered from recombinant cells within immunoprotective capsules . The success of this strategy provides the prototype for engineering nonsecreted gene products for therapy via this novel method of somatic gene therapy.

J Cell Biol, 1994 Nov, 127(3), 737 - 49
The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals; Waterham HR et al.; We describe the cloning of the Hansenula polymorpha PER1 gene and the characterization of the gene and its product, PER1p . The gene was cloned by functional complementation of a per1 mutant of H . polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim- phenotype) . The DNA sequence of PER1 predicts that PER1p is a polypeptide of 650 amino acids with no significant sequence similarity to other known proteins . PER1 expression was low but significant in wild-type H . polymorpha growing on glucose and increased during growth on any one of a number of substrates which induce peroxisome proliferation . PER1p contains both a carboxy- (PTS1) and an amino-terminal (PTS2) peroxisomal targeting signal which both were demonstrated to be capable of directing bacterial beta-lactamase to the organelle . In wild-type H . polymorpha PER1p is a protein of low abundance which was demonstrated to be localized in the peroxisomal matrix . Our results suggest that the import of PER1p into peroxisomes is a prerequisite for the import of additional matrix proteins and we suggest a regulatory function of PER1p on peroxisomal protein support.

Biotechniques, 1994 Nov, 17(5), 944 - 53
Increased protein expression through improved ribosome-binding sites obtained by library mutagenesis; Wilson BS et al.; This report describes a method whereby library mutagenesis combined with drug selection was used to generate unique and efficient ribosome-binding sites (RBS) for expressing recombinant proteins in Escherichia coli . The RBS was deleted from a vector expressing beta-lactamase and replaced with a 16-base sequence containing a library of mutations . Selection of the library with ampicillin yielded several unique RBS sequences that were more efficient than ompA RBS for expressing a bacterial (beta-lactamase) and a mammalian protein (single-chain Fv antibody) . The described approach provides a practical means to improve recombinant protein expression and, also, provides new sequences to further evaluate the complex regulatory mechanism underlying translation initiation.

Protein Sci, 1994 Nov, 3(11), 1953 - 60
Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli; Georgiou G et al.; The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli . The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl) . This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces . When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range . It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates . We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces . This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136) . Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose . These conditions exert a similar effect on the formation of inclusion bodies in vivo.

Gene, 1994 Oct 11, 148(1), 171 - 2
A novel Escherichia coli expression-export vector containing alkaline phosphatase as an insertional inactivation screening system; Guzman CA et al.; An Escherichia coli expression-export vector was constructed (pCGV1, 6.3 kb) containing the alkaline phosphatase structural gene (phoA) located downstream from the phage lambda pR and pL promoters positioned in tandem and the cIts857 gene encoding lambda thermosensitive repressor . The phoA gene is fused to DNA encoding a hybrid signal sequence that contains the N-terminal portion of the beta-lactamase (Bla) signal sequence and the C-terminal region of the PhoA signal sequence . Within the DNA encoding hybrid signal sequence, a unique NheI restriction site is present where polymerase chain reaction (PCR)-amplified genes may be cloned . The 5' PCR primers reconstitute the C-terminal portion of either the PhoA or Bla signal sequences to restore an intact signal peptide . Recombinant phoA- clones are selected on indicator plates containing 5-bromo-4-chloro-3-indolyl phosphate.

EMBO J, 1994 Oct 3, 13(19), 4684 - 94
Bacterial cell wall recycling provides cytosolic muropeptides as effectors for beta-lactamase induction; Jacobs C et al.; A mechanism for bacteria to monitor the status of their vital cell wall peptidoglycan is suggested by the convergence of two phenomena: peptidoglycan recycling and beta-lactamase induction . ampG and ampD, genes essential for beta-lactamase regulation, are here shown to be required for recycling as well . Cells lacking either AmpG or AmpD lose up to 40% of their peptidoglycan per generation, whereas Escherichia coli normally suffers minimal losses and instead recycles 40 or 50% of the tripeptide, L-alanyl-D-glutamyl-meso-diaminopimelic acid, from its peptidoglycan each generation . The ampG mutant releases peptidoglycan-derived material into the medium . In contrast, the ampD mutant accumulates a novel cell wall muropeptide, 1,6-anhydro N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (anhMurNAc-tripeptide), in its cytoplasm . This work suggests that AmpG is the permease for a large muropeptide and AmpD is a novel cytosolic N-acetylmuramyl-L-alanine amidase that cleaves anhMurNAc-tripeptide to release tripeptide, which is then recycled . These results also suggest that the phenomenon of beta-lactamase induction is regulated by the level of muropeptide(s) in the cytoplasm, since an ampD mutation that results in beta-lactamase expression even in the absence of a beta-lactamase inducer coincides with accumulation of anhMurNAc-tripeptide . The transcriptional regulator AmpR is presumably converted into an activator for beta-lactamase production by sensing the higher level of muropeptide(s) . This may be an example of a general mechanism for signaling the progress of external events such as cell wall maturation, cell division or cell wall damage.

J Bacteriol, 1994 Oct, 176(20), 6410 - 3
Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions; Prinz WA et al.; To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions . While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not . This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm.

Wei Sheng Wu Xue Bao, 1994 Oct, 34(5), 360 - 6
{A novel extended-spectrum beta-lactamase in a ceftazidime-resistant isolate of E . coli}; Cheng Y et al.; A novel extended-spectrum beta-lactamase (ESbla) encoded on a plasmid of approximately 7.5kb, conferring resistance to beta-lactams tested except cefoxitin and imipenem, was found in a ceftazidime-resistant isolate of E . coli form our hospital . The resistance to beta-lactams was transferred by conjugation to E . coli JP559 together with the aminoglycosides and sulfonamide resistance . Clavulanate, one of beta-lactamase inhibitors, inhibited its activity . This ESbla gene hybridized neither with an intra-genic fragment of SHV-1 nor with that of TEM-1 . The molecular origin of that novel ESbla needed to be further studied.

J Antimicrob Chemother, 1994 Oct, 34(4), 555 - 64
Inhibition of the renal excretion of tazobactam by piperacillin; Komuro M et al.; A 1:4 by weight of combination of tazobactam, a new beta-lactamase inhibitor, and piperacillin, is now under development in Japan . After bolus iv administration of the combination to beagle dogs, piperacillin both significantly raised the area under plasma concentration time curve (AUC0 approximately infinity) and significantly decreased the total body clearance (Cltot) of tazobactam . The percentage binding of tazobactam and piperacillin to dog and human serum protein was the same for the combination as for the individual compounds . Piperacillin significantly decreased the renal clearance (Clr) and the clearance ratio (Cr) of tazobactam in dogs . Further, probenecid significantly decreased Clr of both tazobactam and piperacillin, and the Cr of tazobactam and piperacillin approximately reached unity . These results indicate that piperacillin inhibits the renal excretion of tazobactam . Both tazobactam and piperacillin are secreted by a tubular anion transport system which is identical to the probenecid secretion system.

Antimicrob Agents Chemother, 1994 Oct, 38(10), 2452 - 3
Sequences of CAZ-3 and CTX-2 extended-spectrum beta-lactamase genes; Chanal C et al.; The nucleotide sequences of blaTEM genes coding for the extended-spectrum beta-lactamases CAZ-3 and CTX-2 were determined . The gene for CAZ-3 is identical to blaTEM-12b . The gene for CTX-2 differs from characterized blaTEM genes for extended-spectrum beta-lactamases by a new combination of already known mutations . We propose for CTX-2 the designation TEM-25.

Antimicrob Agents Chemother, 1994 Oct, 38(10), 2266 - 9
Effect of threonine-to-methionine substitution at position 265 on structure and function of TEM-1 beta-lactamase; Huang W et al.; TEM beta-lactamase variants with the amino acid substitutions R164S, E104K, G238S, and E240K (ABL numbering) display increased activity toward extended-spectrum cephalosporins . The T265M substitution is frequently found to be associated with the above substitutions in extended-spectrum beta-lactamases . However, the residue is located away from the active site in the three-dimensional structure and has been assumed to have no effect on catalysis . To examine the effect of the substitution on the structure and function of TEM beta-lactamase we constructed the following mutants: G238S, T265M, T265M:G238S, and T265M:G238S:E240K . Each enzyme was purified to homogeneity and the kinetic parameters kcat, Km and kcat/Km were determined for cefotaxime, ceftazidime, cephaloridine, and ampicillin . The results indicate that the T265M mutation has little effect on hydrolysis . In addition, we used immunoblotting to show that the substitution has little or no effect on the in vivo steady-state levels of beta-lactamase.

J Toxicol Sci, 1994 Oct, 19 Suppl 2, 233 - 47
{Reproductive and developmental toxicity studies of tazobactam/piperacillin or tazobactam (3)--Perinatal and postnatal study in rats with intraperitoneal administration}; Sato T et al.; Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor and piperacillin (PIPC) is an antibiotics which is used in clinical field widely . The combination of TAZ and PIPC (TAZ/PIPC), which is combined with TAZ and PIPC at rate of 1:4, has been developed because of PIPC is unstable to various beta-lactamases . Perinatal and postnatal toxicity were studied in rats given daily intraperitoneal doses of TAZ/PIPC (200, 800 or 1600 mg/kg/day) or TAZ (40, 320 or 1280 mg/kg/day) . TAZ/PIPC or TAZ were given from day 17 of pregnancy through day 21 of lactation . Total daily doses were administered in two equally divided doses . In this study, evaluation of the late stage of gestation, parturition, lactation and maternal behavior in adult rats and postnatal evaluation of the growth and development, and reproductive performance of the F1 generation occurred . In the TAZ/PIPC, maternal toxicity (decreased food consumption) was observed at 800 and 1600 mg/kg groups during perinatal period . A slight decrease in body weight gain during perinatal period and increased pup mortality and decreased pup weight in lactation period were observed at 1600 mg/kg group . An increase in stillbirths also was observed at 1600 mg/kg group . In the TAZ, maternal toxicity (decreased food consumption) was observed at all dosage groups during perinatal period . A decrease in body weight gain also were observed during perinatal period at 1280 mg/kg group . At maternotoxic doses of 320 and 1280 mg/kg groups, decreased pup weight were observed during lactation period . An increase in stillbirths also was observed at 1280 mg/kg group . Transient, significant decrease in pup body weights at 1280 mg/kg group in early postweaning period . No other effects occurred for the F1 generation rats . In conclusion, perinatal development and postnatal growth and development of offspring were affected only at the intermediate and high doses that caused maternal toxicity in this study . Therefore it is seemed that non-observed effect dose levels (NOELs) of TAZ/PIPC for dams is less than 200 mg/kg/day and that of TAZ is less than 40 mg/mg/day, and NOELs of TAZ/PIPC is 200 mg/kg/day and that of TAZ is 40 mg/kg/day for offspring under the condition of this study.

J Toxicol Sci, 1994 Oct, 19 Suppl 2, 215 - 32
{Reproductive and developmental toxicity studies of tazobactam/piperacillin or tazobactam (2)--Teratological study in rats with intravenously administration}; Sato T et al.; Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor and piperacillin (PIPC) is an antibiotics which is used in clinical field widely . The combination of TAZ and PIPC (TAZ/PIPC), which is combined with TAZ and PIPC at rate of 1:4, has been developed because of PIPC is unstable to various beta-lactamases . Teratogenic potential were studied in rats given daily intravenous doses of TAZ/PIPC (625, 1250, 2500 or 3750 mg/kg/day) or TAZ (125, 500 or 3000 mg/kg/day) . TAZ/PIPC or TAZ were given from day 7 to day 17 of pregnancy . Total daily doses were administered in two equally divided doses . The study includes postnatal evaluation of the growth and development and reproductive performance of the F1 generation . Maternal deaths occurred in all groups given TAZ/PIPC . The incidence (range of 3 to 6 animals/group) was not dose dependent . Maternal body weight was decreased in rats receiving 3000 mg/kg of TAZ and food consumption was reduced in all drug-treated groups . Slight decreases in fetal body weights were observed at some doses that caused maternal body-weight or food-consumption decreases (2500 or 3750 mg/kg of TAZ/PIPC, 3000 mg/kg of TAZ) . But these depressions of fetal body weights were not significant from control data . There were no fetal malformations or variations attributable to the test articles . Postnatal growth and development, behavior and reproductive performance of the F1 generation were not affected by the administration of TAZ/PIPC or TAZ . In conclusion, TAZ/PIPC or TAZ was not teratogenic in the rats . It is seemed that non-observed effect dose levels (NOELs) of TAZ/PIPC and TAZ for dams is less than 625 and 125 mg/kg/day in general toxicity respectively, however, NOELs of TAZ/PIPC is 3750 mg/kg/day or more and that of TAZ is 300 mg/kg/day or more for their offspring under the condition of this study.

J Toxicol Sci, 1994 Oct, 19 Suppl 2, 199 - 24
{Reproductive and developmental toxicity studies of tazobactam/piperacillin or tazobactam(1)--Fertility and general reproduction study in rats with intraperitoneal administration}; Sato T et al.; Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor and piperacillin (PIPC) is an antibiotics which is used in clinical field widely . The combination of TAZ and PIPC (TAZ/PIPC), which is combined with TAZ and PIPC at rate of 1:4, has been developed because of PIPC is unstable to various beta-lactamases . Fertility and general reproductive performance were studied in rats given daily intraperitoneal doses of TAZ/PIPC (200, 800 or 1600 mg/kg/day) or TAZ (40, 160 or 640 mg/kg/day) . TAZ/PIPC or TAZ were given during premating period (70 days in males and 15 days in females), the pairing period (in males and females) and the gestation and lactation periods (in females) . Total daily doses were administered in two equally divided doses . The study includes evaluation of the F1 generation and the F2 generation through weaning . In the TAZ/PIPC, maternal toxicity (decreased food consumption) was observed at 200 mg/kg and above dosage groups . At maternotoxic doses of 800 and 1600 mg/kg groups, increased resorptions, decreased live litter size, and increased fetal variations (reversible changes in ribs) were observed . Reversible delays in ossification of caudal vertebrae were also observed at 1600 mg/kg group . In the TAZ, maternal toxicities were observed at 160 mg/kg group (decreased food consumption) and 640 mg/kg group (decreased body weight gain and food consumption) . Furthermore, necropsy (raised and/or colored areas present in the cecum) revealed slight increases at 40 mg/kg and above dosage groups . Slight decreases in implantations and resultant slight decreases in live litter size, reversible delays in renal development, and increased stillbirths were observed at 640 mg/kg group . Postnatal growth and development, behavior and reproductive performance of the F1 generation were not affected by the administration of TAZ/PIPC or TAZ . There were no effects on any of the fetal or pup parameters evaluated in the F2 generation . In conclusion, mating behavior and fertility were not affected by TAZ/PIPC or TAZ in this study . TAZ/PIPC or TAZ caused adverse change in reproductive performance of the F0 generation only at doses that caused maternal toxicity . The F1 and F2 generation were not affected.(ABSTRACT TRUNCATED AT 400 WORDS)

J Toxicol Sci, 1994 Oct, 19 Suppl 2, 177 - 97
{A six-month intravenous repeated dose toxicity study of tazobactam/piperacillin and tazobactam in dogs}; Hayashi T et al.; Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor . Tazobactam/Piperacillin (TAZ/PIPC) is a formulation consisting of TAZ and PIPC in a ratio of 1:4 . A six-month intravenous repeated dose toxicity study of TAZ/PIPC and TAZ including a one-month recovery period were carried out using male and female dogs . The doses were 200, 400 and 800 mg/kg/day for TAZ/PIPC, and 40, 80 and 160 mg/kg/day for TAZ . The results were as follows . 1 . No test article-related deaths occurred during the study period . No effects on clinical findings, body weight and food consumption were evident . 2 . No test article-related changes were noted in hematological, serum biochemical and urinalysis evaluations, and opthalmological and electrocardiographic examinations . 3 . There were no test article-related changes in macroscopic findings or organ weight . 4 . The histopathological examination revealed deposition of marked PAS-positive aggregates in liver cells of dogs given 400 mg/kg/day or more of TAZ/PIPC and 80 mg/kg/day or more of TAZ . Electron micrographs of hepatocytes revealed glycogen granules to be accumulated in the cytoplasm, and an increase of smooth endoplasmic reticulum . 5 . After a one-month recovery period, the histopathological changes had generally disappeared, suggesting that they were reversible . 6 . From the histopathological changes of liver, the no-toxic dose levels for TAZ/PIPC and TAZ were 200 mg/kg/day and 40 mg/kg/day, respectively.

J Toxicol Sci, 1994 Oct, 19 Suppl 2, 155 - 76
{A six-month intraperitoneal repeated dose toxicity study of tazobactam/piperacillin and tazobactam in rats}; Hayashi T et al.; Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor . Tazobactam/Piperacillin (TAZ/PIPC) is a formulation consisting of TAZ and PIPC in a ratio of 1:4 . A six-month intraperitoneal repeated dose toxicity study of TAZ/PIPC and TAZ including a one-month recovery period were carried out using male and female rats . The doses were 200, 400 and 800 mg/kg/day for TAZ/PIPC, and 40, 80 and 160 mg/kg/day for TAZ . The results were as follows . 1 . No test article-related deaths occurred during the study period . No effect on clinical finding of survival rats was evident . 2 . There was no dose-related increases of food consumption in both the males and females given TAZ/PIPC and PIPC . Slight reductions in body weight gain occurred in males given 800 mg/kg/day of TAZ/PIPC . 3 . Decreases in erythrocyte, hemoglobin and hematocrit, and increases in reticulocytes were seen only at study termination in the group given 800 mg/kg/day of TAZ/PIPC . Increases in reticulocytes were seen only at study termination in the females given 80 or 160 mg/kg/day of TAZ . 4 . A decrease in triglyceride levels was observed in the males given 800 mg/kg/day of TAZ/PIPC or 160 mg/kg/day of TAZ . 5 . The ophthalmoscopic examination or urinalysis show no test article-related changes . 6 . Enlarged ceca in all groups of animals given TAZ/PIPC and in the females given 160 mg/kg/day of TAZ were observed . 7 . An increase of relative organ weight in liver was noted in the males and females given 800 mg/kg/day of TAZ/PIPC, in the males given 80 or 160 mg/kg/day of TAZ and in the females given 160 mg/kg/day of TAZ . 8 . In the hepatocytes, accumulation of PAS-positive materials which was identified histochemically and ultrastructurally as glycogen, was present in the males given 800 mg/kg/day of TAZ/PIPC and in the males given 80 or 160 mg/kg/day of TAZ . 9 . After a one-month recovery period, the changes of liver had generally disappeared, suggesting that they were reversible . 10 . From the histopathological changes of liver, the no-toxic dose level in both the males and females was 400 mg/kg/day and 40 mg/kg/day for TAZ/PIPC and TAZ, respectively.

What Is Nitrification?, What Is Prokaryote?, What is Food Microbiology?, What Is Environmental Microbiology?, What Is Genome?, o, Microbe, s, Bacteria, i, Microbiology, r, Microorganisms, r, Bacterium, n, Salmonella typhimurium, i, Bioreactor, r, Bacillus, n, Yeasts, e, Bacteroides, n, Bioremediation, r, Bacteriophage, s, Botulin, s, B. anthracis, o, Microorganisms, o, Pseudomonas aeruginosa, c, E coli O157, n, Escherichia coli, c, Bacteriological, o, Staphylococcus aureus, e, Bacteroides, a, Bacteriophage, a, Bacillus, n, Escherichia coli, n, Escherichia coli, a, Lactobacillus

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005