Beta lactamase

J Toxicol Sci, 1994 Oct, 19 Suppl 2, 145 - 53
{Single-dose toxicity studies of tazobactam/piperacillin and tazobactam}; 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 . Singe-dose toxicity studies in TAZ/PIPC and TAZ were carried out using mice and rats of both sexes and male dogs . The results were as follows . 1 . A common clinical sign in mice and rats administered TAZ/PIPC or TAZ by all routes was soft stool . Other signs in mice and rats included a decrease in spontaneous motor activity and/or a decreased respiratory rate for the intraperitoneal (i.p.), subcutaneous (s.c.) or intravenous (i.v.) route . The animals administered by the i.v . route showed tremor for mice and clonic convulsion for rats before death . Hyperemia, hemorrhage or edema of the lung, and hemorrhage of the digestive tract were observed in these animals at necropsy . An enlargement of the spleen was seen in some of the surviving animals treated with TAZ/PIPC . 2 . In dogs, TAZ/PIPC caused vomiting, and TAZ caused vomiting, respiratory abnormality, soft stool and diarrhea by the intravenous (i.v.) administration . 3 . TAZ/PIPC or TAZ caused clinical signs such as the loss of hair at the injection site for the s.c . route, and necrosis of the tail for the i.v . route in mice and rats, also caused limping of the injected anterior limb in dogs . Necrosis and hemorrhage at the injection site, and peritonitis by the i.p . injection were observed at necropsy . These findings were due to the irritation of TAZ/PIPC or TAZ.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Membr Biol, 1994 Oct-Dec, 11(4), 271 - 7
Structure and function of the class C tetracycline/H+ antiporter: three independent groups of phenotypes are conferred by TetA (C); Griffith JK et al.; The class C tetracycline/H+ antiporter, TetA(C), confers nine distinct phenotypes in Escherichia coli: resistance to tetracycline, reduced culture density at stationary phase (growth yield), increased supercoiling of plasmid DNA, delayed growth in succinate minimal medium, complementation of potassium uptake defects, increased susceptibility to cadmium, increased susceptibility to fusaric acid, increased susceptibility to bleomycin and increased susceptibility to several classes of cationic aminoglycoside antibiotics . These nine phenotypes were resolved into three 'linkage' groups based on their patterns of suppression by mutations of the tetA(C) gene of plasmid pBR322 . Group I includes resistance to tetracycline, increased susceptibility to cadmium and reduced growth yield . Group II includes delayed growth in succinate minimal medium and complementation of potassium uptake defects . Group III includes increased supercoiling of plasmid DNA and increased susceptibilities to fusaric acid, bleomycin and cationic aminoglycosides . Phenotypes of Groups II and III, but not Group I, also were conferred by a chimeric gene encoding a fusion between the N-terminal 34 residues of TetA(C) and the C-terminal 429 residues of a structurally-similar protein, the E . coli galactose/H+ symporter, GalP . In contrast, none of these phenotypes was conferred by a chimeric gene encoding a fusion between the N-terminal 34 residues of TetA(C) and a structurally-dissimilar protein, TEM beta-lactamase . These results demonstrate that the three groups of linked phenotypes are dependent on different elements of the TetA(C) amino acid sequence, implying that TetA(C) confers these phenotypes by at least three independent mechanisms.

J Biol Chem, 1994 Sep 23, 269(38), 23444 - 50
Characterization of TEM-1 beta-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime; Venkatachalam KV et al.; Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins such as cefotaxime and ceftazidime . To identify other amino acid substitutions that alter the activity of TEM-1 toward extended-spectrum cephalosporins, a random library was constructed that contained all possible amino acid substitutions over the 3-residue window of 238-241 (ABL numbering) . Mutants were selected for 100-fold greater ceftazidime resistance than wild-type . All mutants had a serine substitution at position 238, a lysine or arginine at position 240, and a small amino acid at position 241 . The role of each substitution was investigated by constructing individual G238S, E240K, and R241G substitutions as well as the G238S:E240K double mutant . Each enzyme was purified to homogeneity and the kinetic parameters kcat and Km were determined using several substrates . The G238S substitution increases catalytic efficiency for both ceftazidime and cefotaxime . However, to achieve large increases in catalytic efficiency, both G238S and the E240K substitutions are required . The R241G substitution results in a small increase in catalytic efficiency for only ceftazidime . The contribution of each residue to the transition-state stabilization energy was found to be additive indicating that the substitutions act independently to change the catalytic properties of the enzyme.

J Mol Biol, 1994 Sep 16, 242(2), 165 - 74
Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL . II . GroEL recognizes thermally unfolded mature beta-lactamase; Zahn R et al.; By thermal equilibrium measurements we found a three-state folding behavior of mature Escherichia coli beta-lactamase TEM2 . The thermodynamically stable intermediate H had no enzymatic activity, but a native-like secondary structure . State H was 9 kcal mol-1 less stable than the native state N and 4 kcal mol-1 more stable than the totally unfolded state U, which is consistent with urea equilibrium measurements of mature beta-lactamase measured under similar conditions . Between 38 degrees C and 50 degrees C there was a decrease in the apparent equilibrium constant for dissociation K'D of the complex between GroEL and mature beta-lactamase, at least partially caused by a decrease in the thermodynamic stability of the native form of mature beta-lactamase . GroEL-bound beta-lactamase was released either after addition of ATP, or in the presence of a competing substrate (i.e . a single-chain antibody), or after lowering the temperature . Whereas at 10 degrees C the folding reaction of mature beta-lactamase was rate limiting, at 37 degrees C the release reaction was the rate-determining step for the regain of beta-lactamase activity, consistent with a decrease of the equilibrium constant for dissociation KD of the complex with temperature . A temperature dependent behavior of GroEL was also observed, when measuring the anilinonaphthalene sulfonic acid (ANS) fluorescence of the chaperone . Similar to all other substrate proteins studied so far, the maximal tryptohan fluorescence of GroEL-bound beta-lactamase was observed at 342 nm . Our results are compatible with a hydrophobic binding pocket of GroEL and confirm the suggested thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL.

J Mol Biol, 1994 Sep 16, 242(2), 150 - 64
Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL . I . GroEL recognizes the signal sequences of beta-lactamase precursor; Zahn R et al.; From equilibrium measurements with urea we found a three-state thermodynamic and kinetic folding behavior for the precursor and mature form of Escherichia coli beta-lactamase TEM2 . The thermodynamic intermediate H of Escherichia coli beta-lactamase and its precursor had no enzymatic activity, and a quenched tryptophan fluorescence intensity, but a native-like wavelength of maximum intensity . State H of mature beta-lactamase was 8.7 kcal mol-1 less stable than the native state N and about 4.2 kcal mol-1 more stable than the unfolded state U, extrapolated to absence of urea . In contrast, state H of precursor beta-lactamase was even more stable than N by about 0.5 kcal mol-1 and about 6.9 kcal mol-1 more stable than U . Native pre-beta-lactamase could be stabilized by lowering the pH value from 7.0 to 5.5, probably by protonating a histidine residue leading to an improved solubility of the signal sequence . Synthetic peptides, containing 23 or 38 N-terminal amino acid residues of pre-beta-lactamase, were unable to compete with pre-beta-lactamase for binding to GroEL . However, GroEL prevented the inactivation of mature beta-lactamase by p38, consistent with competition between GroEL and mature beta-lactamase for binding to p38 . The equilibrium constant for dissociation KD of the complex between GroEL and p23, a peptide containing exclusively the signal sequence of pre-beta-lactamase, was measured with the BIAcore instrument to be in the range 10(-7) to 10(-8) M . Our results are consistent with co-operative binding of GroEL to the mature part and to the signal sequence of pre-beta-lactamase . We suggest a thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL.

FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 91 - 6
High pressure conditions stimulate expression of chloramphenicol acetyltransferase regulated by the lac promoter in Escherichia coli; Kato C et al.; Recombinant plasmids with the chloramphenicol acetyltransferase (CAT) structural gene behind several kinds of promoters were tested for expression in Escherichia coli during growth at atmospheric pressure (0.1 MPa) and at high pressure (30 MPa) . Expression of the CAT gene from the lac promoter was remarkably activated (approx . 78-fold) by high pressure in the absence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) . The stimulation of the CAT activity by the lac promoter at high pressure did not simply result from an increased plasmid copy number, because the CAT activities from the other promoters and beta-lactamase activities were unaffected at high pressure.

FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 159 - 64
The negative regulator of beta-lactamase induction AmpD is a N-acetyl-anhydromuramyl-L-alanine amidase; Holtje JV et al.; Construction of a malE-ampD gene fusion allowed purification of biologically active fusion protein by affinity chromatography . The cloned malE-ampD gene fusion complemented a chromosomal ampD mutation . Purified MalE-AmpD fusion protein was found to have murein amidase activity with a pronounced specificity for 1,6-anhydromuropeptides, the characteristic murein turnover products in Escherichia coli . Being a N-acetyl-anhydromuranmyl-L-alanine amidase AmpD is likely to be involved in recycling of the turnover products . It is suggested that the negative regulatory effect of AmpD is due to the hydrolysis of anhydro-muropeptides which may function as signals for beta-lactamase induction.

J Antibiot (Tokyo), 1994 Sep, 47(9), 1030 - 40
Synthesis and beta-lactamase inhibitory activity of 6-{(1-heteroarylthioethyl-1,2,3-triazol-4-yl)-methylene}penam sulfones; Im C et al.; The synthesis of beta-lactamase inhibitory activity of a series of sodium 6-{(1-heteroarylthioethyl-1,2,3-triazol-4-yl)methylene}pe nicillanate, 1,1-dioxides are described . Their activity was compared with tazobactam and sulbactam . The Z-isomers were more active than the E-isomers . The in vitro activity of the Z-isomers of the phenylthiadiazole derivatives (13a and 15a) was better than sulbactam against the tested beta-lactamases and comparable to tazobactam especially against TEM-2 and cephalosporinase . But their synergistic activity with five antibiotics was inferior to tazobactam.

Ned Tijdschr Geneeskd, 1994 Aug 20, 138(34), 1708 - 11
{Prevalence of syphilis and gonorrhea in a family practice in Curaçao,1987-1991}; Braakman-Bonder IM; OBJECTIVE . To determine the prevalence of syphilis and gonorrhoea in a general practice from 1987 to 1991 . DESIGN . Contact registration . SETTING . Sentro Mediko Kas Chikitu in Curacao . METHOD . The numbers of patients having a syphilis or gonorrhoea infection were recorded . Using the chi 2-test prevalence was related to age and (or) sex . RESULTS . Syphilis infections (n = 96) were not age-related (chi 2 = 2.21; df = 4; p = 0.70) and occurred significantly more often among men (chi 2 = 19.70; p < 0.001); 6 out of 31 infected women contracted a syphilis infection during pregnancy; 9% of the men and 3% of the women between the ages of 15 and 64 contracted syphilis during this period . Between the ages of 15 and 24 there were significantly more and between the ages of 45 and 64 significantly fewer gonorrhoea infections (chi 2 = 21.99; df = 4; p < 0.001) . Gonorrhoea infections (n = 75) were significantly more frequent among men (chi 2 = 61.3; p < 0.001); 37% of the men had had a previous infection . Of the cultured N . gonorrhoea 27% proved to be beta-lactamase-positive . Of the male population between 15 and 64 years 6% contracted gonorrhoea, of the female population 0.4%.

Biochem J, 1994 Aug 15, 302 ( Pt 1), 1 - 4
The role of lysine-67 in a class C beta-lactamase is mainly electrostatic; Monnaie D et al.; By using site-directed mutagenesis, the conserved Lys-67 residue situated three positions after the active-site Ser of a class C beta-lactamase was replaced by Arg or Gln . The Lys-67-Gln protein was nearly inactive . Although severely impaired, the Lys-67-Arg mutant exhibited an appreciable activity above pH 7.5 and, for some poor substrates of the wild-type enzyme, the kcat . values were even increased . The properties of the Lys-67-Arg mutant were studied by both steady-state and transient-state kinetic methods with a variety of compounds representing distinct classes of available substrates . With beta-lactam substrates, the kcat./Km values reflecting the efficiency of the acylation step (k+2/K) were decreased 25-100-fold . When the individual values could be measured, k+2 was not significantly altered, but K was found to be strongly increased, a result most likely explained by a corresponding increase in the k+1/k-1 ratio . These results, combined with the much stronger impairment of the Lys-67-Gln mutant, can be interpreted by attributing an electrostatic role to the positive ammonium group of the Lys-67 side chain.

Bioorg Med Chem, 1994 Aug, 2(8), 757 - 71
Functionalized depsipeptides, substrates and inhibitors of beta-lactamases and DD-peptidases; Cabaret D et al.; A series of derivatives of phenyl phenylacetylglycinates (aryl phenaceturates) with a carboxylate substituent meta to the oxygen of the phenoxide leaving group and a functionalized methylene group in the ortho- or para-position have been synthesized . These molecules possess a latent o- or p-quinone methide electrophile which could be unmasked during enzymic turnover and could react with an active site nucleophile . This chemistry does seem to occur in solution where a common hydrolysis product, independent of the benzylic leaving group, presumably o- or p-hydroxymethylphenol, was observed . These depsipeptides are substrates of class A and C beta-lactamases, particularly of the latter, comparable with the parent m-carboxyphenyl phenaceturate . They also have modest inhibitory activity against these enzymes and against the serine DD-peptidase of Streptomyces R61 . The inhibition of a class C beta-lactamase was turnover dependent, as expected of mechanism-based inhibitor, but the small leaving group dependence of the inhibition suggested that the quinone methide, if it was in fact responsible for the inhibition, was generated in solution subsequent to release of the product phenol from the active site.

Biochemistry, 1994 Jul 19, 33(28), 8577 - 86
The role of tyrosine 150 in catalysis of beta-lactam hydrolysis by AmpC beta-lactamase from Escherichia coli investigated by site-directed mutagenesis; Dubus A et al.; The kinetics of beta-lactam hydrolysis by wild-type AmpC beta-lactamase from Escherichia coli and three mutant proteins created by substitution of tyrosine 150 have been examined . The catalytic efficiency was decreased 10- to 1000-fold according to the substrate and mutant being studied . The effect of the mutation was much stronger with rapidly hydrolyzed substrates (e.g., cephalothin) than it was with slowly hydrolyzed substrates (e.g., ceftriaxone) . With the latter substrates, the mutagenesis had a much stronger effect on apparent affinity than it did on rates of catalysis . Indeed, the enzyme appeared to be more reactive toward certain of the slowly hydrolyzed substances (e.g., methicillin, aztreonam, and ceftriaxone) . These observations were not compatible with an obligatory role of tyrosine 150 in catalysis . The analysis of the effects of the mutation on activity was complicated by the observation of at least two, kinetically distinct, forms of the enzymes . It appeared that mutation of tyrosine 150 influenced the kinetic properties of one state and that this residue is involved in the partitioning of the enzyme between the different reactive states.

EMBO J, 1994 Jul 15, 13(14), 3272 - 7
Disruption of the gene encoding p12 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature; Nishiyama K et al.; The Escherichia coli cytoplasmic membrane protein, p12, stimulates the protein translocation activity reconstituted with SecY, SecE and SecA . The gene encoding p12, which is located at 69 min on the E . coli chromosome, was deleted to examine the role of p12 in protein translocation in vivo . The deletion strain exhibited cold-sensitive growth . Pulse-chase experiments revealed that precursors of outer membrane protein A, maltose binding protein and beta-lactamase accumulated at 20 degrees C but not at 37 degrees C . The deletion strain harboring a plasmid which carries the gene encoding p12 under the control of the araBAD promoter was able to grow in the cold when p12 was expressed with the addition of arabinose . Furthermore, the accumulated precursors were rapidly processed to the mature forms upon the expression of p12 . Immunoblot analysis revealed the steady-state accumulation of precursor proteins at 20 degrees C, whereas the accumulation was only marginal at 37 degrees C, indicating that the function of p12 is more critical at 20 degrees C than at 37 degrees C . Finally, proteoliposomes were reconstituted with or without p12 to demonstrate that the stimulation of the activity by p12 increases with a decrease in temperature . From these results, we concluded that p12 is directly involved in protein translocation in E . coli and plays a critical role in the cold . We propose the more systematic name, SecG, for p12.

FEMS Microbiol Lett, 1994 Jul 1, 120(1-2), 75 - 80
Nucleotide sequences of the genes coding for the TEM-like beta-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2); Belaaouaj A et al.; Two blaTEM-like genes were characterized that encoded IRT beta-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with beta-lactamase inhibitors . Plasmids carrying this resistance were isolated from E . coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers . The gene for IRT-1 beta-lactamase resembled the blaTEM-1B gene, and that for IRT-2 resembled blaTEM-2 . However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1 . The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929 . The consequence of C to T transition in the blaIRT-1 gene and C to A transversion in the blaIRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants . Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to beta-lactamase inhibitors . Furthermore, these basic to neutral amino acid replacements explain the more acidic pI (pI = 5.2) of these IRT enzymes compared to that of TEM-1 (pI = 5.4) . The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this beta-lactamase to inhibition by p-chloromercuribenzoate.

FEMS Microbiol Lett, 1994 Jul 1, 120(1-2), 111 - 7
Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to beta-lactamase inhibitors; Brun T et al.; The clinical isolate Escherichia coli PEY was highly resistant to amoxycillin, ticarcillin and piperacillin associated to beta-lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam and brobactam but susceptible to cephalosporins, aztreonam and imipenem . The susceptibility to mecillinam indicated that this phenotype was not related to hyperproduction of the TEM-1 beta-lactamase . E . coli PEY produced a new plasmid-mediated inhibitor-resistant beta-lactamase of pI 5.2, which was named IRT-4 . The determination of the amino acid sequence (Swiss-Prot accession number, P00810) of the purified protein indicated that IRT-4 differed from TEM-1 by two substitutions: Leu for Met-69 (ABL numbering) and Asp for Asn-276 . A Met-69-Leu variant of TEM-1, obtained by site-directed mutagenesis, has been described as resistant to clavulanate . The Asp for Asn-276 substitution has not been reported previously . The side chains of Asp-276 and Arg-244 were expected to interact . Determinations of 50% inhibitory concentrations of beta-lactamase inhibitors and substrate profile of IRT-4 suggested that such an ionic bond was implicated in the alteration of the mechanistic process of TEM-1 beta-lactamase.

Postgrad Med, 1994 Jul, 96(1), 67 - 70, 73-5, 79 passim
Dermatologic emergencies . When early recognition can be lifesaving; Gannon T; Early recognition and treatment of life-threatening dermatoses can reduce morbidity and mortality . Pemphigus vulgaris can usually be brought under control with high doses of corticosteroids . In cases of necrotizing fasciitis, early, extensive debridement of involved tissue is essential, since antibiotic therapy alone has little effect . Patients with toxic epidermal necrolysis and occasionally those with Stevens-Johnson syndrome may need care similar to that required for a major burn . Therapy for toxic shock syndrome includes aggressive fluid replacement and beta lactamase-resistant antistaphylococcal antibiotics . Treatment of urticaria and acquired angioedema includes histamine receptor blockers, prednisone (for intractable cases), and epinephrine (for respiratory compromise); danazol (Danocrine) or stanozolol (Winstrol) may be useful for prophylaxis of hereditary angioedema.

Antimicrob Agents Chemother, 1994 Jul, 38(7), 1608 - 14
Use of the chromosomal class A beta-lactamase of Mycobacterium fortuitum D316 to study potentially poor substrates and inhibitory beta-lactam compounds; Galleni M et al.; Sixteen different compounds usually considered beta-lactamase stable or representing potential beta-lactam inhibitors and inactivators were tested against the beta-lactamase produced by Mycobacterium fortuitum . The compounds exhibiting the most interesting properties were BRL42715, which was by far the best inactivator, and CGP31608 and ceftazidime, which were not recognized by the enzyme . These compounds thus exhibited adequate properties for fighting mycobacterial infections . Although cloxacillin, dicloxacillin, cefoxitin, and CP65207-2 exhibited poor inhibitory efficiency against the enzyme, they were also rather poor substrates and might be considered potential antimycobacterial agents . By contrast, CGP31523A and ceftamet were good substrates.

J Infect, 1994 Jul, 29(1), 23 - 31
Branhamella catarrhalis in children and adults . A study of prevalence, time of colonisation, and association with upper and lower respiratory tract infections; Ejlertsen T et al.; The colonisation rate of Branhamella catarrhalis in patients from 0 to 45 years of age was examined . Of 561 women admitted to hospital in labour, 6 (1%) carried B . catarrhalis in their throats but none carried the organism in their vaginas . None of 534 newborn babies became colonised at birth or during their 5 days' stay in hospital . Neither were 102 neonates < 1 month of age in hospital colonised . The maximum colonisation rate during childhood was observed in children 1-48 months of age with 143 of 266 (54%) children colonised . Among children 4-15 years of age, four of 57 (7%) children with healthy respiratory tracts were colonised . Significantly more children with upper or lower respiratory tract infections (RTI) were colonised (68%) than were children without such infections (36%), (P < 0.001) . After recovery from RTI, the isolation rate in the RTI group fell to that of the non-RTI group . A seasonal variation in prevalence was not observed . Of all the strains of B . catarrhalis isolated, 84% produced beta-lactamase.

Arzneimittelforschung, 1994 Jul, 44(7), 856 - 8
Synthesis and beta-lactamase inhibitory activity of new 6 beta-sulfonamidopenicillanic acids; Changov LS et al.; New 6 beta-aryl(alkyl)sulfonamidopenicillanic acids and their sulfoxides were synthesized by sulfonylation of 6 beta-aminopenicilanic acid or its beta-sulfoxide with an appropriate sulfonyl chloride . The corresponding 6 beta-sulfonamidopenicillanic acids sulfones were prepared by oxidation of the sulfoxides with potassium permanganate in aqueous medium . The obtained compounds reduced the minimum inhibitory concentrations of ampicillin against 8 reference and 7 clinically isolated strains.

Gene, 1994 Jun 24, 144(1), 31 - 6
Transcriptional analysis and heterologous expression of the gene encoding beta-lactamase inhibitor protein (BLIP) from Streptomyces clavuligerus; Paradkar AS et al.; Transcription of bli, the gene encoding beta-lactamase (Bla) inhibitor protein (BLIP) of Streptomyces clavuligerus, was analyzed by promoter-probe studies, Northern hybridization and high-resolution S1 nuclease mapping . The 1-kb SalI DNA fragment immediately upstream from the bli open reading frame (ORF) showed promoter activity when tested using the xylE-based promoter-probe vector, pIJ4083 . The promoter activity was approx . 36-fold higher in S . clavuligerus than in S . lividans . Northern hybridization analysis of S . clavuligerus RNA revealed that bli was expressed as a 0.7-kb monocistronic transcript . High-resolution S1 nuclease mapping identified the transcription start point as an A residue 47 bp upstream from the bli start codon . When the bli ORF, along with 111 bp of upstream sequence including the promoter, was introduced into S . lividans, the transformants produced BLIP, but in amounts approx . 12-fold lower than that produced by S . clavuligerus . Involvement of some additional regulatory element that is present in S . clavuligerus, but absent in S . lividans, could explain the difference in the promoter activities and therefore the difference in the overall expression of bli in the two hosts.

J Cell Biochem, 1994 Jun, 55(2), 209 - 17
Signal peptide hydrophobicity is finely tailored for function; Rusch SL et al.; In order to titrate the dependence of individual steps in protein transport on signal peptide hydrophobicity, we have examined a series of mutants which involve replacement of the hydrophobic core segment of the Escherichia coli alkaline phosphatase signal peptide . The core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues . Thus, a nonfunctional polyalanine-containing signal peptide is titrated with the more hydrophobic residue, leucine . Analysis of this series identified a midpoint for rapid precursor processing between alanine to leucine ratios of 6:4 and 5:5 {Doud et al . (1993): Biochemistry 32:1251-1256} . Examination of precursors that are processed more slowly indicates a lower limit of signal peptide hydrophobicity that permits membrane association and translocation . Analysis of precursors that are processed rapidly defines an intermediate range of hydrophobicity that is optimum; above this level precursors become insensitive to transport inhibitors such as sodium azide and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in parallel with substantial inhibition of beta-lactamase processing . Our data indicate that there is a surprisingly narrow range of signal peptide hydrophobicity which both supports transport of the protein to which it is attached and which does not have such a high affinity for the transport pathway that it disrupts the appropriate balance of other secreted proteins.

FEMS Microbiol Lett, 1994 Jun 1, 119(1-2), 7 - 12
Selective release of the periplasmic enzyme beta-lactamase from Escherichia coli with tetradecyl betainate; Ahlstrom B et al.; The periplasmic enzyme beta-lactamase was selectively released from Escherichia coli K12 by the amphiphilic quaternary ammonium compound tetradecyl betainate at certain concentration intervals . At low concentrations little enzyme was released, and at high concentrations enzyme inactivation occurred . Greater effects of tetradecyl betainate were seen both with respect to release and inactivation at higher pH . At intermediate concentrations of tetradecyl betainate high yields of beta-lactamase were obtained with no detectable contribution of the cytoplasmic marker beta-galactosidase . The highest yields of beta-lactamase activity were obtained when high concentrations of salt were added 1 min after permeation of the bacteria with tetradecyl betainate.

Genomics, 1994 Jun, 21(3), 583 - 7
Isolation and regional localization of cosmid linking clones from human chromosome 12; Hoglund M et al.; We have developed a new method for constructing cosmid linking libraries . The method is based on the insertion of a selection gene, beta-lactamase, in genomic cosmid clones containing recognition sites for rare-cutting enzymes . The selection gene is maintained as a gene cassette in a plasmid and may be excised by the enzymes NotI, SacII, SplI, MluI, BssHII, and NarI or combinations of these enzymes . Using this gene cassette and a genomic cosmid library made from a human-hamster cell line containing the human chromosome 12 as its only human component, a chromosome 12-specific NotI linking library was constructed . The NotI linking clones contained recognition sites for other rate-cutting enzymes, SacII and BssHII, at high frequency, indicating the presence of CpG islands . Thirty cosmid linking clones were regionally localized by FISH and were found to be clustered to chromosome bands 12p13, 12q13, and 12q24.

J Antimicrob Chemother, 1994 Jun, 33(6), 1117 - 26
Clinical isolates of Escherichia coli producing multiple TEM mutants resistant to beta-lactamase inhibitors; Sirot D et al.; Twenty clinical isolates of Escherichia coli resistant to amoxycillin and ticarcillin, both in combination with clavulanic acid, were studied . The ranges of MICs for these strains as determined by the agar dilution method were as follows: amoxycillin, 2048- > 4096 mg/L; ticarcillin, 512- > 4096 mg/L; piperacillin, 32-256 mg/L; mecillinam, 0.5-8 mg/L; and cephalothin 4-16 mg/L . Combining amoxycillin with beta-lactamase inhibitors, each at a fixed concentration of 4 mg/L, had only modest potentiating effects on the activities of this agent, the ranges of MICs falling to 256- > 2048 mg/L in the presence of clavulanic acid or sulbactam and to 64-1024 mg/L and 128-2048 mg/L in the presence of tazobactam and brobactam respectively . The pI values for the beta-lactamases produced by the 20 isolates were 5.2 for 15 strains, 5.4 for four strains and 7.4 for a single strain . Colony hybridization with oligonucleotides was performed in order to detect substitutions of arginine at position 241 (Arg-241) and methionine at position 67 (Met-67) . Based on this technique, the four beta-lactamases with pI values of 5.4 were grouped into two oligotypes (+ = hybridization, - = non-hybridization)-Arg-241+, Met-67- (n = 3) and Arg-241+, Met-67+ (n = 1); in one of the three mutants which did not hybridize with the Met-67 probe, leucine had been substituted for methionine at position 67 . The beta-lactamases with pI values of 5.2 which were identified in 15 strains were grouped into the following three oligotypes: Arg-241-, Met-67+ (n = 7); Arg-241-, Met-67- (n = 6); and Arg-241+, Met-67- (n = 2) . In three of the 13 mutants which failed to hybridize with the Arg-241 probe, serine residues had replaced arginine residues at position 241 . Substitutions of Arg-241 or Met-67 led to reduced affinities of the mutant enzymes for the beta-lactams tested . The results of the hybridization studies demonstrate that, amongst E . coli clinical isolates, there is a diversity of mutant TEM enzymes mediating resistance to beta-lactamase inhibitors.

EMBO J, 1994 May 15, 13(10), 2289 - 96
An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY; Luirink J et al.; In Escherichia coli, a signal recognition particle (SRP) has been identified which binds specifically to the signal sequence of presecretory proteins and which appears to be essential for efficient translocation of a subset of proteins . In this study we have investigated the function of E . coli FtsY which shares sequence similarity with the alpha-subunit of the eukaryotic SRP receptor ('docking protein') in the membrane of the endoplasmic reticulum . A strain was constructed which allows the conditional expression of FtsY . Depletion of FtsY is shown to cause the accumulation of the precursor form of beta-lactamase, OmpF and ribose binding protein in vivo, whereas the processing of various other presecretory proteins is unaffected . Furthermore, FtsY-depleted inverted cytoplasmic membrane vesicles are shown to be defective in the translocation of pre-beta-lactamase using an in vitro import assay . Subcellular localization studies revealed that FtsY is located in part at the cytoplasmic membrane with which it seems peripherally associated . These observations suggest that FtsY is the functional E . coli homolog of the mammalian SRP receptor.

J Biol Chem, 1994 May 13, 269(19), 13887 - 92
The role of the carrier protein and disulfide formation in the folding of beta-lactamase fusion proteins in the endoplasmic reticulum of yeast; Simonen M et al.; We have studied the relationship between folding and secretion competence of hsp150-beta lactamase fusion proteins in Saccharomyces cerevisiae . hsp150 is a secretory protein of yeast, and beta-lactamase was chosen, since its folding can be monitored by assaying its enzymatic activity . The hsp150 pre-pro-protein consists of a signal peptide, subunit I, a repetitive region, and a unique C terminus . Fusion of beta-lactamase to the C terminus of hsp150 produced Cla-bla protein, which was secretion-competent but inactive . The Pst-bla protein, where beta-lactamase was fused to subunit I, was also inactive and mostly secreted, but part of it remained in the pre-Golgi compartment . When beta-lactamase was fused to the C-terminus of the repetitive region, the fusion protein (Kpn-bla) was translocated to the endoplasmic reticulum, acquired disulfide bonds, and adopted an enzymatically active conformation . Kpn-bla was secreted to the medium without decrease of specific activity or retention in the cell . Folding of Kpn-bla to an active and transport-competent form required co-translational disulfide formation, since treatment of cells with dithiothreitol resulted in endoplasmic reticulum-retained inactive Kpn-bla . When dithiothreitol was removed, Kpn-bla resumed transport competence but remained inactive . Reduction of prefolded Kpn-bla did not inhibit enzymatic activity or transport . The repetitive hsp150 carrier may have use in heterologous protein production by conferring secretion competence to foreign proteins in yeast.

Gene, 1994 May 3, 142(1), 113 - 7
High-level secretion of correctly processed beta-lactamase from Saccharomyces cerevisiae using a high-copy-number secretion vector; Castelli LA et al.; We have sought to obtain a convenient system for the high-level production of secreted proteins in yeast . With the aid of a secretion reporter cassette we examined the secretion of beta-lactamase (Bla) as a model protein and found the highest expression in Saccharomyces cerevisiae using a high-copy-number plasmid . We further developed the high-copy-number plasmid introducing a secretion cassette that has a convenient cloning site coinciding with the sequence encoding the KEX2 cleavage site . Large quantities of correctly-processed product can therefore be obtained . We show that 0.3 g/l of correctly processed beta-lactamase can be obtained in fed-batch cultures without the need for selective media or significant loss of the plasmid.

Antimicrob Agents Chemother, 1994 May, 38(5), 1134 - 9
Reversal of clavulanate resistance conferred by a Ser-244 mutant of TEM-1 beta-lactamase as a result of a second mutation (Arg to Ser at position 164) that enhances activity against ceftazidime; Imtiaz U et al.; The mutation of Arg-244 to Ser (Arg-244-->Ser mutation) in the TEM-1 beta-lactamase has been shown to produce resistance to inactivation by clavulanate in the mutant enzyme and resistance to ampicillin plus clavulanate in a strain of Escherichia coli producing this enzyme . The Arg-164-->Ser mutation in the TEM-1 beta-lactamase (TEM-12 enzyme) is known to enhance the activity of the enzyme against ceftazidime, resulting in resistance to the drug in a strain producing the mutant enzyme (D . A . Weber, C . C . Sanders, J . S . Bakken, and J . P . Quinn, J . Infect . Dis . 162:460-465, 1990) . The doubly mutated derivative of the TEM-1 enzyme (Ser-164/Ser-244) retains the characteristics of the Ser-164 mutant enzyme, i.e., enhanced activity against ceftazidime and sensitivity to inactivation by clavulanate . It also confers the same phenotype as the Ser-164 mutant enzyme, i.e., resistance to ceftazidime and ampicillin, with reversal of this resistance in the presence of clavulanate . Thus, the Arg-164-->Ser mutation in the TEM-1 beta-lactamase suppresses the effect of the Arg-244-->Ser mutation which, by itself, reduces the sensitivity of the enzyme to inactivation by clavulanate.

Antimicrob Agents Chemother, 1994 May, 38(5), 1085 - 9
Emergence of clinical isolates of Escherichia coli producing TEM-1 derivatives or an OXA-1 beta-lactamase conferring resistance to beta-lactamase inhibitors; Zhou XY et al.; Sixteen Escherichia coli clinical isolates which were resistant to ampicillin and amoxicillin-clavulanate but susceptible to cephalothin were studied . Eight strains showed the presence of a beta-lactamase which comigrates with reference OXA-1 enzyme . The eight other strains produced different TEM-1 derivatives which had in common a higher Km for penicillins and a higher 50% inhibitory concentration for the beta-lactamase inhibitors . By oligotyping and sequencing of PCR products, it was shown that Ser (AGC) (TEM-30; also called TRI-1) in three strains and Cys (TGC) (TEM-31; also called TRI-2) in one strain were substituted for Arg-241 (CGC), that Leu (CTG) (TEM-33) and Val (GTG) (TEM-34) in one strain each were substituted for Met-67 (ATG), and that in other mutants the two latter substitutions occurred together with the substitution of Asp (GAT) (TEM-35 and TEM-36) for Asn-272 (AAT) . Therefore, different sets of amino acid substitutions of TEM-1 can be found in clinical isolates and lead to resistance to beta-lactamase inhibitors.

Mol Microbiol, 1994 May, 12(3), 491 - 504
Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer; Timm J et al.; The gene encoding a class A beta-lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high-level amoxicillin-resistant mutant that produces large amounts of beta-lactamase (blaF*) . The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence . Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high-level production of the beta-lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter . These analyses also showed that transcription and translation start at the same position . A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N-terminal amino acid sequence indicated the presence of a typical signal peptide . The fusion of blaF (or blaF*) to the E . coli gene phoA resulted in the production of BlaF-PhoA hybrid proteins that had alkaline phosphatase activity . These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.

J Nat Prod, 1994 May, 57(5), 654 - 7
SB-202742, a novel beta-lactamase inhibitor isolated from Spondias mombin; Coates NJ et al.; SB-202742 {1}, an anacardic acid derivative possessing beta-lactamase inhibitory activity, has been isolated from a hexane extract of the plant, Spondias mombin . Its isolation, structure determination, and biological activity are reported herein.

Infection, 1994 May-Jun, 22(3), 193 - 6
Moraxella catarrhalis in upper respiratory tract of healthy Yemeni children/adults and paediatric patients: detection and significance; Sehgal SC et al.; A highly variable carriage rate of Moraxella catarrhalis has been reported in the literature . In order to assess the reasons for this variability, detection rates of this organism from various sites of the upper respiratory tract of children and adults were studied . Throat swabs, oral swabs and nasal swabs from 131 children, 96 adults and 64 paediatric patients with upper respiratory tract infections were cultured on a selective medium . Detection rates of 31.4% in children less than three years of age, 38.5% in children between 4 and 12 years, 11.7% in adults and 21.9% in patients were found, respectively . The reasons for high variability in the carriage rates were many including the number and site of specimen collection, media used for isolation and identification criteria . All isolates were sensitive to amoxycillin-clavulanic acid and co-trimoxazole . A significantly higher share of M . catarrhalis isolates from patients were beta-lactamase producers (12/14, 85.7%) as compared to normal healthy subjects (41.9%), suggesting a cautious approach in the use of beta-lactam antibiotic in respiratory tract infections.

Biofactors, 1994 May, 4(3-4), 173 - 5
Cloning of promoter-active DNA sequences from Chainia (NCL 82.5.1) in Escherichia coli; Chauthaiwale VM et al.; The ability of Escherichia coli cells to recognise and use the regulatory signals of genes from Chainia (NCL 82-5-1) was determined in vivo using gene fusions . DNA fragments from Chainia were cloned in E . coli using the promoter probe plasmid pJAC4 . Four of the randomly selected recombinants exhibited varying strengths of promoter activity as assessed by the concentration of ampicillin required to kill 50% of the colonies (LD50 values) and by beta-lactamase activity . The origin of the inserts was confirmed by colony hybridization of clones with labelled genomic DNA of Chainia . The beta-lactamase activity of recombinant colonies was substantially higher (> 10-fold) than that of colonies transformed with pJAC4 without any insert . The results show that a few Chainia DNA sequences are recognized by E . coli as transcription initiation signals for the expression of beta-lactamase gene, inspite of the high guanine/cytosine content of the Chainia genome.

Antimicrob Agents Chemother, 1994 Apr, 38(4), 905 - 7
Penetration of piperacillin-tazobactam into cancellous and cortical bone tissues; Incavo SJ et al.; The penetration characteristics of piperacillin-tazobactam into cortical and cancellous bone tissues were investigated in 10 patients undergoing total hip replacement . The concentration ratios of piperacillin/tazobactam were 9.4 +/- 1.8 in cancellous bone tissue and 8.0 +/- 2.2 in cortical bone tissue, which were close to the 8:1 ratio of drugs administered . The mean ratios of drug concentrations in bone and plasma for cancellous and cortical tissue were 23 and 18%, respectively, for piperacillin and 26 and 22%, respectively, for tazobactam . The concentrations of tazobactam achieved are sufficient to exert anti-beta-lactamase activity and supportive of clinical trials involving bone and joint infections, including those caused by beta-lactamase-producing pathogens.

Yeast, 1994 Apr, 10(4), 497 - 508
Use of beta-lactamase as a secreted reporter of promoter function in yeast; Cartwright CP et al.; K1 preprotoxin is the 316 residue precursor of the K1 killer toxin secreted by the yeast Saccharomyces cerevisiae . The SP beta la reporter consists of the mature, secreted form of beta-lactamase (beta la) fused to S and P, two fragments of preprotoxin . S is the N-terminal 34 residues, including the secretion signal . P, a 67 residue 'processing' segment with three sites for N-glycosylation, terminates in a Lys Arg site for cleavage by the Kex2 protease . Expression of SP beta 1a in yeast results in efficient secretion, processing by signal peptidase and glycosylation in the endoplasmic reticulum, producing pro beta la . Kex2 cleavage of pro beta la in the lumen of a late Golgi compartment releases beta la, which accumulates stably in culture media buffered at pH 5.8-7 . The half-life of secretion is 11 min at 30 degrees C; 10-12% of the total activity in exponential-phase cells is intracellular, mostly in the form of pro beta la, indicating that transit from the endoplasmic reticulum to the Golgi is rate limiting . We have used SP beta la expression in single- and multi-copy vectors to compare the PGK, GAL1, GAL10, PHO5 and CUP1 promoters under varying nutritional conditions . In exponential-phase cells, secretion of beta la over a 40-fold range and up to several micrograms/ml was proportional to transcript level, demonstrating that SP beta la can be employed as a convenient secreted reporter of promoter function in yeast.

J Biotechnol, 1994 Mar 31, 33(2), 195 - 204
Effect of growth rate and cultivation environments on cloned gene stability and the cloned gene product formation in Streptomyces lividans; Lee JH et al.; The growth rate and environmental effects on the stability of recombinant plasmid, pDML6 containing beta-lactamase gene, and the cloned gene product formation in Streptomyces lividans were studied . A maximum production rate of the cloned gene product was obtained at a specific growth rate 0.106 h-1 in glucose-limited chemostat cultivations without genetic selection pressure . Optimum environmental conditions for the recombinant plasmid stability and maximum formation rates of the cloned gene product were determined using continuous cultivations at the optimum specific growth rate . The fractions of plasmid harboring mycelium in prolonged cultivation up to 50 generations were varied from 77 to 95% . The recombinant plasmid was stably maintained in the host cells grown in different temperatures (24 to 36 degrees C) and pH (6.0 to 8.5) . The formation of the cloned gene product was optimum at pH 7.0 and 27 degrees C, at which the maximum enzyme production rate was 0.82 kU g-1 h-1 . Continuous cultivations varying the dissolved oxygen tension (10 to 80% air saturation) showed that the plasmids were maintained stably and the specific enzyme production rates were increased with increasing dissolved oxygen levels.

Biochemistry, 1994 Mar 15, 33(10), 2782 - 91
"Partly folded" state, a new equilibrium state of protein molecules: four-state guanidinium chloride-induced unfolding of beta-lactamase at low temperature; Uversky VN et al.; Guanidinium chloride- (GdmCl-) induced unfolding of beta-lactamase has been investigated by a combination of size-exclusion chromatography (SEC-FPLC) and usual optical methods . It has been shown that at low temperatures this protein unfolds through two equilibrium intermediates . The first of these intermediates is the molten globule state, while the other (which we have called a "partly folded" state) is less compact than the molten globule but much more compact than the unfolded state . It also preserves a substantial part of secondary structure of the native or molten globule state . We suggest that this new "partly folded" state of a protein molecule can be the equilibrium counterpart of the first kinetic intermediate of protein folding, formed within a few milliseconds, i.e., after the "burst" stage of folding.

Appl Environ Microbiol, 1994 Mar, 60(3), 1029 - 32
Characteristics of beta-lactamase-inhibiting proteins from Streptomyces exfoliatus SMF19; Kim MK et al.; Streptomyces exfoliatus SMF19 produced two types of extracellular beta-lactamase-inhibiting proteins, 48 (beta LIP-I) and 33 (beta LIP-II) kDa . The inhibition mode of beta LIP-I on Bacto Penase and benzylpenicillin as substrates was uncompetitive (i.e., both Km and Vmax were changed), while that of beta LIP-II was noncompetitive (i.e., only Vmax was changed) . The inhibition constants of beta LIP-I and beta LIP-II were 0.62 x 10(-4) and 2.74 x 10(-1) mumol, respectively . The N-terminal amino acid sequence of beta LIP-I was NH3-*-S-T-V-F-D-L-V-*-L-G, and that of beta LIP-II was NH3-D-F-*-V-F-D-L-E-A-T-D-E.

Mol Microbiol, 1994 Mar, 11(5), 819 - 31
Reversible topology of a bifunctional transmembrane protein depends upon the charge balance around its transmembrane domain; Kim H et al.; Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminal OmpA signal sequence was fused with TEM beta-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker . The hybrid proteins were found to be inserted in the membrane . Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the beta-lactamase remained in the cytoplasm . This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor . In the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal beta-lactamase domain across the membrane and subsequently as a membrane anchoring signal . The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence . Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the amino-terminal nuclease domain across the membrane, leaving the carboxyl terminal beta-lactamase domain in the cytoplasm . When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation . A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence . Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.

Clin Chem, 1994 Mar, 40(3), 347 - 57
Selected strategies for improving sensitivity and reliability of immunoassays; Kricka LJ; Selected recent advances in immunoassay are reviewed . Development has continued on new labels (beta-lactamase, pyrophosphatase, luciferases, photoproteins, pyridopyridazines, europium cryptates, metal carbonyl complexes, porphines, phosphors) and label-detection methods (e.g., chemiluminescence assays, thermometric assays, NADP(+)- and FADP-based coupled assays) . Various methods have been explored to increase assay sensitivity, including label amplification via catalyzed reporter deposition (peroxidase label) and immuno-polymerase chain reaction (DNA label) . The focus of new immunoassay strategies has been on improved reliability (bispecific antibodies), assay simplification (piezoelectric and surface plasmon resonance immunosensors, phase-modulation fluorescence spectroscopy, polymerized bilayer assemblies), and simultaneous multianalyte testing (e.g., quadruple labeling with lanthanides, one-step test devices for drugs of abuse).

J Biol Chem, 1994 Feb 18, 269(7), 5218 - 24
Involvement of FtsH in protein assembly into and through the membrane . I . Mutations that reduce retention efficiency of a cytoplasmic reporter; Akiyama Y et al.; To identify cellular factors that assist in membrane protein biogenesis, we looked for mutants affected in the "stop transfer" anchoring process . Using a SecY-PhoA fusion protein in which alkaline phosphatase (PhoA) mature sequence is attached to the last cytoplasmic domain following the 10th transmembrane segment of SecY, we isolated a mutation (std101) that allowed significant export of the PhoA moiety across the membrane . The mutation did not cause nonspecific leakage of cytoplasmic proteins . The mutation was identified as a single base change in the ftsH gene, causing an amino acid substitution in the proposed periplasmic region of FtsH, a putative membrane-bound ATPase . In addition, the ftsH1 temperature-sensitive mutation caused a similar phenotype . Disruption of the chromosomal ftsH in combination with a lac promoter-controlled copy of ftsH on a plasmid rendered the cell viability dependent on lac induction . Repression of this system resulted in a strong Std phenotype as well as significant export defects of beta-lactamase and OmpA . Thus, the loss of ftsH function enhances translocation of normally anchored protein segments and retards that of normally translocated proteins . These results suggest that FtsH participates in assembly of proteins into and through the membrane . It is needed for the cell to assure efficient stop-transfer of some transmembrane proteins.

J Bacteriol, 1994 Feb, 176(3), 563 - 8
Selection of functional signal peptide cleavage sites from a library of random sequences; Palzkill T et al.; The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide . After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I . A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site . To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences . Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library . The sequences of 15 mutants indicated a bias towards small amino acids . The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues . Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position . The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons . Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.

G Batteriol Virol Immunol, 1994 Jan-Dec, 86(1-12), 121 - 5
{Etiology and rational therapy of acute otitis media in adults}; Serra A et al.; Acute otitis media (AOM) is an infection frequently found in children, but tends to be less frequent with age and its frequency in adults is only about 1% . The etiology of AOM in children is prevalently bacterial; numerous studies have shown the most common etiological agents . But the etiology in adults has not been well studied . The authors examined 40 cases of AOM in adults, the pathologic material was obtained by needle aspiration; in 32 samples there was bacterial growth . In the majority of the cases (94%) the bacteria isolated were the same as those found in children: S . pneumoniae, H . influenzae and B . catarrhalis; much rarer were S . pyogenes and S . aureus . On the potential beta-lactamase producing strains, this activity was measured . From our findings we believe that there is the necessity to have a rational antibiotic therapy (due to the difficulty in obtaining pathologic material) with active drugs for the probable etiological agents of AOM.

Protein Eng, 1994 Jan, 7(1), 131 - 6
Secondary structure characterization of beta-lactamase inclusion bodies; Przybycien TM et al.; The secondary structure of proteins in E . coli inclusion bodies was investigated via Raman spectroscopy . Inclusion bodies were purified from cells expressing different forms of RTEM beta-lactamase and grown at either 37 or 42 degrees C . All of the solid phase inclusion body samples examined gave amide I band spectra that were perturbed from that of the native, purified protein in both solution and powder forms; secondary structure estimates indicated significant decreases in alpha-helix and increases in beta-sheet contents in the inclusion body samples . The structure estimates for inclusion bodies isolated from 37 degrees C cultures were similar, regardless of aggregate localization in the E . coli cytoplasmic or periplasmic spaces or beta-lactamase precursor content . Inclusion bodies obtained from 42 degrees C cells exhibited a further reduction of alpha-helix and augmentation of beta-sheet contents relative to those from 37 degrees C cultures . These results are consistent with the paradigm for inclusion body formation via the self-association of intra-cellular folding intermediates having extensive secondary structure content . Further, the overall secondary structure content of inclusion bodies is not significantly affected by subcellular compartmentalization, but may be altered at increased temperatures.

FEMS Microbiol Lett, 1993 Dec 15, 114(3), 349 - 54
Complementation of growth defect in an ampC deletion mutant of Escherichia coli; Bishop RE et al.; beta-Lactamase genes of class-A (Rtem) and class-C (ampC) were placed under control of an inducible tac-promoter and expressed in Escherichia coli . Expression of RTEM had no observable effect on the growth properties of E . coli strains HB101 (ampC+) or MI1443 (delta ampC) . E . coli MI1443 exhibited a decline in growth rate at mid-exponential phase which could be delayed by expression of AmpC at early-exponential phase . AmpC expression otherwise inhibited growth, particularly during the transition into exponential phase where growth was prevented altogether . We suggest that the AmpC beta-lactamase, but not RTEM, may have an additional cellular function as a peptidoglycan hydrolase.

Biophys J, 1993 Dec, 65(6), 2304 - 12
Differential stability of beta-sheets and alpha-helices in beta-lactamase: a high temperature molecular dynamics study of unfolding intermediates; Vijayakumar S et al.; beta-Lactamase, which catalyzes beta-lactam antibiotics, is prototypical of large alpha/beta proteins with a scaffolding formed by strong noncovalent interactions . Experimentally, the enzyme is well characterized, and intermediates that are slightly less compact and having nearly the same content of secondary structure have been identified in the folding pathway . In the present study, high temperature molecular dynamics simulations have been carried out on the native enzyme in solution . Analysis of these results in terms of root mean square fluctuations in cartesian and {phi, psi} space, backbone dihedral angles and secondary structural hydrogen bonds forms the basis for an investigation of the topology of partially unfolded states of beta-lactamase . A differential stability has been observed for alpha-helices and beta-sheets upon thermal denaturation to putative unfolding intermediates . These observations contribute to an understanding of the folding/unfolding processes of beta-lactamases in particular, and other alpha/beta proteins in general.

J Protein Chem, 1993 Dec, 12(6), 783 - 9
Critical amino acids responsible for converting specificities of proteins and for enhancing enzyme evolution are located around beta-turn potentials: data-based prediction; Murakami M; Various reports have described that amino acid substitutions can alter substrate, positional, inhibitory, and target gene specificities of proteins . By using the method of Chou and Fasman, the present work predicts that critical amino acids for converting these substrate specificities of trypsin, L-lactate dehydrogenase, aspartate aminotransferase, beta-lactamase, and cytochrome P-450 are found to exist within regions predicted as beta-turns . The ratios of hydroxylation and oxygenation positions of substrates by cytochrome P-450 and lipoxygenase, respectively, are varied by changes of the protein structures, probably around turn conformations . Inhibitory specificities of bovine pancreatic trypsin inhibitor and alpha 1-antitrypsin and target gene specificity of glucocorticoid receptor are converted by changing turn structures . Occurrence of beta-turn probabilities can be predicted around the amino acid alteration positions of an evolutionally antecedent protein of a nylon degradation enzyme . These findings will have relevance to work on protein engineering and enzyme evolution.

Antimicrob Agents Chemother, 1993 Dec, 37(12), 2760 - 1
Sequences of MGH-1, YOU-1, and YOU-2 extended-spectrum beta-lactamase genes; Rice LB et al.; Genes for MGH-1, YOU-1, and YOU-2 extended-spectrum beta-lactamases have been cloned and sequenced . The gene for MGH-1 has the sequence of blaTEM-10, YOU-2 has that of blaTEM-12, and YOU-1 has that of blaTEM-26 . All have evolved from blaTEM-1b but have the strong dual promoter sequence of blaTEM-2.

Bioconjug Chem, 1993 Nov-Dec, 4(6), 434 - 9
Synthesis of bulky beta-lactams for inhibition of cell surface beta-lactamase activity; Karunaratne DN et al.; Procedures are described for the preparation of a series of compounds consisting of methicillin linked to beta-cyclodextrin through variable hydrophilic linkers . beta-Cyclodextrin was coupled to the antibiotic methicillin to prevent the antibiotic from permeating the outer membranes of bacteria . Stoichiometric oxidation of the beta-cyclodextrin with sodium metaperiodate provided a functional group for coupling to the linker . Methicillin was coupled to the linker via its carboxyl group . These compounds were tested for activity toward purified beta-lactamase . The length of the spacer arm between beta-cyclodextrin and methicillin was crucial in binding beta-lactamase and inhibiting activity . Compounds with longer spacers were effective inhibitors of beta-lactamase . We have deduced that the length of the spacer should be greater than 16 A for optimum inhibition of beta-lactamase.

Antimicrob Agents Chemother, 1993 Nov, 37(11), 2438 - 42
Critical hydrogen bonding by serine 235 for cephalosporinase activity of TEM-1 beta-lactamase; Imtiaz U et al.; The role of Ser-235 in the catalytic mechanism of the TEM-1 beta-lactamase has been explored by the study of a mutant enzyme in which Ser-235 has been substituted by alanine (Ala-235 mutant enzyme) . A comparative kinetic analysis of both the wild-type and the Ala-235 TEM-1 enzymes revealed little effect of this substitution of residue 235 on the turnover of penicillins but a greater effect on the turnover of cephalosporins . Susceptibility testing of Escherichia coli strains harboring the wild-type TEM-1 beta-lactamase and the Ala-235 mutant enzyme revealed an effect of the mutation similar to that observed in the enzymological studies . The MICs of two representative cephalosporins for the strain containing the mutant enzyme were much lower than those for the isogenic strain bearing the wild-type TEM-1 beta-lactamase . On the other hand, the strain with the mutant enzyme was still highly resistant to penicillins.

Biochem J, 1993 Nov 1, 295 ( Pt 3), 871 - 8
Substrate-induced inactivation of the OXA2 beta-lactamase; Ledent P et al.; The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied . Among these, only three appeared to correspond to the integrated Henri-Michaelis equation . 'Burst' kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem . Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates . Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour . The hydrolysis of imipenem was characterized by the occurrence of two 'bursts', and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product.

Biochem J, 1993 Nov 1, 295 ( Pt 3), 705 - 11
Interactions between active-site-serine beta-lactamases and mechanism-based inactivators: a kinetic study and an overview; Matagne A et al.; The interactions between three class A beta-lactamases and three beta-lactamase inactivators (clavulanic acid, sulbactam and olivanic acid MM13902) were studied . Interestingly, the interaction between the Streptomyces cacaoi beta-lactamase and clavulanate indicated little irreversible inactivation . With sulbactam, irreversible inactivation was found to occur with the three studied enzymes, but no evidence for transiently inactivated adducts was found . Irreversible inactivation of the S . albus G and S . cacaoi enzymes was particularly slow . With olivanate, irreversible inactivation was also observed with the three enzymes, but with the S . cacaoi enzyme, no hydrolysis could be detected . A tentative summary of the results found in the literature is also presented (including 6 beta-halogenopenicillanates), and the general conclusions underline the diversity of the mechanisms and the wide variations of the rate constants observed when class A beta-lactamases interact with beta-lactamase inactivators, in agreement with the behaviours of the same enzymes towards their good and poor substrates.

FEBS Lett, 1993 Sep 27, 331(1-2), 159 - 61
Assembly of eukaryotic class III (N-out, C-in) membrane proteins into the Escherichia coli cytoplasmic membrane; Hennessey ES et al.; Class III membrane proteins lack cleavable signal peptides but adopt an N-out, C-in topology with respect to their native membranes . We have analysed the fate of two eukaryotic class III plasma membrane proteins, human erythrocyte glycophorin C and influenza A virus M2 protein, in Escherichia coli . The N-terminal domains of both proteins were efficiently localised to the extracytoplasmic side of the bacterial cytoplasmic membrane . When beta-lactamase was fused to the C-terminus of glycophorin C it was localised to the cytoplasm, and protease treatment of spheroplasts caused a reduction in size of the fusion protein consistent with glycophorin C adopting its native topology in E . coli.

FEMS Microbiol Lett, 1993 Sep 15, 112(3), 343 - 8
Cloning, nucleotide sequence and expression of a beta-lactamase gene from Streptomyces lavendulae; Sendouda A et al.; A hybridized DNA fragment was cloned as a 7.6-kb fragment from Streptomyces lavendulae KCCS0263 using a 1.9-kb SacI-XbaI DNA fragment from S . cellulosae as a probe . The latter fragment encoded a beta-lactamase which can bind blue dextran . The hybridized region was reduced to a 2.8-kb KpnI-BclI fragment and the nucleotide sequence was determined . The nucleotide sequence indicated one open reading frame whose amino acid sequence is very similar to that of the beta-lactamase from S . cellulosae . The gene produced beta-lactamase enzyme at a low but significant amount.

Res Microbiol, 1993 Sep, 144(7), 575 - 80
Secretion of bacterial beta-lactamase into culture broth by a mutant strain of Saccharomyces cerevisiae; Broker M; The secretion of Escherichia coli beta-lactamase was studied in a Saccharomyces cerevisiae ts1 mutant strain . The signal sequence of pre-beta-lactamase was recognized by the yeast cell and the precursor protein was processed to an enzymatically active mature beta-lactamase . In contrast to conventional strains, the mutant strain was able to secrete bla-gene-encoded beta-lactamase into the culture broth . These results indicate the potential usefulness of ts1 mutant strains for biotechnological purposes.

Infection, 1993 Sep-Oct, 21(5), 336 - 7
Panaritium ossale et articulare caused by Moraxella nonliquefaciens; Piontek K et al.; Only little is known about soft tissue, bone or joint infections caused by Moraxella spp . A case of panaritium ossale et articulare caused by Moraxella nonliquefaciens in an 80-year-old patient immunocompromised by diabetes mellitus and liver insufficiency is reported here . Surgery, local antiseptic treatment, and therapy with aminopenicillins plus beta-lactamase-inhibitors led to complete cure of the infection within 10 days.

Plasmid, 1993 Sep, 30(2), 155 - 8
Target sequence specificity of transposon Tn5 in the absence of major hotspots in the plasmid pBR322: identification of a new hotspot; Boyd LA et al.; The plasmid pLB11-1 is a pBR322 derivative in which part of the tetracycline resistance (tet) gene (basepair coordinates 23 to 375), containing five hotspots for Tn5 insertion (D . E . Berg et al., Genetics 105, 813-828, 1983), has been replaced with a 5.1-kb fragment of Escherichia coli genomic DNA encoding an osmoregulatory function . Restriction mapping of 40 pLB11-1::Tn5 derivatives, chosen at random from 240 Tn5 insertion derivatives with an unaltered osmotolerant phenotype, placed Tn5 in the vector portion of 20 clones . The majority of these insertions (16/20) were located in a 0.09-kb region immediately downstream of the beta-lactamase (bla) gene . Nucleotide (nt) sequence analysis of seven insertions from this group revealed an identical site of insertion within pBR322, representing a hitherto unidentified hotspot for Tn5 insertion . The target sequence, 5'GTCTGACGC, was found to be duplicated in these cases.

Biotechnol Prog, 1993 Sep-Oct, 9(5), 539 - 47
Molecular characterization of beta-lactamase inclusion bodies produced in Escherichia coli . 1 . Composition; Valax P et al.; We have determined the macromolecular composition of inclusion bodies formed by overexpressing beta-lactamase from three different expression systems as a function of the growth conditions . The inclusion bodies were purified by differential gradient centrifugation and detergent extraction . Both the expression system and the growth conditions were shown to have a pronounced effect on inclusion body composition . Specifically, contaminating polypeptides ranged from less than 5% to over 50% of the total protein content . Phospholipids composed 0.5-13% of the inclusion bodies . Nucleic acids represented a minor impurity for both cytoplasmic and periplasmic inclusion bodies . Cytoplasmic inclusion bodies of the mature beta-lactamase had the lowest amount of impurities, irrespective of the growth conditions . On the other hand, large amounts of outer membrane proteins and phospholipids were observed in periplasmic inclusion bodies from cells grown at basic pH . Our results show that, at least under some growth conditions, protein aggregation in vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.

J Clin Microbiol, 1993 Aug, 31(8), 2244 - 7
Comparison of extracellular enzymes of Fusobacterium necrophorum subsp . necrophorum and Fusobacterium necrophorum subsp . funduliforme; Amoako KK et al.; A total of 10 strains each of Fusobacterium necrophorum subsp . necrophorum and Fusobacterium necrophorum subsp . funduliforme were tested for the production of 13 extracellular enzymes . DNase, alkaline phosphatase, and lipase were predominantly associated with all the strains of F . necrophorum subsp . necrophorum, with DNase not detected in any of the strains of F . necrophorum subsp . funduliforme . In addition, the strains of F . necrophorum subsp . necrophorum were generally more hemolytic than those of F . necrophorum subsp . funduliforme . Lecithinase, beta-lactamase, elastase, hyaluronidase, chondroitin sulfatase, and coagulase were not detected in any of the strains . DNase may be used to differentiate between the two subspecies.

J Nat Prod, 1993 Aug, 56(8), 1373 - 96
Synthesis and reaction of potential alternate substrates and mechanism-based inhibitors of clavaminate synthase; Iwata-Reuyl D et al.; Clavaminate synthase is an FeII/alpha-ketoglutarate-dependent enzyme central to the biosynthesis of the beta-lactamase inhibitor clavulanic acid . In the presence of dioxygen it catalyzes the oxidative cyclization/desaturation of proclavaminic acid to clavaminic acid in a two-step process . Samples of (4'R)- and (4'S)-D,L-{4'-2H}proclavaminic acid have been prepared and used to demonstrate that oxazolidine ring formation occurs with retention of configuration . The stereochemical course of oxygen insertion from substrate that takes place in this oxidative cyclization is the same as that observed from molecular oxygen in several hydroxylation reactions catalyzed by other FeII/alpha-ketoglutarate-dependent enzymes . The ferryl (FeIV = O) species thought to be transiently involved in each of these processes was investigated in the present work with clavaminate synthase and three structural analogues of proclavaminic acid bearing vinyl or ethynyl groups at C-4' or a cyclopropyl at C-4 . In the synthesis of the former two derivatives and proclavaminic acid stereoselectively labeled with deuterium at C-4', introduction of the unsaturated substituents in a stereochemically defined manner at C-4' relied upon ready access to (4R)-4-thiophenyl-2-azetidinone . Trimethylsilyl substitution could be easily achieved at C-3 of the optically pure starting material to give the readily separable cis and trans diastereomers . In radical chain reactions in which the thiophenyl was replaced by deuterium or in anionic reactions in which the thiophenyl was eliminated as its sulfone and replaced by addition of carbanions, the steric bulk of the trimethylsilyl group at C-3 governed the approach of incoming reagents to give the trans product . The enzymatic fate, however, of these derivatives was disappointing, yielding neither detectable reaction nor hoped-for inactivation of clavaminate synthase . Finally, as mixed competitive/noncompetitive inhibitors of catalysis, they gave unexceptional inhibition constants in the range 2-10 mM.

Bioorg Med Chem, 1993 Aug, 1(2), 151 - 4
beta-Lactamase inhibitors derived from N-tosyloxy-beta-lactams; Teng M et al.; Electrophilic N-tosyloxy-beta-lactams, N-tosyloxy-4-phenyl-2-azetidinone (2b) and N-tosyloxy-3-(S)-phthalimido-4-(S)-2-azetidinone (2c), are described . These agents are novel potent beta-lactamase inhibitors.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7084 - 8
Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo; Missiakas D et al.; We have identified and characterized the Escherichia coli gene dsbB, whose product is required for disulfide bond formation of periplasmic proteins, by using two different approaches: (i) screening of a multicopy plasmid library for clones which protect E . coli from the lethal effects of dithiothreitol (DTT), and (ii) screening of insertion libraries of E . coli for DTT-sensitive mutants . Mapping and characterization of mutations conferring a DTT-sensitive phenotype also identified the dsbA, trxA, and trxB genes, whose products are involved in different oxidation-reduction pathways . Null mutations in dsbB conferred pleiotropic phenotypes such as sensitivity to benzylpenicillin and inability to support plaque formation of filamentous phages, and they were shown to severely affect disulfide bond oxidation of secreted proteins such as OmpA and beta-lactamase . These phenotypes resemble the phenotype of bacteria carrying either a null mutation in the dsbA gene or the double mutation dsbA dsbB . Sequencing and expression of the dsbB gene revealed that it encodes a 20-kDa protein predicted to possess an "exchangeable" disulfide bond in -Cys-Val-Leu-Cys- . The dsbB gene maps at 26.5 min on the genetic map of the E . coli chromosome, and its transcription is directed from two promoters, neither of which resembles the canonical E sigma 70-recognized promoter.

Biochem J, 1993 Jul 1, 293 ( Pt 1), 195 - 201
Mechanism of action of DD-peptidases: role of asparagine-161 in the Streptomyces R61 DD-peptidase; Wilkin JM et al.; The role of residue Asn-161 in the interaction between the Streptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators was probed by site-directed mutagenesis . The residue was successively replaced by serine and alanine . In the first case, acylation rates were mainly affected with the peptide and ester substrates but not with the thiol-ester substrates and beta-lactams . However, the deacylation rates were decreased 10-30-fold with the substrates yielding benzoylglycyl and benzoylalanyl adducts . The Asn161Ala mutant was more generally affected, although the acylation rates with cefuroxime and cefotaxime remained similar to those observed with the wild-type enzyme . Surprisingly, the deacylation rates of the benzoylglycyl and benzoylalanyl adducts were very close to those observed with the wild-type enzyme . The results also indicate that the interaction with the peptide substrate and the transpeptidation reaction were more sensitive to the mutations than the other reactions studied . The results are discussed and compared with those obtained with the Asn-132 mutants of a class A beta-lactamase.

J Bacteriol, 1993 Jul, 175(13), 4129 - 36
Use of the "blue halo" assay in the identification of genes encoding exported proteins with cleavable signal peptides: cloning of a Borrelia burgdorferi plasmid gene with a signal peptide; Giladi M et al.; We have recently reported a phoA expression vector, termed pMG, which, like TnphoA, is useful in identifying genes encoding membrane-spanning sequences or signal peptides . This cloning system has been modified to facilitate the distinction of outer membrane and periplasmic alkaline phosphatase (AP) fusion proteins from inner membrane AP fusion proteins by transforming pMG recombinants into Escherichia coli KS330, the strain utilized in the "blue halo" assay first described by Strauch and Beckwith (Proc . Natl . Acad . Sci . USA 85:1576-1580, 1988) . The lipoprotein mutation lpp-5508 of KS330 results in an outer membrane that is leaky to macromolecules, and its degP4 mutation greatly reduces periplasmic proteolytic degradation of AP fusion proteins . pMG AP fusions containing cleavable signal peptides, including the E . coli periplasmic protein beta-lactamase, the E . coli and Chlamydia trachomatis outer membrane proteins OmpA and MOMP, respectively, and Tp 9, a Treponema pallidum AP recombinant, diffused through the leaky outer membrane of KS330 and resulted in blue colonies with blue halos . In contrast, inner membrane AP fusions derived from E . coli proteins, including leader peptidase, SecY, and the tetracycline resistance gene product, as well as Tp 70, a T . pallidum AP recombinant which does not contain a signal peptide, resulted in blue colonies without blue halos . Lipoprotein-AP fusions, including the Borrelia burgdorferi OspA and T . pallidum Tp 75 and TmpA showed halo formation, although there was significantly less halo formation than that produced by either periplasmic or outer membrane AP fusions . In addition, we applied this approach to screen recombinants constructed from a 9.0-kb plasmid isolated from the B31 virulent strain of B . burgdorferi . One of the blue halo colonies identified produced an AP fusion protein which contained a signal peptide with a leader peptidase I cleavage recognition site . The pMG/KS330r- cloning and screening approach can identify genes encoding proteins with cleavable signal peptides and therefore can serve as a first step in the identification of genes encoding potential virulence factors.

FEMS Microbiol Lett, 1993 Jun 15, 110(2), 239 - 42
The biosynthetic genes for clavulanic acid and cephamycin production occur as a 'super-cluster' in three Streptomyces; Ward JM et al.; The cosmid cloning vector pHC79 has been used to clone fragments of chromosomal DNA from the Streptomyces: S . clavuligerus, S . jumonjinensis and S . katsurahamanus . These strains all produce both the beta-lactam antibiotic, cephamycin and the beta-lactamase inhibitor, clavulanic acid . Although structurally related these two beta-lactams are known to be derived from different biosynthetic precursors . Hybridisation studies and restriction mapping have shown that the gene clusters encoding the two biosynthetic pathways are chromosomally adjacent in these strains, thus creating a 'super-cluster' of genes involved in both the production and enhancement of activity of a beta-lactam antibiotic.

Biochem J, 1993 Jun 1, 292 ( Pt 2), 555 - 62
A comparative study of class-D beta-lactamases; Ledent P et al.; Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity . 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover . Labelling of the same beta-lactamase with 6 beta-iodo{3H}penicillanate allowed the identification of Ser-70 as the active-site serine residue . In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer . The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms . A first overview of the enzymic properties of the three 'oxacillinases' is presented . With the OXA2 enzyme, 'burst' kinetics, implying branched pathways, seemed to prevail with many substrates.

J Antimicrob Chemother, 1993 May, 31(5), 655 - 64
Selection of variants of the TEM-1 beta-lactamase, encoded by a plasmid of clinical origin, with increased resistance to beta-lactamase inhibitors; Thomson CJ et al.; A TEM-1 derived variant beta-lactamase with increased resistance to beta-lactamase inhibitors was selected in vitro . The variant beta-lactamase was obtained from the parent strain Escherichia coli J62-2 containing resistance plasmid R1 which encodes the TEM-1 beta-lactamase . The variants were obtained by repeated subculture of E . coli J62-2 (R1) for five days in the presence of sub-inhibitory concentrations of amoxycillin plus clavulanic acid . Comparison of this variant beta-lactamase with a clinically isolated TEM derived beta-lactamase, with increased resistance to beta-lactamase inhibitors, suggests that they are the same enzyme . Both beta-lactamases have the same pI at 5.25 and have similar ID50 values for the beta-lactamase inhibitors clavulanic acid, sulbactam and tazobactam.

Mol Microbiol, 1993 May, 8(3), 615 - 23
The arsD gene encodes a second trans-acting regulatory protein of the plasmid-encoded arsenical resistance operon; Wu J et al.; The plasmid-encoded arsenical resistance (ars) operon produces resistance to trivalent and pentavalent salts of arsenic and antimony . The first gene in the operon, arsR, was previously shown to encode a repressor protein . A newly identified gene, arsD, is shown here to encode a regulatory protein, the ArsD protein . The gene was identified by construction of an in-frame fusion between the C-terminally truncated arsD gene and the coding region for the mature form of beta-lactamase (blaM) . The native arsD gene product was overexpressed and radioactively labelled as a 13 kDa polypeptide . A frameshift mutation within the arsD gene resulted in elevated levels of expression of downstream ars genes . Co-expression of a wild-type arsD gene in trans with the operon containing the mutated arsD gene reduced expression of the downstream genes to wild-type levels . The presence of the arsD gene had no effect on the basal level of operon expression set by the arsR gene product, and the repression produced by the arsD gene product was not affected by inducers of the operon . The results indicate that the ArsD protein is an inducer-independent trans-acting regulatory protein.

FEMS Microbiol Lett, 1993 Apr 15, 108(3), 353 - 9
A lipoprotein signal peptide plus a cysteine residue at the amino-terminal end of the periplasmic protein beta-lactamase is sufficient for its lipid modification, processing and membrane localization in Escherichia coli; Oudega B et al.; By genetic exchange and in vitro mutagenesis a hybrid beta-lactamase was constructed that contained the pCloDF13-encoded bacteriocin release protein signal peptide plus a cysteine residue coupled to the mature portion of beta-lactamase . Immunoblotting, labelling with {3H}palmitate in the presence and absence of globomycin, and pulse-chase experiments revealed that this hybrid construct is modified with lipid and processed into a lipid-modified beta-lactamase . Subcellular localization studies revealed that this hybrid is localized both in the cytoplasmic and outer membranes of Escherichia coli cells . A mutant derivative with an incomplete lipobox (LVG instead of LVAC+1) was not processed and was found in the cytoplasmic membranes.

J Antibiot (Tokyo), 1993 Apr, 46(4), 641 - 6
(6R)-6-(substituted methyl)penicillanic acid sulfones: new potent beta-lactamase inhibitors; Adam S et al.; A series of (6R)-6-(substituted methyl)penicillanic acid sulfones has been prepared starting from the corresponding 6-(substituted methylene)penicillanates . The new sulfones 9a, 9b, 9c and 9d have been shown to be potent beta-lactamase inhibitors.

Pathol Biol (Paris), 1993 Apr, 41(4), 337 - 42
{Multifactorial analysis of the phenotypes for beta-lactams of 1044 Escherichia coli strains}; Chardon H et al.; 1,044 E . coli strains were randomly collected by the beginning of 1992 . Their susceptibility for seven beta-lactam antibiotics: amoxycillin, augmentin, ticarcillin, claventin, cephalothin, cefoxitin and cefotaxime, was studied routinely by the agar diffusion method . The datas were analyzed by the CERIB multifactorial analysis package which yields to homogeneous populations . This analysis showed four well defined populations: 1) 588 strains (56.4%) susceptible to all antibiotics; 2) 410 strains (39.3%) present a penicillinase phenotype; 3) 11 strains (1.05%) are cephalosporinase producer; 4) 7 strains (0.67%) were identified as producing an extended-spectrum beta-lactamase . The remaining strains: 28 (2.68%) had a reduced susceptibility to all antibiotics, which suggests the combination of few resistance mechanisms or other hypothesis.

Chemotherapy, 1993 Mar-Apr, 39(2), 88 - 95
Characterization of BRO enzymes and beta-lactamase transfer of Moraxella (Branhamella) catarrhalis isolated in Japan; Ikeda F et al.; Of the 68 strains of beta-lactamase-producing Moraxella (Branhamella) catarrhalis isolated in Japan that were studied, 62 (91%) produced the BRO-1-type beta-lactamase and 6 (9%) produced the BRO-2 type . There were no strains containing the BRO-3-type beta-lactamase . We compared the susceptibility of BRO-1- and BRO-2-producing strains to various oral beta-lactam antibiotics . We found that the BRO-1-producing strains were less susceptible than the BRO-2-producing strains . Although the BRO-1 and BRO-2 types showed a similar hydrolysis pattern, the specific activity of BRO-1 was 3-fold that of BRO-2 . We examined the transfer of the BRO-1 and BRO-2 genes to non-beta-lactamase-producing M . catarrhalis No . 4020 and found that of the 13 donor strains producing BRO-1, 11 (85%) were able to transfer the gene for BRO-1 production by conjugation . Of the 6 donor strains producing BRO-2, 2 (33%) were able to transfer the gene for BRO-2 production by conjugation . For 3 of the 13 (23%) BRO-1-producing strains and 1 of the 6 (17%) BRO-2-producing strains, about 13 Mdalton of plasmids were detected . These plasmid-containing strains were used as donors, and in beta-lactamase-producing transconjugants the same size of plasmids was detected . However, when the total DNA is extracted from strains with the ability to transfer by conjugation, the transformation of the beta-lactamase-producing gene can occur regardless of the presence or absence of plasmids . Furthermore, even if purified plasmids are transformed, beta-lactamase-producing transformants are not obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1993 Feb 14, 124(1), 111 - 4
Sequence of a gene encoding beta-lactamase from Streptomyces cellulosae; Ogawara H; The nucleotide sequence of beta-lactamase (Bla)-encoding gene, bla, from Streptomyces cellulosae KCCS0127 was determined . The deduced amino acid sequence was very close to that of class-A Bla, especially those from Streptomyces species, but completely different from class-D Bla . This is contrary to the result expected from its substrate specificity and its property of binding blue dextran and NADP+.

Chem Pharm Bull (Tokyo), 1993 Feb, 41(2), 400 - 2
Use of high performance liquid chromatography for increased assay sensitivity of beta-lactamase activity in bile; Kunieda S et al.; A study to develop a sensitive method for measuring beta-lactamase activity in bile was conducted . Since separation of substrate from biological components is required to increase the assay sensitivity and to achieve an accurate assay of beta-lactamase activity, high performance liquid chromatography (HPLC) was used for separation and analysis of the substrates (cephaloridine for cephalosporinase, benzylpenicillin for penicillinase and cefuloxime for cefuloximase) . In addition, conditions for increased assay sensitivity were also studied and optimal substrate concentrations and reaction times were determined . beta-Lactamase activity of 0.05 munit/ml in bile was detected using the HPLC assay method which is a significant improvement when compared to the direct spectrophotometric method which has a detection limit of approximately 10 munit/ml.

J Antimicrob Chemother, 1993 Feb, 31(2), 237 - 44
A membrane-bound precursor beta-lactamase in strains of Moraxella catarrhalis and Moraxella nonliquefaciens that produce periplasmic BRO-1 and BRO-2 beta-lactamases; Steingrube VA et al.; By employing the non-ionic detergent Triton X-100, a membrane-bound beta-lactamase was extracted from strains of Moraxella (Branhamella) catarrhalis and Moraxella nonliquefaciens that produce BRO-1 and BRO-2 beta-lactamases . Unlike BRO-1 and BRO-2, which exhibit multiple major bands on isoelectric focusing (IEF), the membrane-bound enzyme focused as a single IEF band at a pI of 6.20, which was not present with either of the other two enzymes . The membrane-bound beta-lactamase could be extracted from all strains producing BRO-1 and BRO-2, including recombinant strains constructed by transformation or conjugation . The enzyme was also recovered from Escherichia coli strain HB101 carrying vector plasmid pLQ521 into which the BRO-1 beta-lactamase gene from M . catarrhalis had been cloned . All three beta-lactamases were indistinguishable by inhibitor profiles with clavulanic acid, BRL 42715, sulbactam and tazobactam . These data suggested that all three beta-lactamases were the product of a single gene in Moraxella spp., and that the membrane-bound beta-lactamase serves as a precursor of both BRO-1 and BRO-2 . Species differences in cellular processing of the membrane-bound enzyme could explain the minor differences in IEF patterns that occurred with BRO-1 and BRO-2 beta-lactamases when present in different species.

Ann Surg, 1993 Feb, 217(2), 115 - 21
Efficacy of a beta-lactamase inhibitor combination for serious intraabdominal infections; Walker AP et al.; A double-blind trial was conducted in 385 patients with suspected bacterial intra-abdominal infections to compare the efficacy and safety of ampicillin-sulbactam with cefoxitin . Patients were randomized to receive either 3 g ampicillin-sulbactam (2 g ampicillin-1 g sulbactam), or 2 g cefoxitin, every 6 hours . To be evaluable, patients had to demonstrate positive culture evidence of peritoneal infection at the time of operation . A total of 197 patients were evaluable for clinical efficacy . The two treatment groups were comparable in demographic features and in the presence of risk factors for infection . Clinical success (absence of infection and of adverse drug reaction) was observed in 86% of patients in the ampicillin-sulbactam group and 78% in the cefoxitin group . Eradication of infection occurred in 88% of the ampicillin-sulbactam group and 79% of the cefoxitin group . There were no differences in the nature or frequency of side effects observed in the two groups . Ampicillin-sulbactam demonstrated no difference in safety or efficacy when compared with cefoxitin in the treatment of serious intra-abdominal infections of bacterial origin.

J Med Microbiol, 1993 Feb, 38(2), 114 - 7
Stability of cefdinir (C1-983, FK482) to extended-spectrum plasmid-mediated beta-lactamases; Payne DJ et al.; Fourteen plasmid-encoded extended-spectrum beta-lactamases were purified from Escherichia coli transconjugants of original clinical isolates . The Vmax, Km and Vmax/Km were each determined for ampicillin, carbenicillin, cephaloridine, cephalexin, cefuroxime, cefixime, cefdinir, ceftazidime and cefotaxime as substrates with eight of these enzymes and with the narrow-spectrum beta-lactamase, TEM-1 . The relative rates of hydrolysis of ampicillin, cephaloridine, cephalexin, cefuroxime, cefixime, cefdinir, ceftazidime and cefotaxime were also determined for the remaining six enzymes . Cefdinir had Vmax/Km or relative rates of hydrolysis values either equal to or lower than ampicillin, cephaloridine, cephalexin and cefotaxime for all the enzymes tested . Overall, cefdinir was more stable to the 15 beta-lactamases tested than either cefuroxime or cefixime; however, ceftazidime was more stable than cefdinir to hydrolysis by eight of the enzymes tested.

Appl Environ Microbiol, 1993 Feb, 59(2), 561 - 6
Overexpression of bacterial hemoglobin causes incorporation of pre-beta-lactamase into cytoplasmic inclusion bodies; Rinas U et al.; The expression of Vitreoscilla hemoglobin (VHb) in Escherichia coli JM101 (pRED2) causes the incorporation of the TEM beta-lactamase precursor into cytoplasmic inclusion bodies (IBs) . Less pre-beta-lactamase is translocated and processed to its mature, periplasmic form in the strain coexpressing VHb than in the control strain E . coli JM101(pUC19) not expressing VHb . When cells are grown in a special fed-batch procedure, the formation of cytoplasmic IBs consisting of pre-beta-lactamase is also inducible in the control strain . Comparative microscopic and compositional analyses of IBs generated in E . coli JM101(pUC19) and JM101(pRED2) under identical growth conditions strongly suggest that pre-beta-lactamase and VHb coaggregate into common IBs in E . coli JM101 (pRED2).

J Clin Lab Anal, 1993, 7(2), 95 - 9
Stability studies of the components of a prototype penicillinase (beta-lactamase)-linked ELISA kit; Khatkhatay MI et al.; Penicillinase (beta-lactamase) enzyme-linked immunosorbent assay (ELISA) for various reproductive hormones developed in the laboratory were found to have wide applicability in the fertility check clinic of the Institute . A need was thought to transform these assays into ready-to-use kit forms . Therefore, prototype ELISA kits for these hormones were developed and stability of the individual component was ascertained at various temperatures (room temperature, 37 degrees C and 2-8 degrees C) . Stability studies were conducted on previously validated assay for pregnanediol-3 alpha-glucuronide (PdG) . The studies showed that immunosorbents (antibody coated plates) are stable at room temperature for a period of 2 weeks, at 37 degrees C for 1 week and at 2-8 degrees C for a period of 9 months when preserved after treatment with glycerol solution . The lyophilised conjugate, standard and immunoassay buffer, colour reagent, and its substrate were stable at 37 degrees C up to 1 week and at room temperature up to 2 weeks and at 2-8 degrees C for a period of 6 months, during which the stability was studied.

J Antimicrob Chemother, 1993 Jan, 31 Suppl A, 1 - 8
Meeting the challenges of beta-lactamases; Moellering RC Jr; A wide variety of beta-lactamases are found in clinical isolates of bacteria and, when present, these enzymes often result in resistance to one or more beta-lactam antibiotics . The prevalence of organisms with these enzymes has increased as beta-lactams have been increasingly used in clinical practice . This paper defines the nature of these enzymes and details the attempts to overcome the problem of resistance mediated by beta-lactamase, efforts which have culminated in the development of a series of effective beta-lactamase inhibitors which can be combined with beta-lactam antibiotics . The currently available compounds, clavulanic acid, sulbactam, and tazobactam are very effective inhibitors of many types of beta-lactamases, but there are additional enzymes which are resistant even to these inhibitors and which pose continuing challenges for the pharmaceutical chemist and clinician alike.

Pediatr Infect Dis J, 1993 Jan, 12(1), 24 - 8
Comparative study of sultamicillin and amoxicillin-clavulanate: treatment of acute otitis media; Chan KH et al.; Sultamicillin is a mutual prodrug of ampicillin and sulbactam that is chemically linked by a diester bond . This investigational agent has beta-lactamase-inhibiting activity by virtue of sulbactam, a novel beta-lactamase inhibitor . A double blind randomized study was conducted to evaluate the safety, efficacy and tolerance of sultamicillin for treatment of acute otitis media compared with amoxicillin-clavulanate . A total of 144 subjects were included (96 randomly assigned to the sultamicillin and 48 to the amoxicillin-clavulanate groups) . No safety concerns for sultamicillin were identified during the study . The clinical efficacy in effusion clearance between the two groups was found not to be statistically different at 10 days (P = 0.23) and 30 days (P = 0.72) . Similar rates of side effects, primarily gastrointestinal, were reported in both study groups . Sultamicillin may be an alternative for the treatment of acute otitis media when persistence and recurrence of disease become an issue.

Nucleic Acids Symp Ser, 1993, (29), 153 - 4
A possible function of DNA curvature in transcription; Ohyama T et al.; The region preceding the beta-lactamase promoter of Escherichia coli plasmid pUC19 has a DNA curvature (curved DNA or bent DNA) . Studies using mutant plasmids showed that the curved DNA structure is required for efficient transcription of the beta-lactamase gene and suggested that the requisite shape is a small part of a right-handed coil . Based on these results, the possible functional significance of DNA curvature in transcription will be discussed.

J Pharm Pharmacol, 1993 Jan, 45(1), 25 - 9
A semi-empirical study of some clavulanic acid derivatives in relation to their activity as beta-lactamase inhibitors; Fernandez B et al.; Clavulanic acid and some of its derivatives are inhibitors of beta-lactamases and hence enhance the activity of antibiotics such as cephaloridine, amoxycillin and ampicillin against beta-lactamase-producing bacteria . Several empirical structure-activity relationships have been proposed for these compounds; bearing these findings in mind, we have carried out a semi-empirical structural study on clavulanic acid and twelve of its derivatives lacking the carboxyl group . We relate our results to the available activity data, focusing on features such as planarity at the N atom, charge distribution and the orientation of the substituents, all of which are related to the activity of these systems.

Tuber Lung Dis, 1992 Dec, 73(6), 337 - 44
Isoelectric focusing patterns of beta-lactamases in the rapidly growing mycobacteria; Zhang Y et al.; beta-lactamases from 259 strains of rapidly growing mycobacteria that included the third biovariant complex of Mycobacterium fortuitum, M . peregrinum, M . abscessus, M . chelonae, the M . chelonae-like organisms (MCLO), and M . smegmatis were analyzed by isoelectric focusing (IEF) . All isolates produced acidic beta-lactamases with major band isoelectric points (pIs) between 4.4 and 6.0 . Each of the 6 taxonomic groups exhibited 1 or 2 characteristic beta-lactamase IEF patterns . Heterogeneity among IEF patterns was evident in 5 of the 6 groups, however, and was greatest among the third biovariant complex of M . fortuitum . beta-lactamase patterns correlated with previously identified taxonomic subgroups of M . smegmatis and the third biovariant complex of M . fortuitum . beta-lactamase IEF analysis of MCLO strains isolated from two outbreaks demonstrated its possible usefulness for epidemiologic evaluation.

FEBS Lett, 1992 Dec 7, 314(1), 89 - 92
'All-or-none' mechanism of the molten globule unfolding; Uversky VN et al.; The Gdm-HCl-induced unfolding of bovine carbonic anhydrase B and S . aureus beta-lactamase was studied at 4 degrees C by a variety of methods . With the use of FPLC it has been shown that within the transition from the molten globule to the unfolded state the distribution function of molecular dimensions is bimodal . This means that equilibrium intermediates between the molten globule and the unfolded states are absent, i.e . the molten globule unfolding follows the 'all-or-none' mechanism.

Anal Biochem, 1992 Dec, 207(2), 329 - 34
Chromogenic redox assay for beta-lactamases yielding water-insoluble products . II . Heterogeneous sandwich assay for hCG; Bieniarz C et al.; A very sensitive and rapid heterogeneous sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) is described . The assay is based on the application of the novel chromogenic redox substrate system for beta-lactamase which is used as label . The chromogen system consists of a thioacetylcephalosporin beta-lactamase substrate, which upon turnover by the enzyme label releases the thiolate with the concomitant reduction of the tetrazolium salt to a colored formazan . The concentration of the formazan is directly related to the amount of the hormone in the sample and is read spectrophotometrically . The enzyme-antibody conjugates, produced through use of heterobifunctional maleimide crosslinker, maintain 90% of the enzyme activity after 30 days at 25 degrees C . Concentrations of the hormone as low as 5 mIU/ml, equivalent to 25 fmol/ml, are detectable in 3 h.

Biosci Biotechnol Biochem, 1992 Dec, 56(12), 1985 - 90
Overproduction of biologically-active human nerve growth factor in Escherichia coli; Fujimori K et al.; A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E . coli . The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide . The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene . E . coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide . The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth . This biological activity was comparable to that of authentic mouse NGF . E . coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein . Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.

J Biol Chem, 1992 Nov 25, 267(33), 23916 - 21
Protein translocation in the endoplasmic reticulum . Ultraviolet light induces the noncovalent association of nascent peptides with translocon proteins; Anderson L et al.; We have photolyzed cell-free translation systems synthesizing beta-lactamase with 254-nm ultraviolet light . In the presence of canine rough microsomes (RM), incomplete chains of beta-lactamase became enriched relative to the full-length molecule in pellet fractions obtained following photolysis and alkaline carbonate extraction . In addition, high molecular weight aggregates were present on SDS-polyacrylamide gels and occurred only when translocation-competent microsomal membranes were used in translation mixtures . The incomplete chains and high molecular weight aggregates were not obtained when RM were inactivated by reaction with N-ethylmaleimide . The incomplete chains did not bind to concanavalin A-Sepharose, indicating that they had not sedimented as a result of being covalently cross-linked to membrane glycoproteins . Both photolysis and alkaline carbonate extraction were required to produce the results . Nascent peptides that were not exposed to alkaline carbonate following photolysis did not appear as high molecular weight bands on SDS-polyacrylamide gels . The high molecular weight aggregates therefore represent denatured protein complexes that contain nascent peptides and microsomal translocon proteins . The results suggest that the translocon is a large proteinaceous complex and that at least a portion of it, when denatured, migrates at a molecular mass of approximately 205 kDa.

J Mol Biol, 1992 Nov 20, 228(2), 359 - 65
General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli . Production of the Bence-Jones protein REIv via fusion to beta-lactamase; Kolmar H et al.; A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli . This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner . The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure . The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage . After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture . A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops . It exploits a colony color genetic screen and can be applied to any DNA sequence replacement . Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.

FEMS Microbiol Lett, 1992 Nov 15, 78(1), 101 - 5
Two different beta-lactamase genes are present in Streptomyces cacaoi; Magdalena J et al.; Two beta-lactamase genes called blaL and blaU have been cloned independently in Liege and in Umea, from Streptomyces cacaoi . Genes blaL and blaU were found to differ largely in their nucleotide sequences, although the encoded proteins both belonged to the class A of beta-lactamases (active-site serine penicillinases) . DNA-hybridization and polymerase chain reaction assays have now demonstrated that both blaL and blaU genes were present in the S . cacaoi strains used in Liege and in Umea.

J Bacteriol, 1992 Nov, 174(21), 6771 - 9
A heterologous membrane protein domain fused to the C-terminal ATP-binding domain of HlyB can export Escherichia coli hemolysin; Thomas WD Jr et al.; The hydrophobic-rich NH2-terminal 34 amino acids of a tetracycline resistance determinant (TetC) were fused to the COOH-terminal 240 amino acids of the hemolysin transporter, HlyB, which contains a putative ATP-binding domain . This hybrid protein replaced the NH2-terminal 467-amino-acid portion of HlyB and could still export the Escherichia coli hemolysin (HlyA) . Export by the hybrid protein was approximately 10% as efficient as transport by HlyB . Extracellular secretion of HlyA by the TetC-HlyB hybrid required HlyD and TolC . The extracellular and periplasmic levels of beta-galactosidase and beta-lactamase in strains that produced the hybrid were similar to the levels in controls . Thus, HlyA transport was specific and did not appear to be due to leakage of cytoplasmic contents alone . Antibodies raised against the COOH terminus of HlyB reacted with the hybrid protein, as well as HlyB . HlyB was associated with membrane fractions, while the hybrid protein was found mainly in soluble extracts . Cellular fractionation studies were performed to determine whether transport by the hybrid occurred simultaneously across both membranes like wild-type HlyA secretion . However, we found that HlyA was present in the periplasm of strains that expressed the TetC-HlyB hybrid . HlyA remained in the periplasm unless the hlyD and tolC gene products were present in addition to the hybrid.

Gene, 1992 Nov 2, 121(1), 87 - 94
Mutagenic analysis of the promoter of the Streptomyces fradiae beta-lactamase-encoding gene; Forsman M et al.; The Streptomyces fradiae beta-lactamase promoter (PblaF) was sequenced and characterized by promoter probing, primer extension, and exonuclease III-mediated deletions . The transcription start point (tsp) was the same in both S . lividans and S . fradiae . Oligodeoxyribonucleotide-directed random mutations and site-specific mutations were introduced in the promoter region . The effects of these mutations on transcription were assayed by an RNA colony hybridization method . This analysis identified cis-acting sequence determinants located similarly to the -10 and -35 regions of a typical Escherichia coli promoter . Also, a change in the distance between these regions from 19 to 17 bp drastically reduced promoter activity . PblaF was shown not to be recognized by sigma-whiG or by sigma-hrdA, hrdC, or hrdD . Sequence alignment of PblaF to sigma factor-classified Streptomyces promoters revealed little homology . Thus, PblaF is probably recognized by an as yet unidentified sigma factor.

Mol Gen Genet, 1992 Nov, 235(2-3), 269 - 78
Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha; Hansen H et al.; Dihydroxyacetone synthase (DAS) and methanol oxidase (MOX) are the major enzyme constituents of the peroxisomal matrix in the methylotrophic yeast Hansenula polymorpha when grown on methanol as a sole carbon source . In order to characterize their topogenic signals the localization of truncated polypeptides and hybrid proteins was analysed in transformed yeast cells by subcellular fractionation and electron microscopy . The C-terminal part of DAS, when fused to the bacterial beta-lactamase or mouse dihydrofolate reductase, directed these hybrid polypeptides to the peroxisome compartment . The targeting signal was further delimited to the extreme C-terminus, comprising the sequence N-K-L-COOH, similar to the recently identified and widely distributed peroxisomal targeting signal (PTS) S-K-L-COOH in firefly luciferase . By an identical approach, the extreme C-terminus of MOX, comprising the tripeptide A-R-F-COOH, was shown to be the PTS of this protein . Furthermore, on fusion of a C-terminal sequence from firefly luciferase including the PTS, beta-lactamase was also imported into the peroxisomes of H . polymorpha . We conclude that, besides the conserved PTS (or described variants), other amino acid sequences with this function have evolved in nature.

J Biol Chem, 1992 Oct 15, 267(29), 20600 - 6
Site-directed mutagenesis at the active site of Escherichia coli TEM-1 beta-lactamase . Suicide inhibitor-resistant mutants reveal the role of arginine 244 and methionine 69 in catalysis; Delaire M et al.; Arginine 244 is a highly conserved residue in Class A beta-lactamases, while methionine 69 is not . Informational suppression experiments show that replacement of M69 by a leucine, or that of R244 by most other amino acids lead to clavulanic acid-resistant phenotypes . The arginyl 244 side chain is tightly held in a network of interactions within the active site . Its replacement by a glutamine or a threonine perturbs the enzyme kinetics but to a smaller extent than would have been predicted if it were directly involved in substrate binding . Clavulanic acid and sulbactam still interact specifically with the mutant enzymes but are much less efficiently metabolized . Substitutions at position 244 also unveil interactions between the C6 substituent of substrates and the Asn132/Glu104 region of the active site . Methionine 69 is located in a region of strong structural constraints and presents an unusual conformation . Molecular dynamics simulation showed that its replacement by a leucine does not release the strain in this area and induces only minor structural changes . Accordingly, the kinetic behavior of the mutant is only marginally perturbed, except for suicide inhibitors . Both clavulanic acid and sulbactam are well degraded by the mutant enzyme, while irreversible inactivation is dramatically decreased . The contribution of both residues to catalysis is discussed in the light of the kinetic and structural data.

J Biochem (Tokyo), 1992 Oct, 112(4), 492 - 4
Effect of the potential triplex DNA region on the in vitro expression of bacterial beta-lactamase gene in superhelical recombinant plasmids; Kato M et al.; The effect of a pyrimidine/purine-biased stretch which has the potential to form an unusual triplex DNA structure on gene expression has been analyzed by measuring the activity of beta-lactamase as a reporter gene in recombinant plasmids . The Escherichia coli transformant carrying the plasmid p7ERS which has a potential triplex DNA region expressed about twofold more beta-lactamase activity than that carrying the plasmid pUC19 . Since the expression of beta-lactamase has been shown to be affected by template topology in vitro, this in vivo observation suggests that the inserted pyrimidine/purine-biased stretch modulates the topology of flanking regions by forming unusual DNA structure to keep the template at the superhelicity favorable for the expression of beta-lactamase.

J Antimicrob Chemother, 1992 Oct, 30(4), 449 - 62
Clinical isolates of Escherichia coli producing TRI beta-lactamases: novel TEM-enzymes conferring resistance to beta-lactamase inhibitors; Vedel G et al.; Two different strains of Escherichia coli exhibiting unusual patterns of resistance to beta-lactam antibiotics were isolated from patients at Cochin Hospital . Both isolates showed a low level of resistance to amoxycillin, ticarcillin and ureidopenicillins but were susceptible to cephalosporins, aztreonam and imipenem; beta-lactamase inhibitors potentiated the activities of the beta-lactams to only a limited extent . All resistance characteristics of the strains were transferable by conjugation to E . coli K12 . Resistance was shown to be due to beta-lactamases of pI 5.20 and relative molecular masses of 24,000 . The hydrolytic and inhibition profiles of these enzymes were similar to each other but differed from those of broad-spectrum beta-lactamases (TEM-1) . The rates of hydrolysis (Vmax) of amoxycillin (c . 200%) were higher than that for TEM-1 (84%) . Ticarcillin, ureidopenicillins and cephaloridine were hydrolyzed slowly . However, as for TEM-1, no hydrolysis was observed with cefoxitin, third generation cephalosporins, aztreonam and imipenem . The high Km values demonstrated the poor affinity of these enzymes for their substrates . Unlike TEM-1, they were poorly inhibited by beta-lactamase inhibitors . These two enzymes differed from each other as follows: (i) the concentrations of clavulanic acid required for 50% beta-lactamase inhibition were 31 mumol/L for one enzyme (E-SAL) and 9.4 mumol/L for the other (E-GUER); (ii) p-chloromercuribenzoate was a more active inhibitor of E-SAL then E-GUER . The titration curve method and DNA-DNA hybridization studies demonstrated that both enzymes were structurally related to TEM-1 . The novel plasmid-encoded enzymes produced by the two isolates of E . coli appeared to be almost identical and to be derived from TEM-enzymes . On the basis of their presumed phylogeny and their biological properties, we propose that these beta-lactamases be given the generic name TRI (TEM Resistant to beta-lactamase Inhibitors).

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2173 - 83
Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a beta-lactamase fusion protein; Niviere V et al.; A secretion vector, pVN1, expressing the {NiFe} hydrogenase signal peptide of Desulfovibrio vulgaris Hildenborough fused to beta-lactamase from Escherichia coli was constructed in order to study the unusual characteristics of hydrogenase signal peptides, which share a strictly conserved sequence, the consensus box: R-R-X-F-X-K . Although the hydrogenase signal peptide-beta-lactamase fusion protein was processed much more slowly than the fusion of beta-lactamase with its own signal peptide, the system mimicked several features expected for hydrogenase biosynthesis in E . coli, including increased export under anaerobic conditions . Site-directed mutagenesis of R(-28), the first arginine residue of the consensus box, to a glutamate completely inhibited export and processing of the fusion protein . The same mutation of R(-33), located outside the consensus box, had almost no effect . The data indicate a specific role for the consensus box sequence in the export mechanism for hydrogenase.

Kansenshogaku Zasshi, 1992 Sep, 66(9), 1276 - 82
{Etiology of pneumonia and host defense}; Sato A et al.; To investigate the relationship of efficacy of chemotherapy to host defense, we reviewed the clinical features, treatment and outcome in 648 patients with acute pneumonia (424 males and 224 females; mean age, 65 years) treated between 1984 and 1989 . Pneumonia complicated pulmonary disease in 336 patients (52%) and complicated systemic disease in 258 (40%) . Pneumonia pathogens were diagnosed in 346 patients (53%); the five major pathogens were S . pneumoniae (19%), H . influenzae (16%), S . aureus (14%), K . pneumoniae (14%) and P . aeruginosa (11%) . The incidence of K . pneumoniae infection were decreased from 18% to 3.5% and that of S . aureus increased from 10.9% to 26.3% during the study period . The incidence of S . aureus and of P . aeruginosa infection was much higher in patients with nosocomial pneumonia, systemic disease, or serum protein concentration under 6.5 g/dl . beta-lactamase antibiotics were administered to 70% of all patients, with an efficacy rate of 74.9% of after the first course of antibiotics therapy . The efficacy rate was decreased in patients with nosocomial pneumonia, systemic or pulmonary disease, or malnutrition . The data presented in this study indicate that the risk of pneumonia must be taken into careful consideration in patients with compromising complications.

Antimicrob Agents Chemother, 1992 Sep, 36(9), 1817 - 20
Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 beta-lactamase genes; Chanal C et al.; CAZ-2, CAZ-6, and CAZ-7 are plasmid-mediated beta-lactamases that are markedly active against ceftazidime . The corresponding structural genes were amplified by the polymerase chain reaction . Nucleotide sequences were determined by direct sequencing of the amplified products . Analysis of the nucleotide and the deduced amino acid sequences showed that CAZ-2, CAZ-6, and CAZ-7 are derived from TEM-2 by three, four, and two amino acid substitutions, respectively . All these substitutions are located at positions 102, 162, 235, 236, and 237 (Sutcliffe numbering), which are known to extend the substrate range of beta-lactamases . These substitutions are Lys-102, Ser-162, and Ser-236 in CAZ-2; Lys-102, Ser-162, Thr-235, and Lys-237 in CAZ-6; and Lys-102 and His-162 in CAZ-7 . These results indicate that the nucleotide sequence of CAZ-2 is identical to that of TEM-8 . The nucleotide sequence of CAZ-7 possesses the two mutations described in TEM-16 by the oligotyping method . In contrast, the combination of mutations encountered in CAZ-6 has not yet been described, and this enzyme was designated TEM-24.

Proteins, 1992 Sep, 14(1), 29 - 44
Probing beta-lactamase structure and function using random replacement mutagenesis; Palzkill T et al.; A new analytical mutagenesis technique is described that involves randomizing the DNA sequence of a short stretch of a gene (3-6 codons) and determining the percentage of all possible random sequences that produce a functional protein . A low percentage of functional random sequences in a complete library of random substitutions indicates that the region mutagenized is important for the structure and/or function of the protein . Repeating the mutagenesis over many regions throughout a protein gives a global perspective of which amino acid sequences in a protein are critical . We applied this method to 66 codons of the gene encoding TEM-1 beta-lactamase in 19 separate experiments . We found that TEM-1 beta-lactamase is extremely tolerant of amino acid substitutions: on average, 44% of all mutants with random substitutions function and 20% of the substitutions are expressed, secreted, and fold well enough to function at levels similar to those for the wild-type enzyme . We also found a few exceptional regions where only a few random sequences function . Examination of the X-ray structures of homologous beta-lactamases indicates that the regions most sensitive to substitution are in the vicinity of the active site pocket or buried in the hydrophobic core of the protein . DNA sequence analysis of functional random sequences has been used to obtain more detailed information about the amino acid sequence requirements for several regions and this information has been compared to sequence conservation among several related beta-lactamases.

Gene, 1992 Sep 1, 118(1), 93 - 5
A modified TnphoA useful for single-stranded DNA sequencing; Mielke DL et al.; The TnphoA transposon constructed by Manoil and Beckwith {Proc . Natl . Acad . Sci . USA 82 (1985) 8129-8133} has been modified to permit easy isolation of single-stranded (ss) DNA of target plasmids . The intergenic region (IG) of filamentous phage f1, which consists of the phage origin of replication and packaging signal, was inserted into a nonessential region of TnphoA . This modified transposon should be useful for the analysis of genes cloned in plasmids that lack a filamentous phage IG . Transposition of TnphoA-IG into a plasmid carries the IG with it; subsequently, after infection with a filamentous helper phage, ss plasmid DNA suitable for sequence analysis and useful for oligodeoxyribonucleotide-mediated mutagenesis of TnphoA-generated fusions can be isolated . The utility of TnphoA-IG was confirmed by analysis of 'blue hops' into the bla (encoding beta-lactamase) and pspE (encoding phage shock protein) genes whose products are secreted into the Escherichia coli periplasm.

J Bacteriol, 1992 Aug, 174(16), 5485 - 7
Membrane topology of the Escherichia coli ExbD protein; Kampfenkel K et al.; The ExbD protein is involved in the energy-coupled transport of ferric siderophores, vitamin B12, and B-group colicins across the outer membrane of Escherichia coli . In order to study ExbD membrane topology, ExbD-beta-lactamase fusion proteins were constructed . Cells expressing beta-lactamase fusions to residues 53, 57, 70, 76, 78, 80, 92, 121, and 134 of ExbD displayed high levels of ampicillin resistance, whereas fusions to residues 9 and 19 conferred no ampicillin resistance . It is concluded that the only hydrophobic segment of ExbD, encompassing residues 23 to 43, forms a transmembrane domain and that residues 1 to 22 are located in the cytoplasm and residues 44 to 141 are located in the periplasm.

Mol Microbiol, 1992 Aug, 6(15), 2201 - 8
Disulphide bridge formation in the periplasm of Escherichia coli: beta-lactamase:: human IgG3 hinge fusions as a model system; De Sutter K et al.; We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human IgG3 hinge region (Hi) fused in-phase to the C-terminal end of bacterial TEM1 beta-lactamase (Bla) . In the first fusion gene blahi, TEM1 beta-lactamase (Bla) . In the first fusion gene blahi, the hinge sequence was directly coupled to the 3' end of the beta-lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits . After expression (24 h, 20 degrees C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin-resistance to the cells . These results indicate that beta-lactamase can efficiently direct the export of proteins fused to its C-terminus, and moreover, at least some of the exported fusion proteins must carry the beta-lactamase moiety in a properly folded form . Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab')2-like dimer . The presence in the periplasm of 'oxidized' monomers (with intrachain disulphide bonds) as well as of several high-molecular-mass proteins, probably resulting from the association between monomers and other cysteine-rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds.

Eur J Biochem, 1992 Jul 15, 207(2), 803 - 11
Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY; Swidersky UE et al.; SecY is an integral plasma-membrane protein of Escherichia coli which is essential for the export of periplasmic and outer-membrane proteins containing cleavable signal sequences . We have synthesized SecY in vitro using an E . coli transcription/translation system . In the absence of membranes, SecY remained largely soluble but cosedimented on sucrose gradients with the membrane fraction when inside-out plasma-membrane vesicles (INV) had been added cotranslationally . Membrane association of SecY was unaffected if the endogenous SecY of the INV had been inactivated by either antibodies, a mutation or trypsin treatment . In contrast, inactivation of the INV SecY interfered with membrane targeting and, consequently, the processing of precursors to beta-lactamase and lambda receptor . When SecY-deprived INV were, however, first functionally reconstituted with in-vitro-synthesized SecY, targeting and translocation of the lambda receptor were partially restored . Thus, the assembly of SecY into INV in vitro leads to an active enzyme . In addition, we show that the prlA4 allele of the secY gene suppresses signal-sequence mutations of the lambda receptor in vitro . Collectively, our results demonstrate that SecY, while functioning as a membrane-located receptor for precursors of exported proteins, appears to be virtually independent of pre-existing SecY for its own membrane integration.

Antimicrob Agents Chemother, 1992 Jul, 36(7), 1545 - 52
Use of an enzyme-linked immunosorbent assay to assess penetration of amoxicillin into lung secretions; Hill SL et al.; An enzyme-linked immunosorbent assay (ELISA) was developed to measure total amoxicillin concentrations penetrating lung secretions, which were compared with "active" concentrations measured by conventional bioassay . An antibody was raised in rabbits to amoxicillin conjugated to bovine serum albumin and used in a competitive binding ELISA (sensitivity, 10 ng/ml; precision {coefficient of variation}, 9%) . The measurement of amoxicillin in lung secretions by using the ELISA method was verified by high-performance liquid chromatography . Amoxicillin concentrations were found to be similar in both whole sonicated sputum and sol-phase sputum obtained by ultracentrifugation following single oral doses of 3 g (4.6 mg/liter for sonicated and 4.7 mg/liter for sol-phase preparations) and 250 mg (0.23 mg/liter for both preparations) . Eight patients with bronchiectasis received 500 mg of amoxicillin three times daily . On the second day of therapy (4 h after the morning dose), the mean concentration of amoxicillin in sputum was 0.88 mg/liter (standard error of the mean {SEM}, 0.11) by ELISA and 0.40 mg/liter (SEM, 0.05) by bioassay, suggesting a significant degree of local inactivation . This difference between total and active amoxicillin levels was found to correlate significantly (r = 0.693; P less than 0.05) with beta-lactamase levels (mean, 29.5 mU/ml; SEM, 9.4) . A pharmacokinetic study on day 3 revealed maximum levels in secretions 2 to 4 h after dosing (mean, 1.36 mg/liter; SEM, 0.26) . At the end of successful therapy (day 14), total and active levels were lower (mean, 0.48 mg/liter; SEM, 0.11 {total}; mean, 0.21 mg/liter; SEM, 0.06 {active}); this result was associated with a reduction in lung inflammation (decreased serum-derived albumin in the lung secretions) . In conclusion, antibiotic penetration is partly dependent on the degree of lung inflammation . The differences observed in total and active levels of amoxicillin and the relationship to beta-lactamase activity in sputum suggest why higher doses of antibiotic may be required to produce a therapeutic response in some patients.

Biotechnol Prog, 1992 Jul-Aug, 8(4), 340 - 6
Escherichia coli host cell modifications in continuous culture affecting heterologous protein overproduction: a population dynamics study; Fu J et al.; There are many published studies of plasmid segregational instability in Escherichia coli in the literature . However, the formation of plasmid-free segregants can be controlled by the addition of selective chemical agents like antibiotics . This solution has become commonplace in both the laboratory and industry . On the other hand, host cell modifications, which result in low production of plasmid-encoded protein and lead to loss of culture productivity, have not been adequately addressed . Continuous culture of an inducible (ptac) Escherichia coli vector containing strain, RB791(pKN), was characterized by strong dynamic changes in the cell population and product (beta-lactamase) expression . Long-term cultivation resulted in the loss of high-level production of beta-lactamase . Loss of productivity was not due to the formation of plasmid-free cells or structural modifications to the plasmid; instead, continuous operation resulted in a culture dominated by irreversibly altered, low-producing cells . Two distinct classes of lac- mutants which inhibited induction were identified (Y- and I(s)).

J Biol Chem, 1992 Jun 25, 267(18), 12570 - 6
Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase; Wu J et al.; The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli . The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB . The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions . Random gene fusions with lacZ and phoA were generated using transposon mutagenesis . A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector . To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase . The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera . From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed.

Am J Med, 1992 Jun 22, 92(6A), 65S - 69S
Efficacy and safety of loracarbef in the treatment of pneumonia; Hyslop DL; The treatment of bacterial pneumonia requires an agent with activity against a wide range of bacterial pathogens, including pathogens that produce beta-lactamase . Loracarbef, a member of the carbacephem class of antibiotics, was tested in a series of clinical trials for its efficacy and safety in the treatment of lobar and bronchial bacterial pneumonia . Successful clinical responses were achieved in 97.6% of the evaluable patients receiving 400 mg twice daily of loracarbef . This compared favorably with the respective response rates of 92.3% for patients receiving 500 mg three times a day of amoxicillin/clavulanate and 95.0% for patients receiving 500 mg three times a day of amoxicillin for the same illnesses . Proven or presumed elimination of the pretherapy pathogen was found in 89% of the patients receiving loracarbef, 92.3% of the amoxicillin/clavulanate-treated patients, and 70.0% of those receiving amoxicillin . Loracarbef was also well tolerated, although nausea and vomiting were associated with the use of all three agents . Nevertheless, treatment with loracarbef resulted in the lowest rate of discontinuation of therapy due to drug-related adverse events . Thus, these clinical trials support the conclusion that loracarbef is a safe and effective treatment for bacterial pneumonia.

FEBS Lett, 1992 Jun 15, 304(2-3), 103 - 8
Coordinate regulation of murein peptidase activity and AmpC beta-lactamase synthesis in Escherichia coli; Bishop RE et al.; In the periplasmic space of Escherichia coli, the (L)-m-A2pm-(D)-m-A2pm peptide, the lipoprotein, and the AmpC beta-lactamase are controlled by growth rate . To explain this coordinate regulation, it is proposed that the AmpC protein functions as an LD-endopeptidase in addition to its known function as a beta-lactamase . As LD-peptides, DD-peptides and beta-lactams are structurally similar, LD-peptidases may belong to the larger family of DD-peptidases and serine beta-lactamases . In contrast to E . coli, many related bacteria possess an inducible AmpC protein . Several gene systems necessary for AmpC induction are known to affect various aspects of peptidoglycan metabolism . It is proposed that AmpC induction occurs indirectly via a recyclable cell wall peptide.

APMIS, 1992 Jun, 100(6), 479 - 89
Screening method for beta-lactamase substrate profiles; Schumacher H et al.; A disc diffusion method, based on the idea of Klundert, for screening of substrate profiles of beta-lactamases was developed in order to perform epidemiological studies . The method was tested against 30 different reference beta-lactamases and 59 clinical isolates known to produce TEM-1, SHV-1 and BRO-1 . The reproducibility and discriminating power of the disc diffusion method made it possible to differentiate between eight types of substrate profiles for the 30 reference beta-lactamases and to differentiate between TEM-1, SHV-1 and BRO-1 from clinical isolates . In combination with analytical isoelectric focusing the disc diffusion method gives a reliable identification of beta-lactamases.

Biochem J, 1992 Jun 1, 284 ( Pt 2), 411 - 5
Mutations altering substrate specificity in OHIO-1, and SHV-1 family beta-lactamase; Shlaes DM et al.; The OHIO-1 beta-lactamase does not normally hydrolyse oxyimino-beta-lactam substrates like cefotaxime, ceftriaxone, ceftazidime or aztreonam . We were able to select spontaneous mutants of an OHIO-1-bearing strain of Escherichia coli using the antibiotic substrates listed above by enrichment methods of frequencies of 10(-8)-10(-10) for all antibiotics except ceftazidime (frequency less than 10(-10)) . Most mutants with increased resistance to the other beta-lactams were also more resistant to ceftazidime . Mutations identified by DNA sequencing included a Gly238----Ser238 substitution identical with the SHV-2 mutation previously described, cysteine and valine substitutions at the identical site, and a Gly242----Cys242 substitution . The Cys238 and Cys242 mutant enzymes had less affinity for aztreonam than had the other mutant enzymes . Hydrolysis of cefotaxime, but not cephaloridine, by the cysteine-substituted enzymes was inhibited by p-chloromercuribenzoate . The mutant enzymes had, in general, greater affinity for the mechanism-based inhibitors sulbactam, clavulanic acid and tazobactam . These results suggest two non-mutually exclusive hypotheses for the structural role of substitutions in this area of the enzyme . Either potential hydrogen-bond donors, such as serine and cysteine, interact directly with the beta-lactam molecules, or the steric bulk of these substitutions distorts the beta-pleated sheet such that the beta-lactam is held in a position favourable for stable binding and catalysis . Finally, our data raise questions about a strategy relying on oligonucleotide-probe technology to detect such mutations, because of the variety of substitutions that give rise to similar phenotypes.

Mol Microbiol, 1992 Jun, 6(12), 1693 - 705
Phylogeny of LCR-1 and OXA-5 with class A and class D beta-lactamases; Couture F et al.; The nucleotide sequences of blaLCR-1 and blaOXA-5 beta-lactamase genes have been determined . Polypeptide products of 260 and 267 amino acids with estimated molecular masses of 27 120 Da and 27,387 Da were obtained for the mature form of LCR-1 and OXA-5 proteins . A progressive alignment was used to evaluate the extent of identity between LCR-1 and OXA-5 with 29 other beta-lactamase amino acid sequences . The data showed that both belong to class D . We identified amino acids conserved in 24 positions for class A beta-lactamases and in 28 positions for five class D enzymes . The structural similarities between class A and class D beta-lactamases are more extensive than indicated by earlier biochemical studies with overall 16% identity between both classes . From the alignment, dendograms were constructed with a distance-matrix and parsimony methods which defined three major groups of proteins subdivided into clusters giving insight on beta-lactamase phylogeny and evolution.

Mol Microbiol, 1992 Jun, 6(12), 1681 - 91
In vivo control of promoter and terminator efficiencies at a distance; Jacquet MA et al.; In pBR329, the genes providing resistance to ampicillin (beta-lactamase, bla) and chloramphenicol (chloramphenicol acetyl transferase, cat) are encoded on the same strand . The bla gene lies downstream of the cat gene, separated by an intergenic sequence of 414 bp . The transcription starts of the two genes are 1090 bp apart . We have probed, in vivo, the effect on transcription of the bla gene, of the introduction, in front of the cat gene, of a series of synthetic promoters covering a large (over 60-fold) range of efficiency . The rising efficiency of the cat promoter has several important consequences for transcription of the bla gene . First, a strong (up to sevenfold) stimulation of the bla promoter is observed, together with a shift of the main bla transcription start site, 10 bp upstream . Furthermore, the relative efficiencies of the bla transcription terminators are reduced . Finally, because of a lesser relative efficiency of the cat transcription terminators as well, we observe enhanced intrusion into the bla gene of transcripts initiated at the cat promoter, some of them extending to the bla transcription terminator and beyond . The operon-like expression of the cat-bla gene tandem is controlled by the efficiency of the cat terminator, which in turn depends on that of the cat promoter . This demonstrates a direct link between the efficiencies of promoter and terminator . Upon inhibition of bacterial gyrase activity, i.e . relaxation of negative supercoiling action, bla expression increases sharply in pBR329, but remains almost unchanged in a plasmid (pBRGC-1) in which cat is under the control of a 6.5-fold stronger promoter . Therefore, under normal gyrase activity, the stimulation of the bla promoter in pBRGC-1 (relative to pBR329) appears to be linked to topological relaxation of its template in situ, in keeping with earlier in vitro observations . We propose that the relaxed state of pBRGC-1 in situ could be due to the decrease in the plasmid linking number, introduced by the 10-12 RNA polymerases that simultaneously transcribe the cat gene in that plasmid, compared with only one or two in pBR329 . We find that the negative superhelical densities of both plasmids are almost identical when extracted from the cell . Therefore gyrase would not correct for the relaxed state of plasmid pBRGC-1 observed in situ.

Gene, 1992 May 15, 114(2), 235 - 7
A vector for the removal of deletion mutants from antibody libraries; Seehaus T et al.; To reduce the number of deletion mutants from antibody (Ab) libraries that had been amplified by PCR from peripheral blood lymphocytes, we constructed the Ab expression vector, pLAB, in which DNA coding for a single-chain Ab was inserted into the gene encoding beta-lactamase (Bla) at the 3'-terminus of its signal sequence . After transforming Escherichia coli with this vector, a fusion protein with a functional Bla domain was produced that was able to protect the bacteria from the action of ampicillin (Ap) . Libraries can therefore be usefully propagated with this vector, since only those clones carrying inserts that are in frame with Bla will survive Ap selection, while others that carry out-of-frame deletions or internal stop codons are eliminated.

J Bacteriol, 1992 May, 174(10), 3407 - 10
Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide; Bowden GA et al.; beta-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) {delta(-20,-1) beta-lactamase} was released from Escherichia coli cells by osmotic shock . Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space . Expression of delta(-20,-1) beta-lactamase conferred a 25-fold increase in the 50% lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm . These results suggest that even in the absence of a leader peptide, mature beta-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.

J Bacteriol, 1992 May, 174(9), 2834 - 42
Nucleotide sequence and transcriptional analysis of activator-regulator proteins for beta-lactamase in Streptomyces cacaoi; Urabe H et al.; The nucleotide sequence of the 2.7-kb DNA fragment upstream of the structural gene of beta-lactamase in Streptomyces cacaoi was determined . Computer-aided "FRAME" analysis revealed four possible open reading frames (ORFs), three in one direction and one in the opposite direction . One of them (ORF1, BlaA) encoded an activator-regulator protein whose deduced amino acid sequence was similar to that of other activator-regulator proteins in bacteria . Insertion of an 8-bp BamHI linker into the BlaA region decreased the beta-lactamase activity sharply, from 50 U to 1 U/ml . This protein (BlaA) was found to bind to the nucleotide sequence between the bla (beta-lactamase structural gene) and blaA genes . Another ORF (ORF2, BlaB) in the same orientation had a couple of amino acid sequences similar to that of pBR322 beta-lactamase . However, insertion of the 8-bp BamHI linker indicated that this ORF was functional as an activator-regulator but not as a beta-lactamase . Therefore, there were two activator-regulator proteins in the upstream region of the structural gene of the beta-lactamase . Nuclease S1 mapping predicted that transcription for the activator proteins commenced at the translational initiation codon or within a few nucleotides from the translational start site . Transcription was in the opposite direction to that of the beta-lactamase structural gene.

Chin Med J (Engl), 1992 May, 105(5), 424 - 9
Purification of beta-lactamases and enzyme kinetic studies on aztreonam; Zhao XJ et al.; Standard beta-lactamases K1, P99, TEM-1, SHV-1 and beta-lactamase K-CAZ purified using FPLC through ion-exchange chromatography and filtration chromatography, were identified by determination of isoelectric points, molecular weights and substrate profiles . The results showed that the beta-lactamase stability of domestic aztreonam was very similar to that of cefotaxime, ceftazidime and much better than that of cefoperazone . Aztreonam showed a high affinity to chromosomal-mediated cephalosporinase P99, its Ki for inhibition of P99 with cephaloridine as substrate was 0.0159 microns . Aztreonam and the three third generation cephalosporins tested were not stable to beta-lactamase K-CAZ, which hydrolysed them in different degrees.

Biochemistry, 1992 Apr 21, 31(15), 3847 - 52
Elucidation of the role of arginine-244 in the turnover processes of class A beta-lactamases; Zafaralla G et al.; The highly conserved arginine-244 of beta-lactamases has been postulated to play a role in their initial recognition of substrates, presumably through ion pairing interactions {Moews, P . C., Knox, J . R., Dideberg, O., Charlier, P., & Frere, J . M . (1990) Proteins: Struct., Funct., Genet . 7, 156-171} . However, in the Michaelis enzyme-substrate complex, no direct function has been attributed to this residue . Two mutants with substitutions of this residue in the TEM-1 beta-lactamase (lysine-244 and serine-244) have been prepared to explore whether the guanidinium group of arginine-244 plays a critical role in the turnover processes . The mutant enzymes are effective catalysts for the hydrolysis of both penicillins and cephalosporins, and the lysine mutant enzyme behaves virtually identically to the wild-type beta-lactamase . Comparative kinetic characterization of the serine mutant and wild-type enzymes attributed apparent binding energies of 1.3-2.3 kcal/mol for the penicillins and 0.3-1.0 kcal/mol for the cephalosporins to the transition-state species by arginine-244 . Furthermore, it was shown that arginine-244 also contributes equally well to ground-state binding stabilization . These results were interpreted to indicate the involvement of a long hydrogen bond between arginine-244 and the substrate carboxylate, both in the ground and transition states . A reassessed picture for substrate anchoring involving interactions of the substrate carboxylate with the side chains of Ser-130, Ser-235, and Arg-244 is proposed to accommodate these observations.

J Mol Biol, 1992 Apr 20, 224(4), 1103 - 13
Inhibition of beta-lactamase by clavulanate . Trapped intermediates in cryocrystallographic studies; Chen CC et al.; Crystallographic studies of the complex between beta-lactamase and clavulanate reveal a structure of two acyl-enzymes with covalent bonds at the active site Ser70, representing two different stages of inhibitor degradation alternately occupying the active site . Models that are consistent with biochemical data are derived from the electron density map and refined at 2.2 A resolution: cis enamine, in which the carboxylate group of the clavulanate molecule makes a salt bridge with Lys234 of beta-lactamase; decarboxylated trans enamine, which is oriented away from Lys234 . For both acyl-enzymes, the carbonyl oxygen atom of the ester group occupies the oxyanion hole in a manner similar to that found in inhibitor binding to serine proteases . Whereas the oxygen atom in the trans product is optimally positioned in the oxyanion hole, that of the cis product clashes with the main-chain nitrogen atom of Ser70 and the beta-carbon atom of the adjacent Ala69 . In contrast to cis to trans isomerization in solution that relieves the steric strain inherent in a cis double bond, at the enzyme-inhibitor interface two additional factors play an important role . The salt bridge enhances the stability of the cis product, while the steric strain introduced by the short contacts with the protein reduces its stability.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1617 - 22
Alteration of the curved helical structure located in the upstream region of the beta-lactamase promoter of plasmid pUC19 and its effect on transcription; Ohyama T et al.; The region preceding the beta-lactamase promoter of Escherichia coli plasmid pUC19 has a curved DNA (bent DNA) structure . The center of the curvature was revealed to exist around nucleotide position 2580 of the plasmid, which is just beside RNA polymerase binding region . It was indicated that the identified region is curved even at 60 degrees C . The gross geometry of the curvature was altered by inserting synthetic double-stranded oligonucleotides between positions 2585 and 2586 . Effect of the alteration on strength of the promoter was not detected in vitro . However, in vivo analyses showed that the promoter strength is apparently dependent, in part, on the gross geometry of the curvature . Insertions of 4 and 16 bp, both of which altered the gross geometry of the curvature greatly, caused considerable reductions of in vivo level of beta-lactamase mRNA . In vivo, overall three-dimensional structure of the region covering the promoter and the curvature seems to play some significant role in transcription of the gene.

J Bacteriol, 1992 Apr, 174(7), 2095 - 101
Structural determinants in addition to the amino-terminal sorting sequence influence membrane localization of Escherichia coli lipoproteins; Gennity JM et al.; The lipid-modified nine-residue amino-terminal sequence of the mature form of the major outer membrane lipoprotein of Escherichia coli contains information that is responsible for sorting to either the inner or outer membrane . Fusion of this sorting sequence to beta-lactamase is sufficient for localization of the resultant lipo-beta-lactamase to the outer membrane (J . Ghrayeb and M . Inouye, J . Biol . Chem . 259:463-467, 1984) . Substitution of the serine adjacent to the amino-terminal lipid-modified cysteine residue of the sorting sequence with the negatively charged residue aspartate causes inner membrane localization (K . Yamaguchi, F . Yu, and M . Inouye, Cell 53:423-432, 1988) . Fusion of the aspartate-containing nine-residue inner membrane localization signal to the normally outer membrane lipoprotein bacteriocin release protein does cause partial localization to the inner membrane . However, a single replacement of the glutamine adjacent to the amino-terminal lipid-modified cysteine residue of bacteriocin release protein with aspartate causes no inner membrane localization . Therefore, an aspartate residue itself lacks the information necessary for inner membrane sorting when removed from the structural context provided by the additional eight residues of the sorting sequence . Although the aspartate-containing inner membrane sorting sequence causes an almost quantitative localization to the inner membrane when fused to the otherwise soluble protein beta-lactamase, this sequence cannot prevent significant outer membrane localization when fused to proteins (bacteriocin release protein and OmpA) normally found in the outer membrane . Therefore, structural determinants in addition to the amino-terminal sorting sequence influence the membrane localization of lipoproteins.

Curr Genet, 1992 Apr, 21(4-5), 265 - 8
Bacterial beta-lactamase is efficiently secreted in Saccharomyces cerevisiae under control of the invertase signal sequence; Bielefeld M et al.; The enzyme beta-lactamase, a secretory protein that is located in the Escherichia coli periplasmic space, can be highly expressed in Saccharomyces cerevisiae . Although the protein can cross eukaryotic membranes, it is only inefficiently secreted by yeast . To determine whether the lack of secretion in yeast is due to the nature of the bacterial signal sequence, it was replaced with the signal peptide of yeast invertase . The presence of the invertase signal peptide led to beta-lactamase secretion of up to 75% . The results indicate that the bacterial signal peptide is not functional in yeast, although cleavage can take place at the authentic processing site . The mature enzyme does not interfere with the yeast secretion pathway.

Steroids, 1992 Apr, 57(4), 154 - 61
A sensitive enzyme-linked immunosorbent assay (ELISA) for testosterone: use of a novel heterologous hapten conjugated to penicillinase; Rao PN et al.; A microplate enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of testosterone in plasma . The assay uses a heterologous system consisting of a novel hapten 4-(17 beta-hydroxy-3-oxoestra-4,9-dien-11 beta-yl)butanoic acid (1) conjugated to penicillinase (beta-lactamase) . The key reaction in the synthesis of the hapten was the cuprate-mediated 1,4-conjugate addition on 3,3,17,17-bis-ethylenedioxy-5 alpha,10 alpha-oxido-estr-9(11)-ene by the Grignard reagent derived from trimethyl 4-bromoorthobutyrate; this regiospecifically introduces the 11 beta-butanoate function . The hapten-penicillinase conjugate was used in the assay in conjunction with the immunoglobulin G (IgG) fraction derived from a previously characterized, highly specific, antitestosterone serum raised against a testosterone-19-O-carboxymethyl ether-bovine serum albumin (T-19-O-CME-BSA) conjugate . This unique system represents one incorporating three elements of structural heterology: bridge, site, and ring heterology between the antigen hapten and enzyme-linked hapten . The limit of detection was 10 pg of testosterone with a sensitivity range between 15 and 1,000 pg . A low level of cross-reactivity with 5 alpha-dihydrotestosterone (6.17%) and 11 beta-hydroxytestosterone (1.03%) was noted . No interference was noted with other common androgens, estradiol, or progesterone . The sensitivity and selectivity observed in the assay may be attributable to the selection of penicillinase as the enzyme marker and the elements of conformational heterology between the antigen-linked and enzyme-conjugated steroid haptens.

Yeast, 1992 Apr, 8(4), 261 - 72
Efficient secretion in yeast based on fragments from K1 killer preprotoxin; Cartwright CP et al.; The alpha and beta components of the secreted K1 killer toxin of Saccharomyces cerevisiae are derived from residues 45-147 and 234-316, respectively, of the 316 residue preprotoxin (ppTox) . The beta N-terminus is produced by Kex2 cleavage after Lys Arg233; when beta la (the mature sequence of beta-lactamase) is fused at this site and the fusion is expressed from the PGK promoter in pDT17, a multicopy plasmid, unexpectedly modest levels of beta la secretion resulted . Over-expression of Kex2 failed to increase beta la secretion while a kex2-null mutation reduced secretion by 98% . beta la secretion in a Kex+ strain was not enhanced by inactivation of the alpha toxin component or by deletion of most of its central hydrophobic segments . However SP-beta la, produced by deletion of ppTox residues 35-176, expressed 10-fold higher beta la activity and the precursor was now secreted with similar efficiency in a kex2-null strain . Fusions of beta la to ppTox at Ala34 or Ala46 also led to efficient secretion in both KEX2 and kex2-null strains . Since these beta la fusions differ only in segments well downstream of the signal peptide and all had similar transcript levels, the efficiency of beta la secretion is apparently determined by the efficiency with which these fusions are translocated to the Golgi compartment where Kex2 is active . Efficiency is high for the shorter fusions, but is 10% or less for the longer fusions; even this fraction is apparently diverted to the vacuole if not cleaved by Kex2 . SP-beta la was the most efficient construct tested; secreted beta la reached 4% of total cell protein, modestly exceeding levels produced by fusion to the MF alpha 1-encoded prepro alpha-factor, suggesting potential for the production of foreign proteins in yeast.

Eur J Clin Microbiol Infect Dis, 1992 Apr, 11(4), 313 - 21
Characterization of cell-bound papain-soluble beta-lactamases in BRO-1 and BRO-2 producing strains of Moraxella (Branhamella) catarrhalis and Moraxella nonliquefaciens; Eliasson I et al.; In Moraxella (Branhamella) catarrhalis and Moraxella nonliquefaciens strains isolated from clinical specimens in the south of Sweden two variants of beta-lactamase were distinguished by isoelectric focusing (IEF) . The BRO-1 (Ravasio type) enzyme was the most common in Branhamella catarrhalis, constituting about 90% of the beta-lactamase found in this species, while the BRO-2 enzyme (1908 type) was as common as BRO-1 in Moraxella nonliquefaciens . The determinants mediating the production of BRO-1 and BRO-2 were both transferable by conjugation . Cell-bound beta-lactamase from reference strains producing BRO-1 and BRO-2 could be solubilized by papain digestion . The isoelectric point of the solubilized enzymes differed distinctly between BRO-1 (pI 6.5) and BRO-2 (pI 6.9) . The molecular species of BRO-1 and BRO-2 released by papain digestion were purified by affinity chromatography with phenylboronic acid agarose gel . They had identical molecular weights of approximately 28,000 . Their kinetic constants were indistinguishable for a number of substrates and beta-lactamase inhibitors.

Eur J Clin Microbiol Infect Dis, 1992 Apr, 11(4), 305 - 12
Use of molecular methods to characterize Moraxella catarrhalis strains in a suspected outbreak of nosocomial infection; Morgan MG et al.; Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of whole cell protein, immunoblotting with normal human serum and restriction endonuclease analysis using Taq I enzyme were applied to 38 clinically significant isolates of Moraxella (Branhamella) catarrhalis obtained during a suspected outbreak of nosocomial infection . Each of 18 strains had individual profiles by at least two of the three methods (unique strains) . The remaining 20 strains were assigned to five groups (A-E) on the basis of similarity by at least two of the three methods . Isolates within groups A, D and E were homologous by all three methods . Immunoblot groups B and C had two distinct whole cell protein profiles (B1 and B2) but indistinguishable restriction endonuclease profiles (group B/C) . This emphasizes the need to use more than one technique in characterizing strains from suspected outbreaks of nosocomial infection . Grouped strains were more likely to originate from the same hospital ward than unique strains and were associated with a significantly longer median time from patient admission to strain isolation (14 versus 3.5 days, p less than 0.005) . Furthermore, the beta-lactamase activity was homologous within the groups . The results suggest that nosocomial infection involving several distinct Moraxella catarrhalis strains persisted over a period of months, involving at least 20 patients on three different wards . Such infection is probably common in wards harbouring suitably predisposed patients . The mode of transmission remains to be elucidated, but the above three techniques possess sufficient reproducibility and discriminatory ability to constitute suitable investigative tools.

Antimicrob Agents Chemother, 1992 Apr, 36(4), 740 - 3
Identification of a streptomycin resistance gene and a partial Tn3 transposon coding for a beta-lactamase in a periodontal strain of Eikenella corrodens; Lacroix JM et al.; The beta-lactamase gene from a periodontal strain of Eikenella corrodens, resistant to penicillins and streptomycin, was inserted into pBGS9 and transformed into Escherichia coli DH5 alpha . A 4.7-kb insert of pJML1, one of the transformants, was partially sequenced and found to contain the right section of transposon Tn3 from the middle of the TnpR resolvase gene to the right inverted repeat RI(R), including the TEM-1 gene . Sequences identical to RSF1010 were found on either side of the Tn3 sequence . pJML1 also contained a streptomycin resistance gene, probably identical to that of RSF1010 . A portion of the pJML1 insert was not homologous to either Tn3 or RSF1010 but was homologous to the chromosomal DNA of E . corrodens ATCC 23834 . It is assumed that the insert of pJML1 was derived from the chromosomal DNA of E . corrodens EC-38.

Biochemistry, 1992 Mar 31, 31(12), 3249 - 55
GroE prevents the accumulation of early folding intermediates of pre-beta-lactamase without changing the folding pathway; Zahn R et al.; In folding studies of pre-beta-lactamase in the presence of GroE, we investigated the pH dependence of the folding reaction . Two critical intermediates in the folding pathway were defined kinetically . I1 is an early folding intermediate recognized by GroE; the misfolding of I1 leads to aggregation, and this is prevented by GroE . A second intermediate I2 is released from GroE after ATP hydrolysis . Its pH-dependent misfolding to a nonnative form, which is not an aggregate, is not prevented by GroE . From these results, a model is proposed, in which the crucial role of GroE consists of allowing the change from I1 to I2 to take place in the complex . Fluorescence spectra of the pre-beta-lactamase complexed to GroE are very similar to those of the native state . The pathway of pre-beta-lactamase folding is not changed by GroE as evidenced by the same half-time and pH dependence of the folding reaction . GroE probably recognizes the signal sequence and some portion of the mature protein since mature beta-lactamase does not interact with GroE even under conditions of slow folding.

Biochim Biophys Acta, 1992 Mar 24, 1130(2), 182 - 8
An efficient expression system for a variant form of the cytotoxic protein alpha-sarcin in Escherichia coli; Oka T et al.; An efficient Escherichia coli system for the production of a variant form of the cytotoxic protein alpha-sarcin (delta Ala 1) has been constructed . cDNA encoded alpha-sarcin lacking N-terminal alanine was ligated with the bla signal peptide sequence (the signal sequence of E . coli beta-lactamase localizes the protein in the periplasm) and was inserted into an inducible bacterial expression vector pKTN2-2 . When the plasmid introduced into E . coli was expressed in the presence of IPTG, the recombinant product accumulated in the periplasmic space . The product was purified by Affi-Gel Blue followed by Bio-Rex 70 column chromatography . Recombinant alpha-sarcin (delta Ala 1) displayed the properties similar to those of authentic alpha-sarcin isolated from Aspergillus giganteus with respect to its molecular mass and enzymatic activity in ribosomal inactivation . The amount of alpha-sarcin variant produced in the system was estimated to be 1.2 mg/l of culture.

FEBS Lett, 1992 Mar 9, 299(2), 135 - 42
Beta-lactamase TEM1 of E . coli . Crystal structure determination at 2.5 A resolution; Jelsch C et al.; The crystal structure of beta-lactamase TEM1 from E . coli has been solved to 2.5 A resolution by X-ray diffraction methods and refined to a crystallographic R-factor of 22.7% . The structure was determined by multiple isomorphous replacement using four heavy atom derivatives . The solution from molecular replacement, using a polyalanine model constructed from the C alpha coordinates of S . Aureus PCl enzyme, provided a set of phases used for heavy atom derivatives analysis . The E . coli beta-lactamase TEM1 is made up of two domains whose topology is similar to that of the PCl enzyme . However, global superposition of the two proteins shows significant differences.

Kansenshogaku Zasshi, 1992 Mar, 66(3), 421 - 5
{Pneumonia caused by Moraxella subgenus Moraxella sp}; Yamazaki K et al.; Moraxella subgenus Moraxella sp . was isolated in pure culture from the sputum of a 43-year-old male with pneumonia and congestive heart failure due to idiopathic dilated cardiomyopathy . In this case, we concluded that the patient's bacterial pneumonia was caused by M . (M.) sp . based on a Gram stain of the sputum smear and bacterial findings, increased WBC count, and elevated CRP . A chest X-ray revealed right middle, and left upper and middle lobe infiltrates . This Moraxella strain produced a BRO-type beta-lactamase, a carbenicillinase-type enzyme.

Biochem J, 1992 Feb 15, 282 ( Pt 1), 189 - 95
Streptomyces albus G serine beta-lactamase . Probing of the catalytic mechanism via molecular modelling of mutant enzymes; Lamotte-Brasseur J et al.; In previous studies, several amino acids of the active site of class A beta-lactamases have been modified by site-directed mutagenesis . On the basis of the catalytic mechanism proposed for the Streptomyces albus G beta-lactamase {Lamotte-Brasseur, Dive, Dideberg, Charlier, Frere & Ghuysen (1991) Biochem . J . 279, 213-221}, the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics . The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates . The present study also shows that, upon binding a properly structured beta-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration.

Rev Prat, 1992 Feb 1, 42(3), 298 - 301
{Recurrent pharyngitis . Indications for tonsillectomy}; Astruc J et al.; In the absence of proven facilitating factor, recurrent tonsillitis occurs at all ages but it is particularly frequent in children aged from 6 to 12 years . Anatomical changes in the tonsils and the presence in their tissue of a complex beta-lactamase-producing bacterial flora explain the difficulties and failures of antibiotic therapy and the need for tonsillectomy in many cases . This operation is also increasingly performed for reasons that have nothing to do with infections . It is often necessary in the hypoventilation syndrome due to upper pharyngeal obstruction when the responsibility of the tonsils is either obvious or demonstrated by more or less complex investigations . Constitutional allergy is no longer a formal contra-indication to tonsillectomy.

Mol Microbiol, 1992 Feb, 6(4), 511 - 20
Kex2-dependent processing of yeast K1 killer preprotoxin includes cleavage at ProArg-44; Zhu YS et al.; The K1 killer toxin of Saccharomyces cerevisiae consists of 103- and 83-residue alpha and beta components whose derivation, from a 316-residue precursor preprotoxin, requires processing at the alpha N-terminus (after ProArg-44), the alpha C-terminus (after ArgArg-149) and at the beta N-terminus (after LysArg-233) . These processing events occur after translocation to the Golgi and have been investigated using beta-lactamase fusions . Signal peptidase cleavage of the precursor, predicted to occur after Ala-26, was confirmed by N-terminal sequence analysis of Ala-34 and Ile-52 fusions . Cleavage at all of the other predicted processing sites, including ProArg-44, is dependent on activity of the Kex2 protease . A fourth Kex2-dependent cleavage occurs at LysArg-188 . Implications for the specificity of Kex2 cleavage and preprotoxin processing are discussed.

Biopolymers, 1992 Jan, 32(1), 97 - 106
Conformation and charge distribution of bicyclic beta-lactams: structure-activity relationships; Fernandez B et al.; The structures of 7-oxo-1-azabicyclo{3.2.0}heptane and its 4-oxa, 3-ethylene-4-oxa, and 3-ethylene-6-methyl-4-oxa derivatives, and of 8-oxo-1-azabicyclo{4.2.0}octane and its 5-oxa derivative, were studied by ab initio methods . Conformations were refined without constraints using the 4-21G and the 4-21G* basis sets, and energies and charge distributions were improved by single-point 6-31G*/4-21G* calculations . The results are are interpreted in terms of structural trends related to beta-lactamase inhibitor capability.

Nucleic Acids Symp Ser, 1992, (27), 151 - 2
DNA conformation of the region preceding the beta-lactamase promoter of pUC19 which is required for efficient transcription; Hirota Y et al.; The upstream region of the beta-lactamase promoter of Escherichia coli plasmid pUC19 has a DNA curvature (bent DNA) . This region was replaced with another sequence by using randomly synthesized oligonucleotides . Among the reconstructed plasmids, a plasmid which could endow E . coli cells with the strongest resistance to ampicillin on plates was selected . Nucleotide sequence and DNA conformation of the altered region was investigated.

Chemotherapy, 1992, 38(6), 399 - 404
Escherichia coli resistant to ampicillin/sulbactam; Eng RH et al.; Escherichia coli strains resistant to ampicillin/sulbactam from hospitals in 4 different geographic locations were examined with respect to type and amount of beta-lactamase produced . A total of 5 strains was examined from each region . The isoelectric points of all of the involved beta-lactamases were 5.4, corresponding to TEM-1 . Km and Vmax values of the beta-lactamases among the clinical isolates resembled those from the control TEM-1 strain . In an 18-hour broth culture the highly resistant isolates produced 3 times more beta-lactamase as compared to the ampicillin/sulbactam-susceptible isolates . However, the highly resistant strains contained approximately the same amount of plasmid DNA (size of > 6,500 bp) as the susceptible isolates . In transformation experiments, both the resistance and the degree of resistance appeared to have been transferable by plasmids . The mechanism for resistance is likely to be a baseline overproduction of TEM-1 beta-lactamase due to either an alteration in the control of gene expression or simply to an increase in the number of copies of the beta-lactamase gene in the plasmids.

Microbios, 1992, 72(291), 137 - 42
Reversal of drug resistance in Mycobacterium leprae by ampicillin/sulbactam; Prabhakaran K et al.; The multiplication of Mycobacterium leprae in foot pads of experimentally-infected mice was suppressed by intramuscular administration of ampicillin combined with sulbactam or YTR-830H, two potent inhibitors of beta-lactamase in the bacteria . The antibiotic or the inhibitors by themselves were inactive . Ampicillin/sulbactam also inhibited the growth of drug-resistant M . leprae which grew in the presence of rifampin or dapsone . The finding provides a new approach to treat leprosy and to overcome drug resistance of the mycobacteria.

Eur J Clin Microbiol Infect Dis, 1991 Dec, 10(12), 1066 - 7
Problems in interpretation of piperacillin susceptibility of TEM-1 producing Escherichia coli in the disk diffusion test; Arstila T et al.; The susceptibility of 455 Escherichia coli blood culture isolate to piperacillin was tested with the disk diffusion test . The presence of different beta-lactamase genes in these strains was also studied using DNA hybridization . Of the TEM beta-lactamase producing isolates, 64% (61/95) were interpreted as intermediately susceptible to piperacillin . Because piperacillin is hydrolyzed by TEM-type beta-lactamases, we suggest that the intermediate susceptibility category should be reduced or omitted in testing piperacillin susceptibility of Escherichia coli isolates.

Int J Biol Macromol, 1991 Dec, 13(6), 345 - 8
Kinetics of the reaction between 5,5'-dithiobis{2-nitrobenzoic acid} and the sulphydryl group in Zn(2+)-dependent beta-lactamase II; Benitez MJ et al.; The kinetics of the reaction of the thiol residue in Zn(2+)-dependent beta-lactamase II with 5,5'-dithiobis{2-nitrobenzoic acid}, and the concomitant inactivation revealed that both events take place at the same rate . The inactivation could not be reverted by incubation with Zn2+ or by using a substrate concentration about eight times the Km of the enzyme . EDTA incubation also produced inactivation of the enzyme, although it was reverted by increasing the substrate concentration in the assay . A dual role is proposed for Zn2+ in beta-lactamase . The kinetic analysis of the thiol modification and the concomitant inactivation is in agreement with previous reports on the implication of the metal ion in catalysis . A role in stabilizing the native structure of the enzyme is also suggested.

Anticancer Drug Des, 1991 Dec, 6(6), 569 - 84
The anti-gene strategy: control of gene expression by triplex-forming-oligonucleotides; Helene C; Oligonucleotides are being developed to selectively inhibit gene expression at the translational level (antisense oligonucleotides) and at the transcriptional level (anti-gene oligonucleotides) . This review deals with the anti-gene strategy whereby an oligonucleotide binds to the major groove of double helical DNA where it forms a local triple helix . The molecular mechanisms for DNA recognition by triple helix formation are discussed together with some of the rules presently available to design the sequence and orientation of the triple helix forming oligonucleotide . Triplex stability can be enhanced by covalent attachment of an intercalating agent to the terminal nucleotide of the oligonucleotide . The intercalating agent can be used to induce irreversible reactions in the target sequence: double strand cleavage by a phenanthroline-Cu chelate in the presence of a reducing agent, photo-induced cleavage by ellipticine derivatives, photo-induced cross-linking of the two DNA strands by psoralen.. . Triple helix-forming oligonucleotides can be used to control gene expression at the transcriptional level . Inhibition of binding of transcription activating factors by triplex formation modulates the level of transcription of the target gene . Binding of a triplex-forming oligonucleotide immediately downstream of the RNA polymerase binding site can inhibit transcription initiation as shown with the E . coli beta-lactamase gene . Studies with cells in culture show that triple helix formation may occur in the intracellular environment and consequently leads to transcription inhibition . This inhibitory effect can be made irreversible by using, e.g., psoralen-substituted oligonucleotides . Oligonucleotides synthesized with the alpha-anomers of nucleotide units are resistant to nucleases and still form triple helices with double-stranded DNA . Oligo-{alpha}-deoxynucleotides can be derived by stabilizing (intercalating) agents or reactive groups (cleaving reagents, cross-linkers ...) . The results presently available provide a rational basis for the development of new tools for cellular biology and of new therapeutical approaches to selectively control gene expression at the transcriptional level.

Mayo Clin Proc, 1991 Dec, 66(12), 1238 - 47
Childhood sinusitis; Ott NL et al.; Childhood sinusitis is difficult to diagnose . It is classified on the basis of duration of inflammation--acute or chronic--and cause of inflammation--infectious or noninfectious . Infectious sinusitis is often a result of obstruction of the osteomeatal complex . Inflammation in noninfectious sinusitis is similar to the inflammatory changes detected in respiratory mucosa of patients with asthma . Acute sinusitis is primarily an infectious process similar to a prolonged infection of the upper respiratory tract . Plain radiography has limited value for the diagnosis of acute sinusitis in children . The most effective treatment of acute sinusitis is administration of a beta-lactamase-resistant antibiotic . Chronic sinusitis may be infectious, noninfectious, or both . Coronal computed tomography of the sinuses and nasal endoscopy are the preferred methods for determining the presence of chronic sinusitis . When physicians prescribe therapy for chronic sinusitis, they need to consider whether the underlying cause is infectious, noninfectious, or both . Treatment of chronic infectious sinusitis is most effective when a beta-lactamase-resistant antibiotic is administered . Chronic noninfectious sinusitis may respond to topically intranasally applied corticosteroids . If medical treatment fails to resolve the disease within 3 months, surgical intervention may be necessary . Finally, although an association between asthma and sinusitis exists, a cause-and-effect relationship has not been established.

Biochem J, 1991 Dec 1, 280 ( Pt 2), 553 - 6
Anomalous behaviour of a protein during SDS/PAGE corrected by chemical modification of carboxylic groups; Matagne A et al.; The 29,000-Mr Actinomadura R39 beta-lactamase exhibited a remarkably low electrophoretic mobility on SDS/PAGE, yielding an Mr value almost twice that computed from the corresponding gene sequence . We showed that chemical modification of the carboxylic groups of glutamic acid and aspartic acid residues restored a normal electrophoretic mobility and that the anomalous behaviour of that protein on SDS/PAGE was due to its very large negative charge at neutral pH . We also compared the behaviour of the same enzyme on gel filtration in the presence of SDS with those of other class A beta-lactamases (Mr approx . 30,000) . These experiments suggested that the very low electrophoretic mobility of the Actinomadura R39 beta-lactamase upon SDS/PAGE was more probably due to a low degree of SDS binding rather than to an unusual shape of the SDS-protein complex.

Cell, 1991 Nov 1, 67(3), 581 - 9
Identification of a protein required for disulfide bond formation in vivo; Bardwell JC et al.; We describe a mutation (dsbA) that renders Escherichia coli severely defective in disulfide bond formation . In dsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds . These disulfideless proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms . The dsbA gene codes for a 21,000 Mr periplasmic protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases . The purified DsbA protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases . Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.

EMBO J, 1991 Nov, 10(11), 3263 - 72
Energetically distinct early and late stages of HlyB/HlyD-dependent secretion across both Escherichia coli membranes; Koronakis V et al.; The alternative secretion pathway which exports hemolysin across both Escherichia coli membranes into the surrounding medium is directed by an uncleaved C-terminal targeting signal and the membrane translocator proteins HlyD and HlyB . In order to identify stages and intermediates in this unconventional secretion process we have examined the effect of inhibition of the total proton motive force (delta P) and its components during the in vivo HlyB/HlyD-dependent export of a 22.4 kDa secretion competent HlyA C-terminal peptide (Actp) . Secretion of Actp was severely inhibited by the proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which collapses simultaneously membrane potential delta psi and the proton gradient delta pH, and also by valinomycin/K+, a potassium ionophore which disrupts delta psi . The inhibition of secretion by valinomycin/K+ was ameliorated by imposition of a pH gradient, the second component of the delta P, and selective depletion of delta pH by nigericin also blocked secretion . This indicates that, as in the secretion of beta-lactamase to the periplasm, HlyB/D-directed secretion requires delta P itself and not specifically one of its components . However, inhibition of HlyB/D-dependent secretion was only marked when CCCP, valinomycin/K+ or nigericin were present during the early stage of Actp secretion; at a later stage the secretion was not significantly inhibited . HlyB/D-dependent secretion appears therefore to share with conventional secretion across the cytoplasmic membrane an early requirement for delta P, but comprises in addition a late stage which does not require delta P, delta psi or delta pH . The translocation intermediate identified in the delta P-independent late stage of secretion was associated with the membrane fraction . Analysis of the protease accessibility of this intermediate in whole cells and spheroplasts showed that it was not in the periplasm, nor was it exposed on the cell surface or on the periplasmic faces of either the inner or outer membranes . This may reflect its close association with the inner membrane or a membrane translocation complex.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9127 - 31
Cloning and characterization of a 3-methyladenine DNA glycosylase cDNA from human cells whose gene maps to chromosome 16; Samson L et al.; We described previously the isolation of a Saccharomyces cerevisiae 3-methyladenine (3-MeAde) DNA glycosylase repair gene (MAG) by its expression in glycosylase-deficient Escherichia coli alkA tag mutant cells and its ability to rescue these cells from the toxic effects of alkylating agents . Here we extend this cross-species functional complementation approach to the isolation of a full-length human 3-MeAde DNA glycosylase cDNA that rescues alkA tag E . coli from killing by methyl methanesulfonate, and we have mapped the gene to human chromosome 16 . The cloned cDNA, expressed from the pBR322 beta-lactamase promoter, contains an 894-base-pair open reading frame encoding a 32,894-Da protein able to release 3-MeAde, but not 7-methylguanine, from alkylated DNA . Surprisingly, the predicted human protein does not share significant amino acid sequence homology with the bacterial AlkA and Tag glycosylases or the yeast MAG glycosylase, but it does share extensive amino acid sequence homology with a rat 3-MeAde DNA glycosylase and significant DNA sequence homology with genes from several mammalian species . The cloning of a human 3-MeAde DNA glycosylase cDNA represents a key step in generating 3-MeAde repair-deficient cells and the determination of the in vivo role of this DNA repair enzyme in protecting against the toxic and carcinogenic effects of alkylating agents.

EMBO J, 1991 Oct, 10(10), 2765 - 72
Protein targeting towards the thylakoid lumen of chloroplasts: proper localization of fusion proteins is only observed in vivo; de Boer D et al.; Routeing of fusion proteins to the thylakoid lumen of the chloroplast was compared in vitro and in vivo . The Escherichia coli protein beta-lactamase was used as a passenger to study this intraorganellar sorting process . The first step, translocation of beta-lactamase into the chloroplast stroma, occurs properly both in vitro and in vivo and is dependent on the presence of a transit peptide in the protein construct . The second step, targeting towards the thylakoid lumen, is more complicated as was also observed previously when other passenger proteins were used . In vitro, the presence of a thylakoid transfer domain is not enough for routeing and proper processing . Only when the complete thylakoid lumen precursor plastocyanin was fused to beta-lactamase was the fusion protein processed adequately, but routeing was still incomplete . However, in vivo, the information present in the thylakoid transfer domain was the only requirement for proper transport towards the thylakoid lumen . These data show that in vivo, the only requirement for targeting of passenger proteins towards the thylakoid lumen is the presence of a transit peptide and a thylakoid transfer domain . Furthermore, we demonstrate that the in vitro import system does not necessarily reflect the in vivo situation with respect to intraorganellar sorting.

Protein Eng, 1991 Oct, 4(7), 811 - 9
Arginine 220 is a critical residue for the catalytic mechanism of the Streptomyces albus G beta-lactamase; Jacob-Dubuisson F et al.; Residue Arg220 was found to be important for the acylation of the Streptomyces albus G beta-lactamase by classical penicillins and cephalosporins bearing a carboxylate on C3 or C4 . The R220L mutant exhibited strongly decreased kcat/Km values for those compounds . Conversely the acylation rates by benzylpenicillin methylester and deacetylcephalosporin C lactone were little affected, indicating a direct or indirect role of that positively charged residue in the interaction of the enzyme cavity with the negative charge of the substrate . Surprisingly that residue is not conserved in all class A beta-lactamases but when it is not present it can be seen in the known tertiary structures that the guanidinium group of another arginine side chain (Arg244) is similarly positioned . The mutation affected the behaviour of the enzyme towards cephaloridine much less than towards cephalothin . This might represent an example of substrate-assisted catalysis where the disappearance of a positive charge on the enzyme is partly compensated by the presence of a similarly charged group on one of the substrate side chains . All the experimental results are nicely explained by computer-modelling of the enzyme-substrate interactions.

Protein Eng, 1991 Oct, 4(7), 805 - 10
Site-directed mutagenesis on TEM-1 beta-lactamase: role of Glu166 in catalysis and substrate binding; Delaire M et al.; Class A beta-lactamases are the major cause of bacterial resistance to beta-lactam antibiotics . In these active-site serine hydrolases, glutamic acid 166 has been hypothesized to act as a general acid-base catalyst . Replacing this residue by tyrosine in TEM-1 beta-lactamase yields an enzyme the activity of which is substantially lowered and strongly dependent on pH, thus confirming the alleged role of Glu166 in catalysis . This substitution also resulted in a spectacular change in substrate profile, the mutant enzyme being more active on cephalosporins than on penicillins . In fact, the E166Y enzyme behaves much like a class C enzyme, with high affinity and low hydrolytic activity towards second and third generation cephalosporins . Glu166 therefore seems to play a major part in defining the substrate profile of class A beta-lactamases.

J Biol Chem, 1991 Sep 15, 266(26), 17187 - 94
Replacement of lysine 234 affects transition state stabilization in the active site of beta-lactamase TEM1; Lenfant F et al.; Lysine 234 is a residue highly conserved in all beta-lactamases, except in the carbenicillin-hydrolyzing enzymes, in which it is replaced by an arginine . Informational suppression has been used to create amino acid substitutions at this position in the broad spectrum Escherichia coli beta-lactamase TEM-1, in order to elucidate the role of this residue which lies on the wall at the closed end of the active site cavity . The mutants K234R and K234T were constructed and their kinetic constants measured . Replacement of lysine 234 by arginine yields an enzyme with similar activity toward cephalosporins and most penicillins, except toward the carboxypenicillins for which the presence of the guanidine group enhances the transition state binding . The removal of the basic group in the mutant K234T yields a protein variant which retains a low activity toward penicillins, but losts drastically its ability to hydrolyze cephalosporins . Moreover, these two mutations largely decreased the affinity of the enzyme for penicillins (10-fold for K234R and 50-fold for K234T) . This can be correlated with the disruption of the predicted electrostatic binding between the C3 carboxylic group of penicillins and the amine function of the lysine . Therefore, lysine 234 in the E . coli beta-lactamase TEM-1 is involved both in the initial recognition of the substrate and in transition state stabilization.

Antimicrob Agents Chemother, 1991 Sep, 35(9), 1772 - 6
Pharmacokinetics of ampicillin and sulbactam in patients undergoing heart surgery; Wildfeuer A et al.; The pharmacokinetics of ampicillin and sulbactam, a new beta-lactamase inhibitor, were investigated in 16 patients undergoing prosthetic cardiac valve insertion . The combination of 2 g of ampicillin and 1 g of sulbactam was administered as perioperative prophylaxis intravenously over 3 to 6 days . Several serum pharmacokinetic parameters were similar for the two drugs after three intravenous doses were given to patients following surgery . The half-lives of elimination of ampicillin and sulbactam were 79 +/- 4.9 and 88 +/- 5.9 min, the volumes of distribution were 15.6 +/- 1.4 and 17.7 +/- 1.2 liters/70 kg, and the total plasma clearances were 144.4 +/- 14.5 and 147.2 +/- 14.5 ml/min, respectively . The peak concentrations of ampicillin and sulbactam in serum were calculated to be 134.3 +/- 1.3 and 58.3 +/- 1.2 micrograms/ml, respectively . Ampicillin and sulbactam rapidly penetrated from the blood into various tissues collected during heart surgery, such as sternum, pericardium, myocardium, and endocardium . The concentrations of ampicillin in tissue ranged from 17.8 +/- 9.9 to 50 +/- 29.5 micrograms/g, and those of sulbactam in tissue ranged from 8.8 +/- 6.2 to 19.6 +/- 10.1 micrograms/g . The concentrations of ampicillin and sulbactam in serum and tissue also apparently exceeded the MICs against most beta-lactamase-producing bacteria usually involved in postoperative wound infections and prosthetic valve endocarditis . The ratio of the two compounds was approximately 2:1 in serum and in the various tissues affected by the operation . The pharmacokinetics of ampicillin and sulbactam in serum and investigated tissues suggest that the combination of the two beta-lactams will be effective in the perioperative prophylaxis of patients undergoing heart surgery.

J Antibiot (Tokyo), 1991 Sep, 44(9), 969 - 78
6-(substituted methylene)penems, potent broad spectrum inhibitors of bacterial beta-lactamase . V . Chiral 1,2,3-triazolyl derivatives; Bennett IS et al.; Structure-activity relationships in a series of (5R)-6-triazolylmethylene penems with potent beta-lactamase inhibitory activity are described . In most cases, their in vitro synergistic activity with amoxycillin is superior to that of clavulanic acid, sulbactam and tazobactam (YTR 830) . Against an Escherichia coli TEM-1 infection in mice, the compounds showed a broad range of potencies; an optimum polarity was found, however, which gave maximum potency.

Infection, 1991 Sep-Oct, 19(5), 328 - 30
Beta-lactamase production in Branhamella catarrhalis isolated from lower respiratory tract secretions in Danish children: an increasing problem; Ejlertsen T et al.; Findings in specimens from the lower respiratory tract of children were reviewed retrospectively in order to assess the rate of Branhamella catarrhalis and beta-lactamase production . B . catarrhalis was isolated in 139 of 756 samples (18.4%) in 1986 and 211 of 723 samples (29.2%) in 1989 . Beta-lactamase production was found in 55.6% of B . catarrhalis strains in 1986 and 80.1% in 1989 (p less than 0.001) . Prevalence of beta-lactamase in B . catarrhalis has now reached the same level in Europe as in the USA.

Mol Microbiol, 1991 Sep, 5(9), 2243 - 53
In vivo analysis of integration of membrane proteins in Escherichia coli; Ito K et al.; The in vivo process of membrane protein integration was studied by pulse-labelling Escherichia coli cells, and assessing integral anchoring of labelled proteins to the lipid bilayer based on their resistance to alkali extraction . To conduct this experiment, conditions for extracting E . coli proteins with alkali were refined, and the immunoprecipitation procedures were improved to allow effective detection of integral membrane proteins . Examination of pulse-labelled, integral membrane proteins, including lactose permease (LacY), SecY, cytochrome omicron subunit II and leader peptidase revealed that all were in the alkali-insoluble fraction, indicating that membrane integration of these proteins takes place rapidly in wild-type cells . However, when LacY was synthesized in excess from a multicopy plasmid, significant proportions were found in the alkali-soluble fraction, indicating that the solubility in alkali is not an intrinsic property of the protein, and suggesting that LacY depends on some limited cellular factor for membrane integration . The unintegrated species of LacY sedimented slowly through an alkaline sucrose gradient . The secY24 mutant cells accumulated higher proportions of unintegrated LacY molecules at lower levels of overproduction than the sec+ cells . LacY overproduction in wild-type cells was found to inhibit processing (export) of beta-lactamase but not of OmpA and OmpF . These results are interpreted to mean that integration of LacY depends on multiple cellular components, one of which is also involved in export of beta-lactamase.

Jpn J Genet, 1991 Aug, 66(4), 399 - 409
Random screening of promoters from Escherichia coli and classification based on the promoter strength; Kubota M et al.; Five hundred fifty DNA fragments 100-500 base pairs in length were cloned from total chromosomal DNA of Escherichia coli, each capable of promoting the synthesis of beta-lactamase when inserted upstream of the ampC structural gene without its own promoter in a promoter-probe plasmid . All clones in this library of putative promoters were classified based on the level of resistance to ampicillin, which ranged from 10 to more than 1,500 micrograms/ml . Most of the higher levels of drug resistance (more than 1,000 micrograms/ml) were due not only to an increase in gene expression but also to an increase in plasmid copy number . The DNA fragments which produced the highest level of drug resistance all mapped at 5.7 min on the E . coli chromosome and shared the same nucleotide sequence . In these fragments, a strong promoter was found, which carries an up stream AT-rich sequence in addition to -35 and -10 signals of the promoter consensus.

Biochem J, 1991 Aug 1, 277 ( Pt 3), 647 - 52
Active-site serine mutants of the Streptomyces albus G beta-lactamase; Jacob F et al.; By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine . Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity . The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme . Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values . Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin . In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated . Both mutant enzymes were nearly as thermostable as the wild-type.

Biotechnology (N Y), 1991 Aug, 9(8), 725 - 30
Structure and morphology of protein inclusion bodies in Escherichia coli; Bowden GA et al.; We have studied the structure and characteristics of inclusion bodies formed by the enzyme beta-lactamase in the periplasmic space of Escherichia coli or in the cytoplasm, following expression of the protein without its signal sequence . Electron microscopy of highly purified protein aggregates using a novel sucrose gradient centrifugation procedure revealed striking morphological differences . Periplasmic inclusion bodies were essentially amorphous whereas the protein particles in the cytoplasm were highly regular . Depending on the cellular location, the inclusion bodies exhibited differences in protein composition even though they were formed by the expression of the same polypeptide chain . It was shown that the chaperonins GroEL and SecB are not incorporated into the inclusion bodies . Furthermore, the degree of solubilization of the inclusion bodies in the presence of denaturants and the sensitivity of the aggregated proteins to protease digestion indicated that the differences between cytoplasmic and periplasmic inclusion bodies extend to the conformation of the associated polypeptide chains.

J Biol Chem, 1991 Jun 25, 266(18), 11425 - 8
Escherichia coli exports previously folded and biotinated protein domains; Reed KE et al.; Biotination of proteins is a post-translational modification that requires a folded acceptor domain . We previously showed that an acceptor domain fused to the carboxyl terminus of several cytosolic proteins results in biotinated fusion proteins in vivo . We now show that proteins encoded by translational gene fusions of two periplasmic proteins, alkaline phosphatase and TEM beta-lactamase, to carboxyl-terminal biotin-accepting sequences are biotinated and exported by Escherichia coli . Expression of the alkaline phosphatase fusion protein in wild type strains resulted in inefficient biotination of the fusion product . This result was due to the rapid export of the acceptor protein before biotination could occur since a very large increase in biotinated fusion protein levels was observed in strains lacking the SecB chaperone protein . The beta-lactamase fusion protein was biotinated but was only stable in strains lacking the DegP periplasmic protease . Both biotinated fusion proteins accumulated in the culture medium in strains possessing defective outer membranes . These results indicate that the export machinery can accommodate both a post-translational modification and a protein domain previously folded into its mature conformation in vivo.

J Antimicrob Chemother, 1991 Jun, 27(6), 749 - 59
Activity of clavulanate combinations against TEM-1 beta-lactamase-producing Escherichia coli isolates obtained in 1982 and 1989; Seetulsingh PS et al.; Recent reports of decreased susceptibility to amoxycillin/clavulanate and ticarcillin/clavulanate combinations amongst Escherichia coli isolates have been attributed to an increased frequency of hyperproduction of TEM-1 beta-lactamase . To test this claim we compared the activities of clavulanate combinations against consecutive E . coli isolates producing TEM-1 beta-lactamase that were obtained from clinical material at The London Hospital during 1982 (n = 50) and 1989 (n = 46) . Enzyme production was quantified and related to the level of clavulanate required to potentiate amoxycillin and ticarcillin . The levels of TEM-1 enzyme production varied 150-fold amongst the isolates, partly because of variation in the gene copy number . A clear correlation existed between enzyme quantity and levels of resistance to the clavulanate combinations . However, there was no significant difference (P greater than 0.05, Mann-Whitney U test) between the isolates obtained in 1982 and 1989 in terms of the clavulanate concentrations required to potentiate the penicillins, or in the distribution of beta-lactamase activities present.

Mol Microbiol, 1991 Jun, 5(6), 1331 - 6
The ArsR protein is a trans-acting regulatory protein; Wu J et al.; The arsR gene encodes the regulatory protein of the plasmid-encoded arsenical resistance operon . A series of in-frame fusions was constructed between the C-terminally truncated arsR gene and the coding region for the mature form of beta-lactamase (blaM) . Fusions containing most of the arsR gene were still inducible by arsenicals . Fusions containing less than 102 residues of the 117-residue ArsR protein were constitutive . When a wild-type arsR gene was placed in trans, the constitutive constructs were again inducible . The results demonstrate that the ArsR protein is a trans-acting regulatory protein which controls its own expression.

J Antimicrob Chemother, 1991 Jun, 27(6), 761 - 7
Susceptibility of Escherichia coli isolates with TEM-1 beta-lactamase to combinations of BRL42715, tazobactam or clavulanate with piperacillin or amoxycillin ; Livermore DM et al.; Production of TEM-1 beta-lactamase is the commonest cause of acquired resistance to amoxycillin and piperacillin in Escherichia coli, now occurring in c . 50% of isolates . Consecutive E . coli isolates producing TEM-1 beta-lactamase were collected at The London Hospital in 1982 (n = 50) and 1989 (n = 46) . Enzyme quantities varied 150-fold amongst the isolates . Randomly-selected isolates from both years (n = 36; nine per quartile of the beta-lactamase activity distribution) were tested for susceptibility to combinations of amoxycillin or piperacillin with clavulanate or tazobactam or with BRL42715, a novel penem . The inhibitor concentrations needed to potentiate the penicillins related to the amount of beta-lactamase produced . BRL42715, at 1 mg/l, rendered all the isolates, including TEM-1 hyperproducers, susceptible to the recommended BSAC breakpoints of 8 mg amoxycillin/1 and 16 mg piperacillin/l . At 2 mg/l, BRL42715 almost always reduced amoxycillin and piperacillin MICs to the levels (1-2 mg/l) expected for E . coli isolates that lack TEM-1 enzyme . Tazobactam, at 1-2 mg/l, reduced piperacillin MICs to 1-2 mg/l for strains in the lower half of the beta-lactamase distribution, but greater than 8 mg tazobactam/l was required to reduce piperacillin MICs to 16 mg/l for one-third of the top quartile isolates . Clavulanate was a stronger potentiator of piperacillin than was tazobactam . On the other hand, amoxycillin was a more difficult substrate to potentiate than piperacillin, and isolates with enzyme levels in the top half of the distribution generally required greater than or equal to 8 mg clavulanate/l to reduce amoxycillin MICs to less than or equal to 8 mg/l.

Philos Trans R Soc Lond B Biol Sci, 1991 May 29, 332(1263), 149 - 56
The energetics of intramolecular reactions and enzyme catalysis; Page MI; The relative rates of reactions should always be examined by an awareness of differential effects . The magnitude and variation of the relative rates of intramolecular reactions can be rationalized by the differences in entropy and strain energy . The relative rates of enzyme-catalysed reactions are sometimes due to groundstate effects . The beta-lactamase-catalysed hydrolysis of beta-lactam antibiotics may require a unique disposition of catalytic groups owing to an unusual process of bond fission in the four membered ring.

Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4176 - 80
Protein stability: electrostatics and compact denatured states; Stigter D et al.; Globular proteins can be denatured by changing pH and ionic strength . Much recent evidence has led to the surprising conclusion that there are two acid-denatured states: one highly unfolded and the other more compact, sometimes called the "molten globule." Here we describe a molecular theory for electrostatic stability of globular proteins based on the properties of the constituent amino acids: oil/water partition coefficients, pK values of the titratable groups, and their temperature dependences . Predicted denaturation temperatures vs . pH are in good agreement with experiments of other workers on myoglobin . The theory also predicts two populations of denatured species, one open and the other more compact, with densities in the range found experimentally for molten globular states . In addition, it predicts a phase diagram (stability vs . pH, ionic strength) in good agreement with experiments of Goto and Fink {Goto, Y . & Fink, A . L . (1989) Biochemistry 28, 945-952; and Goto, Y . & Fink, A . L . (1990) J . Mol . Biol . 214, 803-805} . The well-known salt destabilization of myoglobin has been generally considered evidence for ion pairing, but the present theory, based on smeared charge repulsion, explains the salt destabilization at low pH without ion pairing . In addition, for myoglobin the theory predicts salt stabilization at high pH, as observed for beta-lactamase by Goto and Fink.

Mol Cell Biol, 1991 May, 11(5), 2620 - 8
In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions; Cartwright CP et al.; Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322 . The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease . Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments . Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells . A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment . Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion . Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter . Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion . Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed . The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable . This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.

Plasmid, 1991 May, 25(3), 198 - 207
The effect of plasmid copy number mutations on pT181 replication initiator protein expression; Wang PZ et al.; Previous studies have shown that plasmid pT181 controls its replication by countertranscript-mediated regulation of the rate of synthesis of the pT181 initiator, RepC . In this study, the relation has been studied between plasmid copy number and RepC synthesis for a series of pT181 copy number mutants . For each mutant plasmid, the repC coding sequence along with its 5' regulatory region was translationally fused to the beta-lactamase structural gene on a vector plasmid unrelated to pT181 . By means of these constructs, the effect of regulatory mutations on the initiator synthesis could be measured at constant copy number . With one exception, the mutant control regions showed elevated beta-lactamase activity in comparison to the wild-type . However, the relative increase was not very well correlated with the copy number of the corresponding mutant plasmid . The possibility is considered that factors such as DNA secondary structure may have important ancillary effects on the regulation mechanism.

Biochem J, 1991 May 1, 275 ( Pt 3), 793 - 5
Phosphonate monoester inhibitors of class A beta-lactamases; Rahil J et al.; Phosphonate monoesters with the general structure: {formula: see text} are inhibitors of representative class A and class C beta-lactamases . This result extends the range of this type of inhibitor to the class A enzymes . Compounds where X is an electron-withdrawing substituent are better inhibitors than the unsubstituted analogue (X = H), and enzyme inhibition is concerted with stoichiometric release of the substituted phenol . Slow turnover of the phosphonates also occurs . These observations support the proposition that the mechanism of action of these inhibitors involves phosphorylation of the beta-lactamase active site . The inhibitory ability of these phosphonates suggests that the beta-lactamase active site is very effective at stabilizing negatively charged transition states . One of the compounds described also inactivated the Streptomyces R61 D-alanyl-D-alanine carboxypeptidase/transpeptidase.

J Antimicrob Chemother, 1991 May, 27(5), 569 - 75
Factors determining resistance to beta-lactam combined with beta-lactamase inhibitors in Escherichia coli; Reguera JA et al.; The influence of inoculum size, beta-lactamase hyperproduction (multicopy plasmid) and modifications in the outer membrane protein profile on the susceptibility of Escherichia coli to combinations of amoxycillin/clavulanate, amoxycillin/sulbactam, amoxycillin/tazobactam and piperacillin/tazobactam were studied . For all combinations the bacterial susceptibility was affected by factors determining an increase in beta-lactamase (inoculum size or hyperproduction) . Clavulanic acid was the most efficient beta-lactamase inhibitor . The absence of the outer membrane proteins, OmpF and OmpC, did not significantly affect susceptibility to the combinations per se but when combined with the presence of beta-lactamase high MICs were observed . Seven out of eight amoxycillin/clavulanate resistant clinical isolates of E . coli had beta-lactamase hyperproduction and a decrease or absence of OmpF.

Biochemistry, 1991 Apr 2, 30(13), 3179 - 88
Substitution of lysine at position 104 or 240 of TEM-1pTZ18R beta-lactamase enhances the effect of serine-164 substitution on hydrolysis or affinity for cephalosporins and the monobactam aztreonam; Sowek JA et al.; By site-directed mutagenesis, TEM-1 beta-lactamase was altered to contain single amino acid changes of E104K, R164S, and E240K, in addition to double changes of E104K/R164S or R164S/E240K and the triple change of E104K/R164S/E240K . Hydrolysis rates for cephaloridine and benzylpenicillin were lowered at least 1 order of magnitude for all enzymes containing R164S substitutions . All mutant enzymes exhibited increased kcat values for beta-lactam antibiotics containing an aminothiazole oxime side chain . Hydrolysis of ceftazidime was most affected, with kcat values increased 3-4 orders of magnitude in all enzymes with the substituted R164S moiety . Km values decreased for all substrates except ceftazidime in the enzymes with multiple mutations . Aztreonam was most affected, with Km values lowered 23-56-fold in the enzymes bearing multiple mutations . When the crystal structures of aztreonam and related monobactams were studied and projected into an active-site model of the PC1 beta-lactamase, it became apparent that the two lysine residues might serve equivalent roles by interacting with the carboxylate of the aminothiazole oxime side chain . Hydrogen-bonding interactions involving the oxime and N7 of the lysine, particularly Lys-104, may also be important in some antibiotics . Ser-164 apparently serves an indirect role, since it is somewhat distant from the active-site cleft.

Gene, 1991 Apr, 100, 51 - 7
Mapping catalytically important regions of an enzyme using two-codon insertion mutagenesis: a case study correlating beta-lactamase mutants with the three-dimensional structure; Zebala J et al.; Two-codon insertion mutants throughout the beta-lactamase (Bla)-encoding gene were characterized . Second site revertants of various mutants were isolated, mapped and sequenced . The activity of the mutants and the ability to get revertants showed a positive correlation with increasing distance from the active site, based on the three-dimensional structure of Bla . This observation is discussed as it may pertain to the generalized use of two-codon insertion mutagenesis in mapping important catalytic regions in enzymes.

J Biotechnol, 1991 Apr, 18(1-2), 29 - 39
Stability of a host-vector system based on complementation of an essential gene in Escherichia coli; Degryse E; Antibiotic selection is the most common selection system for plasmid-containing bacteria . This technique, nevertheless, can be a source of problems during the expression of heterologous genes in Escherichia coli . We have developed an alternative selection system based on the complementation of a chromosomal auxotrophic (dapD2) mutation by the corresponding wild type gene carried on a plasmid . We show that the system effectively selects for the presence of plasmid on solid and liquid medium . In addition, we have observed a loss of viability associated with high levels of gene expression and accumulation of a heterologous protein, but the selective power and improved intrinsic stability of the dap+ plasmid, compared to a beta-lactamase (bla) based vector, excludes overgrowth of the culture by plasmidless cells.

J Biotechnol, 1991 Apr, 18(1-2), 141 - 52
The effect of the partition locus on plasmid stability and expression of a prolonged chemostat culture; Weber AE et al.; The stability, copy number, and gene expression of the pBR322 plasmid containing the par-locus under prolonged cultivation were studied . In the initial stage of the experiment it was observed that the par-locus had a stabilization effect on plasmid maintenance . This observation was consistent with previously reported results . However, after approximately 225 h, a mixed population of plasmid-containing and plasmid-free cells appeared . The mixed culture was stably maintained for approximately 200 h . In addition, the relative plasmid copy number showed an increase as compared to the par- culture . After 100 h the copy number decreased, reached a minimum, then stabilized . The beta-lactamase activity, was not significantly affected by the par-locus.

J Mol Biol, 1991 Mar 20, 218(2), 387 - 96
Anion and pH-dependent conformational transition of an amphiphilic polypeptide; Goto Y et al.; While several proteins, including beta-lactamase, cytochrome c and apomyoglobin, are maximally unfolded at pH 2 by HCl in the absence of salt, the addition of anions, either from salt or acid, co-operatively induces the unfolded proteins to refold to a molten globule state, because anions bind preferentially to the compact molten globule state compared to the extended unfolded state . To study the role of the anion-dependent conformational transition at neutral pH, we synthesized a model polypeptide of 51 amino acid residues, consisting of tandem repeats of a Lys-Lys-Leu-Leu sequence and containing a turn sequence, Asn-Pro-Gly, at the center of the molecule . The model polypeptide showed no significant conformation by circular dichroism under conditions of low salt at neutral pH . However, addition of anions, either from salt or acid, induced the folding transition to an alpha-helical conformational state . The order of effectiveness of various anions in inducing the folding transition was consistent with the series of anions in inducing the molten globule of the acid-denatured protein . This suggests that the helical state of the model polypeptide is equivalent to the molten globule state . At pH values above 9, the model polypeptide also took an alpha-helical conformation, which was very similar to that induced by anions . On the basis of the chloride and pH-dependent conformational transitions, a phase diagram for the conformational states was constructed . The phase diagram was explained simply by assuming that the conformational transition is linked to the proton and the anion bindings to a limited number of amino groups and that anions bind only to the protonated groups.

Biochem J, 1991 Mar 15, 274 ( Pt 3), 855 - 9
Evidence from a mutant beta-lactamase for the mechanism of beta-lactamase-catalysed depsipeptide aminolysis; Mazzella LJ et al.; The Ser-70----Gly mutant of the TEM-1 beta-lactamase, where the active-site serine hydroxy group has been lost, does not catalyse the hydrolysis of either benzylpenicillin or N-(phenylacetyl)glycyl depsipeptides . This is as would be expected for a double-displacement mechanism where the Ser-70 becomes acylated at an intermediate stage . Further, however, the mutant enzyme, unlike the wild-type, does not catalyse aminolysis of depsipeptides by D-phenylalanine . If the active site is not structurally disrupted by the mutation, this result shows that Ser-70 is necessary for the aminolysis reaction and implies that this reaction, like the hydrolysis, proceeds by way of an acyl-(serine)-enzyme intermediate . Although physical evidence suggests that the mutant enzyme does not have a structure in solution identical with that of the wild-type, the mutant does still bind beta-lactam substrates . The latter result suggests sufficient conservation of the active-site structure for the major conclusion above to hold.

FEBS Lett, 1991 Mar 11, 280(1), 27 - 31
Release of periplasmic proteins induced in E . coli by expression of an N-terminal proximal segment of the phage fd gene 3 protein; Rampf B et al.; We used the enzymes beta-lactamase and alkaline phosphatase to quantitatively evaluate the release of periplasmic proteins from E . coli cells transformed by plasmids harboring gene 3 of phage fd . Different deletion mutants of gene 3 released varying fractions of the enzymes . From these results we conclude that essentially the amino-terminal proximal part, upstream of the first glycine-rich region but not this region itself, is responsible for the excretion of periplasmic proteins in E . coli cells expressing the gene 3 protein of phage fd.

Antimicrob Agents Chemother, 1991 Mar, 35(3), 524 - 8
Acquired resistance of Nocardia brasiliensis to clavulanic acid related to a change in beta-lactamase following therapy with amoxicillin-clavulanic acid; Steingrube VA et al.; Previous studies have demonstrated that Nocardia brasiliensis is susceptible to amoxicillin-clavulanic acid and that its beta-lactamases are inhibited in vitro by clavulanic acid . A cardiac transplant patient with disseminated infection caused by N . brasiliensis was treated with this drug combination with good response, but relapsed while still on therapy . The relapse isolate was found to be identical to the initial isolate by using genomic DNA restriction fragment patterns obtained by pulsed field gel electrophoresis, but it was resistant to amoxicillin-clavulanic acid . On isoelectric focusing, the beta-lactamase from the relapse isolate exhibited a shift in the isoelectric point (pI) of its major band from 5.10 to 5.04 compared with the enzyme from the pretreatment isolate . As determined by using values of the amount of beta-lactamase inhibitor necessary to give 50 +/- 5% inhibition of beta-lactamase-mediated hydrolysis of 50 microM nitrocefin, the beta-lactamase of the relapse isolate was also 200-fold more resistant than the enzyme from the pretreatment isolate to clavulanic acid and was more resistant to sulbactam, tazobactam, cloxacillin, and imipenem . The beta-lactamase of the relapse isolate exhibited a 10-fold decrease in hydrolytic activity for cephaloridine and other hydrolyzable cephalosporins compared with that for nitrocefin . Acquired resistance to amoxicillin-clavulanic acid in this isolate of N . brasiliensis appears to have resulted from a mutational change affecting the inhibitor and active site(s) in the beta-lactamase.

J Antibiot (Tokyo), 1991 Mar, 44(3), 338 - 43
6-(substituted methylene)penems, potent broad spectrum inhibitors of bacterial beta-lactamase . IV . Kidney stability, serum binding and additional biological evaluation of racemic derivatives; Bennett IS et al.; Sodium (5RS)-Z-6-(substituted methylene)penem-3-carboxylates (3) are extremely potent inhibitors of bacterial beta-lactamases, but some members of this group of compounds are highly bound to human serum, while others are readily degraded by renal dehydropeptidase I enzyme . Consequently, the stability of a variety of 6-(substituted methylene)penems (3) to human kidney homogenate, their binding to human serum and their activity in a mouse infection model was investigated at an early stage, and were instrumental in the selection of the 1,2,3-triazolylmethylene derivatives (e.g . 3k) as a class of compounds worthy of further evaluation.

J Antibiot (Tokyo), 1991 Mar, 44(3), 331 - 7
6-(substituted methylene)penems, potent broad spectrum inhibitors of bacterial beta-lactamase . III . Structure-activity relationships of the 5-membered heterocyclic derivatives; Bennett I et al.; Sodium (5RS)-Z-6-(heterocyclylmethylene)penem-3-carboxylates (2) are a series of extremely potent inhibitors of bacterial beta-lactamases . A variety of 5-membered heteroaromatic derivatives have been prepared and structure-activity studies reveal a preferred substituent orientation . One of these derivatives, the 1-methyl-1,2,3-triazolyl compound (5m) is a more potent synergist of amoxycillin than clavulanic acid, sulbactam or tazobactam.

J Bacteriol, 1991 Mar, 173(6), 1997 - 2005
Effect of OmpA signal peptide mutations on OmpA secretion, synthesis, and assembly; Tanji Y et al.; In previous investigations, we have examined the effect of OmpA signal peptide mutations on the secretion of the two heterologous proteins TEM beta-lactamase and nuclease A . During these studies, we observed that a given signal peptide mutation could affect differentially the processing of precursor OmpA-nuclease or precursor OmpA-lactamase . This observation led us to further investigate the influence of the mature region of a precursor protein on protein export . Preexisting OmpA signal peptide mutations of known secretion phenotype when directing heterologous protein export (nuclease A or beta-lactamase) were fused to the homologous mature OmpA protein . Four signal peptide mutations that have previously been shown to prevent export of nuclease A and beta-lactamase were found to support OmpA protein export, albeit at reduced rates . This remarkable retention of export activity by severely defective precursor OmpA signal peptide mutants may be due to the ability of mature OmpA to interact with the cytoplasmic membrane . In addition, these same signal peptide mutations can affect the level of OmpA synthesis as well as its proper assembly in the outer membrane of Escherichia coli . Two signal peptide mutations dramatically stimulate the rate of precursor OmpA synthesis three- to fivefold above the level observed when a wild-type signal peptide is directing export . The complete removal of the OmpA signal peptide does not result in increased OmpA synthesis . This finding suggests that the signal peptide mutations function positively to stimulate OmpA synthesis, rather than bypass a down-regulatory mechanism effected by a wild-type signal peptide . Overproduction of wild-type precursor OmpA or precursors containing signal peptide mutations which lead to relatively minor kinetic processing defects results in accumulation of an improperly assembled OmpA species (imp-OmpA) . In contrast, signal peptide mutations which cause relatively severe processing defects accumulate no or only small quantities of imp-OmpA . All mutations result in equivalent levels of properly assembled OmpA . Thus, a strong correlation between imp-OmpA accumulation and cell toxicity was observed . A mutation in the mature region of OmpA which prevents the proper outer membrane assembly of OmpA was suppressed when export was directed by a severely defective signal peptide . These findings suggest that signal peptide mutations indirectly influence OmpA assembly in the outer membrane by altering both the level and rate of OmpA secretion across the cytoplasmic membrane.

Biotechnol Prog, 1991 Mar-Apr, 7(2), 99 - 110
Immobilization of Escherichia coli JM103{pUC8} in kappa-carrageenan coupled with recombinant protein release by in situ cell membrane permeabilization; Ryan W et al.; Immobilization of Escherichia coli JM103{pUC8} was carried out with kappa-carrageenan as the support matrix . Substantial natural excretion of beta-lactamase, attributable to the less intact membrane of plasmid-harboring cells, was observed in immobilized cell cultures . Nevertheless, a significant portion of the beta-lactamase produced was retained in the cells . As compared to suspension cultures, much higher beta-lactamase activities, especially in the extracellular liquid, and much longer retention of plasmid-bearing cells (improved plasmid stability) were observed in immobilized cell cultures . Further enhancement in excretion of the recombinant protein (beta-lactamase) was achieved by permeabilization of cell membrane by periodic exposure of the immobilized cell cultures to ethylenediaminetetraacetic acid (EDTA) . While the presence of EDTA led to some suppression of cell growth in suspension cultures, cell growth in gel beads was not affected by EDTA to the same extent, possibly due to lesser exposure of immobilized cells to EDTA . Exposure of immobilized cell cultures to EDTA presumably inhibited plasmid replication and led in turn to diversion of cellular resources for the support of expression of plasmid genes . Indeed, treatment of the immobilized cell cultures with EDTA resulted in increased production of beta-lactamase when compared to the enzyme production in EDTA-free cultures . More frequent addition of EDTA increased the period of retention of plasmid-bearing cells in these cultures but did not have any noticeable adverse effect on synthesis of beta-lactamase . Improvement in plasmid stability in EDTA-treated immobilized cell cultures was ascribed to the reduction in the growth rate differential between plasmid-free and plasmid-bearing cells, since plasmid-free cells were subject to more reduction in specific growth rate than were plasmid-bearing cells.

Biochemistry, 1991 Feb 26, 30(8), 2281 - 92
Elucidation of the order of oxidations and identification of an intermediate in the multistep clavaminate synthase reaction; Salowe SP et al.; The enzyme clavaminate synthase (CS) catalyzes the formation of the first bicyclic intermediate in the biosynthetic pathway to the potent beta-lactamase inhibitor clavulanic acid . Our previous work has led to the proposal that the cyclization/desaturation of the substrate proclavaminate proceeds in two oxidative steps, each coupled to a decarboxylation of alpha-ketoglutarate and a reduction of dioxygen to water {Salowe, S . P., Marsh, E . N., & Townsend, C . A . (1990) Biochemistry 29, 6499-6508} . We have now employed kinetic isotope effect studies to determine the order of oxidations for CS purified from Streptomyces clavuligerus . By using (4'RS)-{4'-3H,1-14C}-rac-proclavaminate, a primary T(V/K) = 8.3 +/- 0.2 was measured from {3H}water release data, while an alpha-secondary T(V/K) = 1.06 +/- 0.01 was determined from the changing 3H/14C ratio of the product clavaminate . Values for the primary and alpha-secondary effects of 11.9 +/- 1.7 and 1.12 +/- 0.07, respectively, were obtained from the changing 3H/14C ratio of the residual proclavaminate by using new equations derived for a racemic substrate bearing isotopic label at both primary and alpha-secondary positions . Since only the first step of consecutive irreversible reactions will exhibit a V/K isotope effect, we conclude that C-4' is the initial site of oxidation in proclavaminate . As expected, no significant changes in the 3H/14C ratio of residual substrate were observed with {3-3H,1-14C}-rac-proclavaminate . However, two new tritiated compounds were produced in this incubation, apparently the result of isotope-induced branching brought about by the presence of tritium at the site of the second oxidation . One of these compounds was identified by comparison to authentic material as dihydroclavaminate, a stable intermediate that normally remains enzyme-bound . On the basis of the body of information available and the similarities to alpha-ketoglutarate-dependent dioxygenases, a comprehensive mechanistic scheme for CS is proposed to account for this unusual enzymatic transformation.

J Antimicrob Chemother, 1991 Feb, 27(2), 191 - 8
Chromosomally-mediated beta-lactamases produced by Levinea amalonatica with activity against methoxyimino-cephalosporins; Ben Yaghlane Bouslama H et al.; Levinea amalonatica strain A2370 was isolated from the blood culture of a patient hospitalized in Charles Nicolle hospital (Tunis) during July 1986 and had decreased susceptibility to cefotaxime . Isoelectric focusing of crude extracts from this strain demonstrated three bands of beta-lactamase activity which focused at pH 5.4, 5.5 and 6.05 . The three bands were separated and analysed for their kinetic properties . One enzyme (pI 5.4) had the properties of a TEM-1 beta-lactamase and did not confer resistance to third-generation cephalosporins . The two other bands, named B1 and B2, had pIs of 6.05 and 5.5 respectively, hydrolysed cefotaxime and similar cephalosporins, and were produced constitutively . After prolonged storage (six months at -20 degrees C) of the highly purified B1 enzyme, a mixture of B1/B2, with the B2 band predominating, was obtained . This observation suggested that the B2 enzyme was derived from B1 . These enzymes appeared to differ from the MJ-2 beta-lactamase described previously in L . amalonatica.

Biopolymers, 1991 Jan, 31(1), 119 - 28
Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe; Semisotnov GV et al.; Binding of the hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to synthetic polypeptides and proteins with a different structural organization has been studied . It has been shown that ANS has a much stronger affinity to the protein "molten globule" state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, alpha-helical, or beta-structural hydrophilic homopolypeptides . The possibility of using ANS for the study of equilibrium and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, beta-lactamase, and alpha-lactalbumin as examples.

Mol Microbiol, 1991 Jan, 5(1), 117 - 22
Folding in vitro and transport in vivo of pre-beta-lactamase are SecB independent; Laminet AA et al.; The rate of folding of the precursor of beta-lactamase is not influenced by the presence of SecB under conditions in which GroEL/ES retards the folding . Wild-type beta-lactamase and several mutants in the signal or the mature protein, affecting either transport or enzyme kinetics and probably folding, were examined for total expression, total enzymatic activity, and transported beta-lactamase (in vivo resistance) in secB- and secB+ strains . We conclude that there is no indication of any relevant interaction between SecB and pre-beta-lactamase in vitro, nor did the secB- mutation affect the transport of wild-type beta-lactamase or any of the mutant in vivo . Thus, putative Escherichia coli "folding modulators' must be of limited specificity.

Biochem J, 1991 Jan 1, 273(Pt 1), 85 - 91
Inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate . The nature of active-site functional groups and comparisons with the native enzyme; Knap AK et al.; The pH-rate profile for inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate supports previous evidence {Knap & Pratt (1989) Proteins Struct . Funct . Genet . 6, 316-323} for the activation of the active-site thiol group by adjacent functional groups . The enhanced reactivity of iodoacetate, with respect to that of iodoacetamide, suggests the influence of a positive charge in the active site . The reactivity of iodoacetate is not affected by dissociation of an active-site functional group of pKa 6.7, which increases the reactivity of neutral reagents, probably because of a compensation phenomenon; it is, however, lost on dissociation of an acid of pKa 8.1 . It is concluded that the active cysteine beta-lactamase has four functional groups at the active site, one nucleophilic thiolate of Cys-70, one neutral acid (most probably the carboxy group of Glu-166, from the crystal structures) and two cationic residues (most probably Lys-73 and Lys-234) . A comparison of these results with the pH-dependence of reactivity of the native RTEM-2 beta-lactamase suggests that the active form of the latter enzyme is also monocationic, although the nucleophile (Ser-70) is likely to be neutral in this case and the carboxylic acid dissociated . A mechanism of class A beta-lactamase catalysis is discussed where the Glu-166 carboxylate acts as a general base/acid catalyst and Lys-73 is principally required for electrostatic stabilization of the anionic tetrahedral intermediate.

Respiration, 1991, 58 Suppl 1, 43 - 6
Chronic bronchitis in the 1990s: up-to-date treatment; Clarke SW; Prevention is the key to eradicating chronic bronchitis . Smoking is the prime factor involved . Quitting smoking is difficult even with modern aids such as counselling, filters, substitutes, hypnosis and acupuncture, and the success rate is only 20% . Passive smoking is also injurious . The role of atmospheric pollution is less well quantified . Other risk factors include lower social class, occupation, area of residence, housing, temperature, and childhood respiratory illness . Influenza vaccination gives 60-70% immunity . With exacerbations of chronic bronchitis, beta-lactamase-producing bacteria are important, though the overall need for antibiotics is uncertain judging by placebo-controlled studies . Inhaled beta 2-agonist bronchodilators may improve airflow obstruction, as may anticholinergic drugs (e.g . ipratropium), the two being additive . Oral theophyllines have a narrow therapeutic window, serious side-effects and only two thirds of the effect of beta 2-agonists . A corticosteroid trial for 2 weeks may relieve refractory airway obstruction in 17-23% of chronic bronchitis, to be followed by inhaled steroids . Mucolytic drugs remain controversial and are difficult to monitor successfully . Oxygen therapy may be indicated for hypoxic chronic bronchitis, including long-term usage in cor pulmonale . Pulmonary rehabilitation by exercise training has recently been appreciated, and may be used in combination with the above treatment modalities.

Scand J Infect Dis, 1991, 23(1), 115 - 6
Branhamella catarrhalis septicemia in an immunocompetent adult; Alaeus A et al.; A 68-year-old man with otitis media developed signs of disseminated intravasal coagulation (DIC) and shock . Beta-lactamase positive Branhamella catarrhalis grew in all blood cultures and in secretion from the middle ear . The patient was immunocompetent and previously healthy . Severe B . catarrhalis septicemia has so far mainly been described in immunocompromised patients, mostly children, but this report shows that it may occasionally occur in immunocompetent adults.

Genetics, 1991 Jan, 127(1), 39 - 51
The Tn3 beta-lactamase gene acts as a hotspot for meiotic recombination in yeast; Stapleton A et al.; Although genetic distances are often assumed to be proportional to physical distances, chromosomal regions with unusually high (hotspots) or low (coldspots) levels of meiotic recombination have been described in a number of genetic systems . In general, the DNA sequences responsible for these effects have not been determined . We report that the 5' region of the beta-lactamase (ampR) gene of the bacterial transposon Tn3 is a hotspot for meiotic recombination when inserted into the chromosomes of the yeast Saccharomyces cerevisiae . When these sequences are homozygous, both crossing over and gene conversion are locally stimulated . The 5' end of the beta-lactamase gene is about 100-fold "hotter" for crossovers than an average yeast DNA sequence.

Nucleic Acids Symp Ser, 1991, (25), 117 - 8
Functional significance of the DNA curvature located near a promoter: an analysis using the beta-lactamase promoter of pUC19; Hirota Y et al.; The upstream region of the beta-lactamase promoter of pUC19 has a DNA curvature . Whether the curvature plays some role in the transcription from the promoter or not has been studied . The overall three-dimensional structure of the region containing the promoter and the curvature seems to play some significant role in the transcription of the gene . Functional significance of the DNA curvature located in the vicinity of a promoter is discussed.

Antisense Res Dev, 1991 Summer, 1(2), 117 - 40
Photoactivatable antisense DNA: suppression of ampicillin resistance in normally resistant Escherichia coli; Gasparro FP et al.; Antisense oligodeoxyribonucleotides complementary to a segment of the beta-lactamase gene and containing psoralen monoadducts at specific sites were examined for their ability to make normally resistant bacteria sensitive to ampicillin . Irradiation of oligonucleotides and psoralens with long-wavelength ultraviolet radiation (380-400 nm) produced monoadducted antisense molecules . High-performance liquid chromatography was used to purify microgram quantities of photoactivatable antisense DNA . Escherichia coli transformed with a plasmid containing the gene for beta-lactamase were used to test a series of oligonucleotides containing psoralen monoadducts after additional exposure to the photoactivating effects of long-wavelength ultraviolet radiation (320-400 nm) . Normally resistant bacteria treated with this photoactivatable form of antisense DNA (0.4 microM) were specifically sensitized to ampicillin . The reduction in colony formation ranged from 31 to 79% in comparison to control oligonucleotides which did not contain photoactivatable monoadduct moieties . Bacteria treated in a similar manner but in the presence of tetracycline instead of ampicillin were not affected . The activity of beta-galactosidase, whose gene is located on the same plasmid as beta-lactamase, was not affected.

Biomed Biochim Acta, 1991, 50(4-6), 643 - 6
An E . coli expression system which detoxifies the HIV protease; Korant BD et al.; Based on a variety of independent assays, the expression of HIV (human immunodeficiency virus type 1) protease in living bacterial cells results in their loss of viability . Although the mechanism is not proven, we have observed degradation of cellular proteins in E . coli expressing large amounts of active HIV protease . In order to avoid the loss of viability, we devised an expression system in which the viral protease is fused to beta-lactamase and is rapidly secreted to the periplasmic space, thus reducing its duration in the cytosol . Furthermore, we find the periplasmic form of the protease is soluble and enzymatically is several-fold more active than enzyme recovered from intracellular aggregates . The question of whether the viral protease may be toxic to infected cells is discussed.

J Cell Biol, 1990 Dec, 111(6 Pt 1), 2283 - 94
The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents; Gorlich D et al.; Bifunctional cross-linking reagents were used to probe the protein environment in the ER membrane of the signal sequence receptor (SSR), a 24-kD integral membrane glycoprotein (Wiedmann, M., T . V . Kurzchalia, E . Hartmann, and T . A . Rapoport . 1987 . Nature {Lond.} . 328:830-833) . The proximity of several polypeptides was demonstrated . A 22-kD glycoprotein was identified tightly bound to the 34-kD SSR even after membrane solubilization . The 34-kD polypeptide, now termed alpha SSR, and the 22-kD polypeptide, the beta SSR, represent a heterodimer . We report on the sequence of the beta SSR, its membrane topology, and on the mechanism of its integration into the membrane . Cross-linking also produced dimers of the alpha-subunit of the SSR indicating that oligomers of the SSR exist in the ER membrane . Various bifunctional cross-linking reagents were used to study the relation to ER membrane proteins of nascent chains of preprolactin and beta-lactamase at different stages of their translocation through the membrane . The predominant cross-linked products obtained in high yields contained the alpha SSR, indicating in conjunction with previous results that it is a major membrane protein in the neighborhood of translocating nascent chains of secretory proteins . The results support the existence of a translocon, a translocation complex involving the SSR, which constitutes the specific site of protein translocation across the ER membrane.

Gene, 1990 Nov 30, 96(1), 51 - 7
Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli; Zhang YB et al.; A genetic system for directly synthesizing eukaryotic membrane proteins in Escherichia coli and assessing their ability to insert into the bacterial cytoplasmic membrane is described . The components of this system are the direct expression vector, pYZ4, and the mature beta-lactamase (BlaM) cassette plasmid, pYZ5, that can be used to generate translational fusions of BlaM to any synthesized membrane protein . The beta-subunit of sheep-kidney Na,K-ATPase (beta NKA), a class-II plasma membrane protein, was synthesized in E . coli using pYZ4, and BlaM was fused to a normally extracellular portion of it . The fusion protein conferred ampicillin resistance on individual host cells, indicating that the BlaM portion had been translocated to the bacterial periplasm, and that, by inference, the eukaryotic plasma-membrane protein can insert into the bacterial cytoplasmic membrane . A series of 31 beta NKA::BlaM fusion proteins was isolated and characterised to map the topology of the eukaryotic plasma membrane protein with respect to the bacterial cytoplasmic membrane . This analysis revealed that the organisation of the beta NKA in the E . coli cytoplasmic membrane was indistinguishable from that in its native plasma membrane.

Science, 1990 Nov 23, 250(4984), 1111 - 7
An E . coli ribonucleoprotein containing 4.5S RNA resembles mammalian signal recognition particle; Poritz MA et al.; The signal recognition particle (SRP) plays a central role in directing the export of nascent proteins from the cytoplasm of mammalian cells . An SRP-dependent translocation machinery in bacteria has not been demonstrated in previous genetic and biochemical studies . Sequence comparisons, however, have identified (i) a gene in Escherichia coli (ffh) whose product is homologous to the 54-kilodalton subunit (SRP54) of SRP, and (ii) an RNA encoded by the ffs gene (4.5S RNA) that shares a conserved domain with the 7SL RNA of SRP . An antiserum to Ffh precipitated 4.5S RNA from E . coli extracts, implying that the two molecules reside in a complex . The 4.5S RNA can also bind to SRP54 and can replace 7SL RNA in an enzymatic assay . The product of a dominant mutation in the ffs gene (4.5S RNAdl1) is also coprecipitated by the antiserum to Ffh protein and is lethal when expressed from an inducible promoter . After induction of 4.5S RNAdl1, the earliest observed phenotype was a permanent induction of the heat shock response, suggesting that there was an accumulation of aberrant proteins in the cytoplasm . Late after induction, translocation of beta-lactamase was impaired; this may be an indirect effect of heat shock, however, because translocation of ribose binding protein or of the porin, OmpA, was unaffected . An unusual separation of the inner and outer membranes, suggestive of a defect in cell envelope, was also observed . Protein synthesis did not cease until very late, an indication that 4.5S RNA probably does not have a direct role in this process.

J Mol Biol, 1990 Nov 5, 216(1), 39 - 47
Export of altered forms of an Escherichia coli K-12 outer membrane protein (OmpA) can inhibit synthesis of unrelated outer membrane proteins; Ried G et al.; Expression of mutant ompA genes, encoding the 325 residue Escherichia coli outer membrane protein OmpA, caused an inhibition of synthesis of the structurally unrelated outer membrane porins OmpC and OmpF and of wild-type OmpA, but not of the periplasmic beta-lactamase . There was no accumulation of precursors of the target proteins and the inhibitory mechanism operated at the level of translation . So far only alterations around residue 45 of OmpA have been found to affect this phenomenon . Linkers were inserted between the codons for residues 45 and 46 . A correlation between size and sequence of the resulting proteins and presence or absence of the inhibitory effect was not found, indicating that the added residues acted indirectly by altering the conformation of other parts of the mutant OmpA . To be effective, the altered polypeptides had to be channelled into the export pathway . Internal deletions in effector proteins, preventing incorporation into the membrane, abolished effector activity . The results suggest the existence of a periplasmic component that binds to OmpA prior to membrane assembly; impaired release of this factor from mutant OmpA proteins may trigger inhibition of translation . The factor could be a See B-type protein, keeping outer membrane proteins in a form compatible with membrane assembly.

J Bacteriol, 1990 Nov, 172(11), 6427 - 34
Beta-lactamase expression in Streptomyces cacaoi; Urabe H et al.; Plasmids were prepared by inserting genomic DNA fragments from Streptomyces cacaoi within the mel gene of plasmid pIJ702 . The inserted DNA fragments contain the beta-lactamase-encoding bla gene and upstream nucleotide sequences of various lengths . The transcription start point of bla was identified by nuclease S1 mapping . Upstream nucleotide sequences of sufficient lengths had an enhancing effect on beta-lactamase production by the Streptomyces host . The dot blot hybridization assay revealed that this effect was exerted at the transcriptional level . Experimental evidence strongly suggests that the underlying mechanism involves, at least in part, one or several trans-acting elements . In one of the constructs, in which the upstream nucleotide sequence was reduced to 0.3 kb, the bla promoter was present but the bla gene was expressed by readthrough from a promoter, possibly the mel promoter, of the pIJ702 vector.

Biochem J, 1990 Nov 1, 271(3), 729 - 34
Characterization of a beta-lactamase produced in Mycobacterium fortuitum D316; Amicosante G et al.; A beta-lactamase from Mycobacterium fortuitum D316 was purified and some physico-chemical properties and substrate profile determined . On the basis of its N-terminal sequence and of its sensitivity to beta-iodopenicillanate inactivation, the enzyme appeared to be a class A beta-lactamase, but its substrate profile was quite unexpected, since nine cephalosporins were among the eleven best substrates . The enzyme also hydrolysed ureidopenicillins and some so-called 'beta-lactamase-stable' cephalosporins.

Antimicrob Agents Chemother, 1990 Nov, 34(11), 2184 - 92
Discrimination of extended-spectrum beta-lactamases by a novel nitrocefin competition assay; Papanicolaou GA et al.; We describe a nitrocefin competition assay for determining inhibition profiles as a useful adjunct to existing biochemical methods for the discrimination of beta-lactamases . The hydrolysis rate of nitrocefin was measured with a plate photometer as the change in A480 over 45 min in the presence of 17 inhibitors . Fourteen well-established beta-lactamases and 13 extended-spectrum beta-lactamases were tested . Correlations with data from isoelectric focusing and amino acid sequencing suggested that the inhibition profile reflects alterations in the active-site configuration of beta-lactamases . The method was especially useful in measuring the relative affinities of beta-lactamases against poorly hydrolyzed substrates and in screening large numbers of isolates for the detection of new beta-lactamase types.

Plasmid, 1990 Nov, 24(3), 218 - 26
Sequencing and expression of aadA, bla, and tnpR from the multiresistance transposon Tn1331; Tolmasky ME; A fragment of Tn1331 including tnpR, aac, aadA, and a bla gene which encodes lower levels of resistance to ampicillin and carbenicillin as compared to those mediated by the TEM beta-lactamase was sequenced . The polypeptide encoded by the bla gene has homology with the OXA-1, PSE-2, and OXA-2 proteins . Genes aac and bla are upstream and downstream respectively of aadA, and are both flanked by recombinational hot spots . Tn1331 has 520-bp direct repeats which include parts of the tnpR and TEM bla genes . Evolutionary models for the genesis of Tn1331 are proposed.

J Antibiot (Tokyo), 1990 Nov, 43(11), 1483 - 8
Cloning from Streptomyces cellulosae of the gene encoding beta-lactamase, a blue-dextran binding protein; Urabe H et al.; A beta-lactamase gene was cloned from Streptomyces cellulosae as a 2.3-kb DNA fragment using Streptomyces lividans 1326 and PIJ385 as a host-vector system . During the course of cloning, a part of the chromosomal DNA fragment cloned together with a part of the vector plasmid were deleted, indicating instability of this contiguous DNA region . The enzyme from the clone showed similar properties with respect to binding of blue dextran and isoelectric point to the enzyme from S . cellulosae . The cloned gene hybridized not only to DNA of S . cellulosae, the source of DNA, but also to DNAs of several Streptomyces species, irrespective of their formation of beta-lactamase . These results suggest that this gene may have homology to genes other than the one for beta-lactamase.

J Biol Chem, 1990 Oct 5, 265(28), 16760 - 6
Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli; Bowden GA et al.; High level expression of TEM beta-lactamase results in the accumulation of precursor and mature protein in the insoluble fraction of Escherichia coli . The mature polypeptide is sequestered in protein aggregates (inclusion bodies) located within the periplasmic space whereas the insoluble precursor is present in the cytoplasm . With the native beta-lactamase, aggregation is observed when the rate of expression exceeds 2.5% of the total protein synthesis rate . Substitution of the native signal sequence with the outer membrane protein A (OmpA) leader peptide results in extensive aggregation of only the mature protein . Furthermore, for OmpA-beta-lactamase, the accumulation of mature insoluble protein is independent of the rate of protein synthesis . These observations cannot be accounted by the kinetics of export of the OmpA-beta-lactamase and the native precursor, therefore suggesting that the signal sequence affects the conformation of the newly secreted mature polypeptide and in turn, the folding pathway . Previously, we have shown that the aggregation of the mature protein secreted using its own signal sequence can be inhibited by growing the cells in the presence of non-metabolizable sugars such as sucrose (Bowden, G., and Georgiou, G . (1988) Biotechnol . Prog . 4, 97-101) . We show here that this phenomenon is not related to osmotic effects, changes in beta-lactamase translation or precursor processing . It follows that the addition of sugars exerts a direct effect on the in vivo pathway of aggregation and folding, in analogy with the well characterized effect of sugars in vitro.

J Bacteriol, 1990 Oct, 172(10), 5643 - 9
Escherichia coli sec mutants accumulate a processed immature form of maltose-binding protein (MBP), a late-phase intermediate in MBP export; Ueguchi C et al.; Protein translocation across the Escherichia coli cytoplasmic membrane may consist of several temporally or topographically distinct steps . Although early events in the translocation pathway have been characterized to some extent, the mechanisms responsible for the trans-bilayer movement of a polypeptide are only poorly understood . This article reports on our attempts to dissect the translocation pathway in vivo . A processed form of maltose-binding protein (MBP) was detected in the spheroplasts of secY and secA temperature-sensitive mutant cells that had been pulse-labeled at the permissive temperature (30 degrees C) . This species of molecule was found to have an electrophoretic mobility identical to that of the mature MBP, but a considerable fraction of it was inaccessible to externally added protease . It had not attained the protease-resistant conformation characteristically observed for the exported mature protein . The radioactivity associated with this species decreased during chase and was presumably converted into the exported mature form, a process that required energy, probably the proton motive force, as demonstrated by its inhibition by an energy uncoupler . The spheroplast-associated processed form was more predominantly observed in the presence of a low concentration of chloramphenicol . A similar intermediate was also detected for beta-lactamase in wild-type cells . These results suggest that in a late phase of translocation, the bulk of the polypeptide chain can move through the membrane in the absence of the covalently attached leader peptide, and the secA-secY gene products are somehow involved in this process . We termed the processed intermediates processed immature forms.

Mol Microbiol, 1990 Oct, 4(10), 1637 - 44
Beta-lactamase as a probe of membrane protein assembly and protein export; Broome-Smith JK et al.; The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria . The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins . Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane . The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E . coli.

Development, 1990 Oct, 110(2), 539 - 46
Morphological and radiochemical evidence for the metabolism of exogenous proteins by the preimplantation sheep blastocyst; Pullar D et al.; The ability of the trophoblast of the ovine preimplantation blastocyst to take up and metabolise proteins has been investigated using two experimental approaches, microscopical and radiochemical . The ultrastructure of the expanded blastocyst obtained from 14 and 17 day pregnant ewes was examined . The morphology of tissues maintained in culture for 24 h has been compared with that of fresh tissues . After culture, the cellular morphology of the explants was well preserved . Fresh and 24 h cultured tissues were incubated with horse-radish peroxidase and ferritin and these proteins subsequently were found to be localized in coated pits, caveolae and secondary lysosomes of the trophoblast . Comparison of the uptake of {3H}dextran and of 125I-labelled bovine serum albumin indicated that proteins could be taken up by cultured tissue by mechanisms in addition to simple fluid phase endocytosis . During culture of explants of blastocyst with 125I-labelled bovine serum albumin, a large fraction of the radioactivity taken up by the tissue appeared in the TCA-soluble fraction of the culture medium indicating that cultured trophoblast hydrolysed proteins . That amino acids released from captured protein could be used for protein synthesis by the trophoblast was indicated by the labelling of tissue and medium proteins after culturing explants with beta-lactamase labelled with {14C}leucine . A major product (Mr approximately 17 x 10(3) present in the medium was likely to have been ovine trophoblast protein-1 . It is concluded that, during the expansion of the ovine blastocyst, the trophoblast has the ability to take up proteins, transport them to lysosomes and degrade them to amino acids which are used for protein synthesis . Thus proteins, as well as free amino acids, present in the histotrophe may be an important source of nitrogen for the sheep conceptus in the critical period just prior to implantation.

Gene, 1990 Sep 28, 94(1), 103 - 7
A totally synthetic plasmid for general cloning, gene expression and mutagenesis in Escherichia coli; Mandecki W et al.; A first totally synthetic Escherichia coli plasmid has been designed, constructed and shown to be a functional, stable, high-copy cloning vector . The FokI method of gene synthesis {Mandecki and Bolling, Gene 68 (1988) 101-107} was used to assemble the plasmid from 30 oligodeoxyribonucleotides . The plasmid contains synthetic modules for the beta-lactamase-encoding gene (bla), replication origin, lacZ gene fragment and multicloning site . The plasmid is patterned after the pUC-type plasmids and has a copy number similar to that of pUC plasmids . The major changes introduced include the removal of nearly 50% of the restriction sites present in pUC plasmids, reduction of plasmid size to 2050 bp, and introduction of transcription terminators downstream from both the bla gene and lacZ fragment . The changes facilitate a number of techniques, such as cloning, mutagenesis, expression and restriction analysis.

Appl Environ Microbiol, 1990 Sep, 56(9), 2742 - 7
Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase; Kadokura H et al.; The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli . The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E . coli . Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product . Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein . Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there . The signal sequence of alkaline phosphatase was found to be correctly processed in E . coli.

Mol Gen Genet, 1990 Sep, 223(3), 353 - 60
Inhibition of cell growth and stable DNA replication by overexpression of the bla gene of plasmid pBR322 in Escherichia coli; Katayama T et al.; A composite plasmid comprising the mini-F and pBR322 replicons was found to inhibit cell growth of a host with conditional mutations in dnaA and rnh under restrictive conditions, where the normal initiation of replication from oriC was inactivated, but the alternative replication initiation from oriK was active . It was further shown that the composite plasmid inhibited stable DNA replication (SDR) which occurs constitutively in cells mutant for rnh . Neither pBR322 nor mini-F alone produced these inhibitory effects . Deletion analyses revealed that the mini-F segment responsible for the inhibition of both processes was the promoter region of the sopA gene which had been cloned into a site upstream of the bla gene on pBR322 in such an orientation as to cause overexpression of bla . Inserting the promoter of the Escherichia coli lac gene into the same site had the same effect . Introduction of a deletion and a frameshift mutation into bla abolished the inhibition . Thus, the inhibition of growth and SDR appear to be due to overproduction of the bla gene product, beta-lactamase.

Biochim Biophys Acta, 1990 Aug 17, 1035(2), 237 - 41
Selection and application of antibodies modifying the function of beta-lactamase; Bibi E et al.; Nine monoclonal antibodies directed against class A beta-lactamases were detected and selected by a novel screening procedure based on assaying the modifications in the catalytic and stability properties of beta-lactamase in solution . Unlike conventional screening, e.g., ELISA or immunoprecipitation, the present method does not depend on firm binding and thus favors detection of low affinity antibodies . Individual antibodies were found to affect the enzymatic activity in various ways including stimulation, neutralization, protection and stabilization . Class A beta-lactamases show only 20% among members of this class . In contrast, two of our monoclonal antibodies cross-reacted with different beta-lactamases and thus demonstrate the presence of shared structural epitopes in this class of enzymes . One of the cross-reacting antibodies was elicited by sequential immunization with two different beta-lactamases . Taken together, our findings stress the importance of the screening method in antibody selection and illustrate the use of 'functional' monoclonal antibodies in the study of the structure-function relationship in an enzyme.

An Esp Pediatr, 1990 Aug, 33(2), 135 - 9
{Branhamella catarrhalis: its respiratory pathogenicity in childhood}; Moreno Galdo A et al.; Branhamella catarrhalis is a nasopharyngeal commensal which is being increasingly recognised as a pathogen, causing mainly infective exacerbations of chronic lung disease . It can also originate serious infections, like septicaemia, in patients with chronic predisposing conditions . During the period from 1979 to 1987, 22,501 respiratory tract samples from children were evaluated . Ninety nine isolated of Branhamella catarrhalis were identified (0.44%) . Patients' age extended from 12 days to 9 years, with patients younger than two years representing 73% . Sixty three out of 77 cases investigated (82%) were positive for beta-lactamase . The most frequent finding was the recovery of Branhamella catarrhalis in tracheal aspirates from children with a tracheotomy or prolonged nasotracheal intubation . One of these children had a septic episode during which Branhamella catarrhalis was isolated from blood . Also remarkable is a case of pneumonia in a patient with congenital hypogammaglobulinaemia . Branhamella catarrhalis was also recovered in a wide variety of acute upper and lower respiratory tract infections in children without previous predisposing conditions . It is less clear its pathogenic role in these cases.

Antimicrob Agents Chemother, 1990 Aug, 34(8), 1570 - 6
OHIO-1 beta-lactamase is part of the SHV-1 family; Shlaes DM et al.; The OHIO-1 beta-lactamase gene was subcloned in a 1.16-kilobase TaqI fragment in the 2.4-kilobase chimeric plasmid pSK04 . After directional subcloning into M13, the DNA sequence of this fragment was determined . The results showed an open reading frame of 858 base pairs (bp) encoding a protein of 286 amino acids . The structural gene showed 95, 87, and 60% DNA sequence identity with SHV-1, LEN-1, and TEM-1, respectively, and 93, 85, and 62% predicted amino acid sequence identity, respectively . The 87 bp upstream of the OHIO-1 structural gene had 96% identity with the upstream flanking sequence of SHV-1, including the -35 and -10 consensus sequences and the putative ribosomal binding site . A 223-bp DNA probe derived from a PstI-HaeII fragment in the C-terminal sequence of OHIO-1 had predicted 96, 88, and 61% sequence identity with SHV-1, LEN-1, and TEM-1, respectively . This probe hybridized to SHV-1 and poorly to LEN-1, but not to TEM-1 or a variety of other plasmid-mediated beta-lactamase genes, under stringent conditions . Screening of plasmid DNA derived from 40 ampicillin-resistant clinical isolates by Southern hybridization with the 223-bp probe uncovered no strains encoding OHIO-1 . Isoelectric focusing of the same collection did identify two strains producing enzymes resembling SHV-1, however . We have also performed a kinetic comparison of OHIO-1, SHV-1, and TEM-1 . OHIO-1 and SHV-1 were indistinguishable from each other but could be distinguished from TEM-1 . These data clearly place OHIO-1 within the SHV-1 family of beta-lactamases.

Enzyme Microb Technol, 1990 Aug, 12(8), 603 - 11
Recovery of a foreign protein from the periplasm of Escherichia coli by chemical permeabilization; Naglak TJ et al.; We have applied the technique of protein release by chemical permeabilization to recover a foreign protein in active form from the periplasm of a recombinant strain of Escherichia coli . The two agents used in our chemical permeabilization scheme, guanidine hydrochloride and Triton X-100, have different modes of action, allowing selectivity in protein release based on intracellular location under different treatment conditions . Specifically, treatment of E . coli C600-1 cells by guanidine alone resulted in 40-fold purification of recombinant beta-lactamase, which is periplasmically expressed in this host . Achieving such high purification in the cell disruption stage could alleviate some of the problems associated with recovery of intracellular products, such as low expression or the need to solubilize cytoplasmic inclusion bodies . Recovery of periplasmic proteins by chemical permeabilization is simpler than by osmotic shock and is less expensive than using enzymatic digestion.

Biochemistry, 1990 Jul 10, 29(27), 6499 - 508
Purification and characterization of clavaminate synthase from Streptomyces clavuligerus: an unusual oxidative enzyme in natural product biosynthesis; Salowe SP et al.; A pivotal step in the biosynthetic pathway to the beta-lactamase inhibitor clavulanic acid is the conversion of proclavaminic acid to clavaminic acid in a reaction requiring Fe2+, alpha-ketoglutarate, and oxygen {Elson, S . W., Baggaley, K . H., Gillett, J., Holland, S., Nicholson, N . H., Sime, J . T., & Woroniecki, S . R . (1987) J . Chem . Soc., Chem . Commun., 1736-1738} . Clavaminate synthase, the enzyme that catalyzes this oxidative cyclization/desaturation, has been purified to homogeneity from clavulanic acid producing cells of Streptomyces clavuligerus (ATCC 27064) . The enzyme behaved as a monomer during gel filtration and migrated with Mr 47,000 during denaturing gel electrophoresis . After ion-exchange FPLC two active forms of the protein were resolved that differed slightly in kinetic constants and apparent molecular weight . Kinetic comparisons with the four possible diastereomers of proclavaminate confirmed the absolute configuration of the substrate to be 2S,3R . The stoichiometry of the overall transformation was determined to be proclavaminate + 2(alpha-ketoglutarate) + 2O2----clavaminate + 2(succinate) + 2CO2 + 2H2O . In the absence of proclavaminate a slow decarboxylation of alpha-ketoglutarate to succinate and CO2 was observed in an uncoupled reaction which resulted in enzyme inactivation . Steady-state kinetic studies were undertaken for an initial description of the enzyme's catalytic cycle . The double-reciprocal plot with alpha-ketoglutarate as the variable substrate was linear; this supports the proposal that two stepwise oxidations of proclavaminate occur, each with the consumption of alpha-ketoglutarate and oxygen and the release of succinate, CO2, and H2O . The intersecting initial velocity plots obtained from pairwise variation of substrate concentrations were consistent with a sequential kinetic mechanism for the first oxidation . Similarities observed between clavaminate synthase and alpha-ketoglutarate-dependent dioxygenases argue for a common mechanism of oxygen activation . However, the nature of the interactions of the substrates in the active site of clavaminate synthase apparently redirects the conventional hydroxylase activity of dioxygenases to the construction of a strained bicyclic skeleton driven by the overall reduction of dioxygen.

Mol Microbiol, 1990 Jul, 4(7), 1101 - 9
The normally periplasmic enzyme beta-lactamase is specifically and efficiently translocated through the Escherichia coli outer membrane when it is fused to the cell-surface enzyme pullulanase; Kornacker MG et al.; Hybrid proteins were constructed in which C-terminal regions of the bacterial cell surface and extracellular protein pullulanase were replaced by the mature forms of the normally periplasmic Escherichia coli proteins beta-lactamase or alkaline phosphatase . In E . coli strains expressing all pullulanase secretion genes, pullulanase-beta-lactamase hybrid protein molecules containing an N-terminal 834-amino-acid pullulanase segment were efficiently and completely transported to the cell surface . This hybrid protein remained temporarily anchored to the cell surface, presumably via fatty acids attached to the N-terminal cysteine of the pullulanase segment, and was subsequently specifically released into the medium in a manner indistinguishable from that of pullulanase itself . These results suggest that the C-terminal extremity of pullulanase lacks signal(s) required for export to the cell surface . When beta-lactamase was replaced by alkaline phosphatase, the resulting hybrid also became exposed at the cell surface, but exposition was less efficient and specific release into the medium was not observed . We conclude that proteins that do not normally cross the outer membrane can be induced to do so when fused to a permissive site near the C-terminus of pullulanase.

EMBO J, 1990 Jul, 9(7), 2315 - 9
The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the beta-lactamase precursor; Laminet AA et al.; One of the fundamental problems in biochemistry is the role of accessory proteins in the process of protein folding . The Escherichia coli heat shock protein complex GroEL/ES has been suggested to be a 'chaperonin' and be involved in both oligomer assembly as well as protein transport through the membrane . We show here that the folding of the purified precursor of beta-lactamase is inhibited by purified GroEL or the GroEL/ES complex with a stoichiometry of one particle per molecule of pre-beta-lactamase . Purified GroES alone has no effect on folding . After Mg2+ ATP addition folding resumes and the yield of active enzyme is higher than in the absence of GroEL or GroEL/ES . Unexpectedly, GroEL or GroEL/ES, when added to folded pre-beta-lactamase, lead to an apparent net 'unfolding', probably to a collapsed state of the protein, which can be reversed by the addition of Mg2+ ATP . The reversible and Mg2+ ATP-dependent association of GroEL/ES with non-native proteins might explain its postulated role in both protein transport and oligomer assembly.

Biochemistry, 1990 Jun 26, 29(25), 6071 - 81
Chemical reactivity of potassium permanganate and diethyl pyrocarbonate with B DNA: specific reactivity with short A-tracts; McCarthy JG et al.; We have examined the reactivity of B DNA with two chemical probes of DNA structure, potassium permanganate (KMnO4; thymine specific) and diethyl pyrocarbonate (DEPC; purine specific, A greater than G) . The DNA probed is from the beta-lactamase promoter region of the vector pBR322, and from the 3' noncoding region of a chicken embryonic myosin heavy chain gene . The chemical probes display variable reactivity with the susceptible bases in these fragments, suggesting that modification of these bases by KMnO4 and DEPC is quite sequence dependent . In contrast, these probes react with the short A-tracts present in these DNA fragments in a reproducible fashion, generating two related patterns of reactivity . In the majority of the A-tracts, all but the 3'-terminal thymine are protected from KMnO4 attack, while DEPC reacts significantly with all but the 3'-terminal adenine of the A-tracts . Some A-tracts also display a very high DEPC reactivity at the adenine adjacent to the 3'-terminal unreactive adenine . Little qualitative difference in the KMnO4 reactivity of the A-tracts was found between 12 and 43 degrees C . However, at lower temperatures the elevated KMnO4 reactivity at the 3'-terminal A-tract thymine is sometimes lost . Raising the temperature of the KMnO4 reaction can cause relatively large increases in the reactivity of some single thymines, suggesting that significant local changes in stacking occur at these thymines at elevated temperatures . The data presented suggest that many short A-tracts embedded in long fragments of DNA can assume a number of related structures in solution, each of which possess distinct junctions with the flanking DNA . This result is consistent with high-resolution structural studies on oligonucleotides containing short A-tracts . The relevance of these results to current models of A-tract structure and DNA bending is discussed . Our data also indicate that KMnO4 and DEPC are potentially useful reagents for the study of sequence-dependent variations in B DNA structure.

Biochimie, 1990 Jun-Jul, 72(6-7), 495 - 503
Probing the active site of beta-lactamase R-TEM1 by informational suppression; Lenfant F et al.; Using a new extended set of 13 amber suppressors in E coli, systematic amino-acid replacements were performed at positions 104(E) and 238(G) of TEM-1 beta-lactamase from PUC19 . The enzyme is tolerant to most substitutions tested at position 104 . Missense revertants E104K, E104S or E104Y exhibited only minor changes in enzyme activity with respect to wild-type TEM-1 . Several substitutions at position 238 resulted in a new cefotaxime hydrolysing capacity, but to an extent that did not confer cefotaxime resistance for the bacteria producing the mutated enzymes . Only when the mutations at codons 104 and 238 were combined on the same gene, did a true cefotaxime resistant phenotype appear, mimicking the situation encountered with 3rd generation cephalosporins resistant clinical isolates.

Bol Asoc Med P R, 1990 May, 82(5), 234 - 6
Prospective study about the incidence of B lactamase producing bacteriae in otitis media in the population of the emergency room of the University Pediatric Hospital; Colon I et al.; The purpose of the study is to determine the incidence of beta-lactamase producing pathogens causing otitis media (O.M.) in the Emergency Room population of the University Pediatric Hospital . In our first four months of study, 22 patients, between the ages of 6 months to 13 y/o have been evaluated . Middle ear secretion cultures were obtained by tympanocentesis . The organisms recovered from cultures were S . epidermidis 3 (14%), S . pneumoniae 2 (9%) H . influenzae 1 (5%), mix flora 1 (5%) and 13 (59%) with no growth . None of these organisms were beta-lactamase producers . Up to 64% of the patients had history of 2 to 5 OM episodes during the last six months . Interesting is the association of bronchial asthma, sinusitis and allergy history with OM . Final study results will be presented in a near future.

Biol Chem Hoppe Seyler, 1990 May, 371 Suppl, 271 - 5
Expression in Escherichia coli of the AIDS virus aspartic protease through a protein fusion; Korant BD et al.; Fusion of the coding sequence for the aspartic protease of HIV-1, the human AIDS virus, to a bacterial beta-lactamase gene, provides an expression system in E . coli which yields high specific activity HIV protease in a soluble form.

FEMS Microbiol Lett, 1990 May, 57(1-2), 49 - 54
Analysis of OmpC-beta-lactamase hybrid proteins: OmpC appears not to contain discrete localization signals; Saarilahti HT et al.; Fusions to the beta-lactamase (bla) gene were employed to analyze the presence of localization information in the mature part of OmpC, a major pore-forming outer membrane protein in Escherichia coli K-12 . Six translational ompC-bla gene fusions were constructed, the shortest of them containing only part of the ompC signal sequence and the largest approximately 90% of the sequence encoding mature OmpC protein . Export of the hybrid proteins to a non-cytoplasmic location was a prerequisite for ampicillin resistance . Localization of the hybrid proteins by cell fractionation and solid phase iodination of whole cells suggested that the exported hybrid proteins possibly interacted with the outer membrane in vivo . No specific sequence of the mature OmpC protein, however, was found to promote this interaction.

J Biol Chem, 1990 Apr 25, 265(12), 6931 - 5
Nonprotein amino acid furanomycin, unlike isoleucine in chemical structure, is charged to isoleucine tRNA by isoleucyl-tRNA synthetase and incorporated into protein; Kohno T et al.; Nonprotein amino acid furanomycin was found to bind with Escherichia coli isoleucyl-tRNA synthetase (IleRS) almost as tightly as the substrate L-isoleucine . The conformation of furanomycin bound to the enzyme was determined by NMR analyses including the transferred nuclear Overhauser effect method . The conformation of IleRS-bound furanomycin was similar to that of L-isoleucine, although the chemical structure of furanomycin is unlike that of L-isoleucine . By E . coli IleRS, E . coli tRNAIle was charged with furanomycin as efficiently as with L-isoleucine . Furthermore, furanomycyl-tRNAIle was bound to polypeptide chain elongation factor Tu as tightly as isoleucyl-tRNAIle . Furanomycin was found to be incorporated into beta-lactamase precursor by in vitro protein biosynthesis . A newly designed amino acid will probably be incorporated into proteins, provided that the new amino acid takes a similar conformation as a protein-constituting amino acid in the active site of an aminoacyl-tRNA synthetase.

Biochemistry, 1990 Apr 10, 29(14), 3480 - 8
Mechanism of acid-induced folding of proteins; Goto Y et al.; We have previously shown {Goto, Y., Calciano, L . J., & Fink, A . L . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 573-577} that beta-lactamase, cytochrome c, and apomyoglobin are maximally unfolded at pH 2 under conditions of low ionic strength, but a further decrease in pH, by increasing the concentration of HCl, refolds the proteins to the A state with properties similar to those of a molten globule state . To understand the mechanism of acid-induced refolding of protein structure, we studied the effects of various strong acids and their neutral salts on the acid-unfolded states of ferricytochrome c and apomyoglobin . The conformational transition of cytochrome c was monitored at 20 degrees C by using changes in the far-UV CD and in the Soret absorption at 394 nm, and that of apomyoglobin was monitored by changes in the far-UV CD . Various strong acids (i.e., sulfuric acid, perchloric acid, nitric acid, trichloroacetic acid, and trifluoroacetic acid) refolded the acid-unfolded cytochrome c and apomyoglobin to the A states as was the case with HCl . For both proteins neutral salts of these acids caused similar conformational transitions, confirming that the anions are responsible for bringing about the transition . The order of effectiveness of anions was shown to be ferricyanide greater than ferrocyanide greater than sulfate greater than thiocyanate greater than perchlorate greater than iodide greater than nitrate greater than trifluoroacetate greater than bromide greater than chloride.(ABSTRACT TRUNCATED AT 250 WORDS)

J Reprod Med, 1990 Mar, 35(3 Suppl), 307 - 12
Development of beta-lactamase inhibitors; Sutherland R; The resistance of bacteria to beta-lactam antibiotics was first associated with the production of the enzyme beta-lactamase as long ago as 1940 . Since then, increasing numbers of beta-lactamase-producing bacteria capable of inactivating penicillins and cephalosporins have been isolated clinically . One approach to the problem posed by beta-lactamase-producing bacteria has been to seek substances that function as inhibitors of beta-lactamase and that can be used to protect beta-lactam antibiotics from destruction by the bacterial enzymes . The first clinically available inhibitor was clavulanic acid, a metabolite of Streptomyces clavuligerus that was identified in a screening program for naturally occurring beta-lactamase inhibitors . Clavulanic acid is a potent inhibitor of many bacterial beta-lactamases and has been formulated with amoxicillin and ticarcillin to produce broad-spectrum antibiotic combinations active against beta-lactamase-producing bacteria . After the discovery of clavulanic acid, various compounds have been reported to function as inhibitors of bacterial beta-lactamases, but only sulbactam and its prodrug, sultamicillin, have become available commercially . The success of clavulanic acid has confirmed beta-lactamase inhibitors as one solution to the problem of antibiotic-resistant bacteria.

J Gen Microbiol, 1990 Mar, 136 ( Pt 3), 521 - 7
Beta-lactamase of Lysobacter enzymogenes: induction, purification and characterization; von Tigerstrom RG et al.; Lysobacter enzymogenes produces an inducible beta-lactamase and induction with 100 micrograms ampicillin ml-1 resulted in an increase of more than 100-fold in enzyme activity . Various other beta-lactam antibiotics also served as effective inducers . The enzyme was obtained from cells by osmotic shocking to release periplasmic components and it was purified primarily by ion-exchange chromatography and PAGE . The beta-lactamase consists of one polypeptide with a molecular mass of about 28 kDa and an isoelectric point greater than 9.6 . It is strongly inhibited by p-chloromercuribenzoate and clavulanic acid but not by EDTA . The enzyme readily hydrolyses several penicillins and cephalosporins, but not oxacillin or cloxacillin . The enzyme therefore belongs to group 2b of the bacterial beta-lactamases.

FEMS Microbiol Lett, 1990 Feb, 55(3), 319 - 23
Hyperproduction of TEM-1 beta-lactamase in clinical isolates of Escherichia coli serotype O15; Shannon K et al.; During an outbreak of infection with ampicillin-resistant, TEM-1 beta-lactamase-producing Escherichia coli serotype O15, some strains were noted to differ from the majority in that they showed reduced susceptibility to amoxycillin/clavulanic acid (Augmentin), ureidopenicillins and first generation cephalosporins and produced increased amounts of beta-lactamase . The plasmid from one such isolate was compared with that from an isolate that produced normal amounts of beta-lactamase . Restriction analysis with EcoRI revealed extra fragments in the plasmid from the beta-lactamase hyperproducer and use of DNA-DNA hybridisation with a biotinylated TEM-1 probe showed genetic rearrangement in the beta-lactamase hyperproducer so that the TEM gene appeared to be present in larger amounts and was located on a smaller fragment than for the plasmid from the strain that produced normal amounts of beta-lactamase.

J Biol Chem, 1990 Jan 15, 265(2), 1005 - 9
A mutation in the small (alpha) subunit of glycyl-tRNA synthetase affects amino acid activation and subunit association parameters; Toth MJ et al.; A strategy was designed to isolate mutants of glycyl-tRNA synthetase that are altered at the amino acid binding site, including a class with altered amino acid specificity . For this purpose, the plasmid pBR322 was mutated so that the codon (AGC) of the active site Ser-68 in the beta-lactamase gene was changed to the glycine codon GGC to inactivate the encoded enzyme . Suppressors that increase the amount of beta-lactamase activity of the Gly-68 allele of beta-lactamase were isolated and some mapped to the gene encoding glycyl-tRNA synthetase (glyS) . While in vitro misaminoacylation of tRNA(Gly) with serine was not detected for any of the mutants, glycyl-tRNA synthetase activity was altered . One severely affected glyS mutant (N302) was studied in more detail . For this mutant, a single Pro-61----Leu substitution in the alpha chain confers an elevation of the Km values for glycine (25-fold) and for ATP (45-fold) in the aminoacylation reaction, but only a minor perturbation of the Km for tRNA . There also was a severely reduced adenylate synthesis activity (greater than 100-fold) . In addition, a nonlinear dependence between aminoacylation activity and enzyme concentration was observed which implies that the alpha chain Pro-61----Leu mutation has disrupted the functionally essential subunit interactions of the holoenzyme . The results of the preceding paper have shown that the alpha chain and parts of the beta chain are required for aminoacylation and adenylate synthesis activity . The results of this study suggest that the alpha chain specifically contributes to amino acid and to ATP binding in a way that is affected by proper subunit interactions.

J Med Chem, 1990 Jan, 33(1), 344 - 7
Orally effective acid prodrugs of the beta-lactamase inhibitor sulbactam; English AR et al.; Sulbactam (1) is a beta-lactamase inhibitor with limited oral bioavailability . Lipophilic double-ester prodrug sulbactam pivoxil (2) significantly improves the oral absorption of sulbactam, as does the mutual prodrug double ester sultamicillin (3) . We have found that double-ester prodrugs of sulbactam terminating in a carboxyl group (8) also were effective oral-delivery vehicles in rats . Carboxyl-terminated double esters have several potential advantages over their nonionizable lipophilic counterparts, including water solubility, crystallinity, choice of salts for dosage forms, and formation of innocuous byproducts on hydrolysis.

Yao Xue Xue Bao, 1990, 25(6), 406 - 11
Pharmacokinetics of sulbactam and ampicillin in mice and in dogs; Liu CX et al.; Sulbactam is a new beta-lactamase inhibitor . The pharmacokinetic characteristics of sulbactam was similar to ampicillin after a single intravenous injection of 200 mg/kg in mice with a half-life of approximately 50 min . The two drugs appear to equilibrate rapidly between central and peripheral compartments . The Vc and Vd suggest that they are widely distributed in the extracellular fluid and into tissues . Co-administration of sulbactam and ampicillin in mice and in dogs showed essentially no change on the kinetics of either ampicillin or sulbactam.

Yao Xue Xue Bao, 1990, 25(5), 340 - 4
{Synthesis and inhibition on beta-lactamase of 4-(3-amido-4-substituted phenyl-2-oxo-azetidinonyl-1)methyl-cyclohexane carboxylic acids and -benzoic acids}; Yin SF et al.; Eleven title compounds have been synthesized from trans-4-amino-methyl-cyclohexylic acid and 4-aminomethyl-benzoic acid, and were identified via elemental analysis, IR, 1HMNR and MS . Their beta-lactamase inhibition activity was determined and contrasted with penicillanic acid S-dioxide . The results of the preliminary test show that all the products have beta-lactamase inhibition activity to some extent.

Appl Environ Microbiol, 1990 Jan, 56(1), 104 - 11
Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor; Chalmers JJ et al.; The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated . E . coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C . The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90% . Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production . However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations) . When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C . Both total soluble and total human epidermal growth factor increased with decreasing temperature . These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures . Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.

Proc Natl Acad Sci U S A, 1990 Jan, 87(2), 573 - 7
Acid-induced folding of proteins; Goto Y et al.; The addition of HCl, at low ionic strength, to the native state of apomyoglobin, beta-lactamase, and cytochrome c caused these proteins to adopt an essentially fully unfolded conformation in the vicinity of pH 2 . However, contrary to expectation, the addition of further acid resulted in refolding to a compact conformation with the properties of a molten globule . The major factor responsible for the refolding is believed to be the binding of the anion, which minimizes the intramolecular charge repulsion that initially brought about the unfolding.

Mol Microbiol, 1989 Dec, 3(12), 1813 - 7
A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli; Broome-Smith JK et al.; The feasibility of using a beta-lactamase fusion approach for maximizing the levels of periplasmic or membrane-bound proteins expressed in Escherichia coli was investigated . The coding region for mature TEM beta-lactamase was fused after the signal peptide and aminoterminal portion of the coding region of a weakly expressed periplasmic protein, PBP3* . The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected . The PBP3* gene of the unmutagenized beta-lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3* . The applications of a beta-lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E . coli are considered.

Antimicrob Agents Chemother, 1989 Dec, 33(12), 2160 - 3
Structural features related to hydrolytic activity against ceftazidime of plasmid-mediated SHV-type CAZ-5 beta-lactamase; Peduzzi J et al.; Tryptic peptides of the novel ceftazidimase CAZ-5 were sequenced by manual Edman degradation and aligned according to strong homology (more than 98%) with SHV-1 and SHV-2 beta-lactamase sequences . CAZ-5 differed from SHV-1 by five amino acid substitutions . Unusually high activity of CAZ-5 towards ceftazidime was imputed to substitution of a Lys for a Glu at position 214 of the mature protein.

J Appl Toxicol, 1989 Dec, 9(6), 427 - 31
The effect of 2,4-dichlorophenol on growth and plasmidic beta-lactamase activity in Escherichia coli; Espigares M et al.; The toxic effects of 2,4-dichlorophenol on two strains of Escherichia coli harbouring plasmids with beta-lactamase activity were studied . The toxicant had a significant effect on growth at concentrations of 12 micrograms ml-1 for the JA 221 (pBR322) strain, as well as at 24 micrograms ml-1 for the J5-3 (RP4) strain . beta-Lactamase production was affected at concentrations of 96 micrograms ml-1 in both strains . However, beta-lactamase production does not seem to constitute a reliable toxicity test in view of its variability and relatively low sensitivity.

Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9094 - 8
An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase; Oliphant AR et al.; Random-sequence or highly degenerate oligonucleotides have been useful for defining functionally important sequences both in proteins and in nucleic acids . In this approach, such oligonucleotides are used to replace a segment of DNA required for a desired function, and functional sequences are identified by an appropriate genetic or biochemical selection . Here, a collection of 500,000 {corrected} altered beta-lactamase proteins was generated by cloning a mixed-base oligonucleotide in place of the sequences coding for a 17-amino acid portion of the enzyme's active site . Approximately 2000 enzymes from this collection were able to confer ampicillin resistance on Escherichia coli . Fifty-eight of these were chosen for further study after characterization with various beta-lactam substrates . beta-Lactamases having altered specificity against different antibiotics, resistance to the suicide inhibitors clavulanic acid and sulbactam, and temperature-dependent activities were obtained . The amino acid residues responsible for these altered properties as well as for basic enzyme activity are defined . This approach should prove to be an effective and general tool for creating proteins with novel properties, especially in situations in which a high-resolution structure of the protein is not known.

J Bacteriol, 1989 Dec, 171(12), 6423 - 9
Evolutionary perspectives on multiresistance beta-lactamase transposons; Lafond M et al.; A series of intragenic DNA probes, encoding the major part of the transposase resolvase and inverted repeats of transposons Tn3, Tn21, and Tn2501, were used in hybridization assays for homologous DNA sequences in 18 transposons studied . The tnpA and tnpR probes detected extensive homology with Tn3-like and Tn21-like elements for 11 transposons . This high degree of homology was confirmed with the 38- and 48-base-pair inverted-repeat oligonucleotide probes of Tn3, Tn21, and Tn2501 . The Southern-type gel hybridization experiments localized the tnpA-homologous sequences on the physical DNA maps constructed . The genetic and physical maps of the transposons were compared, as were their nucleic acid sequence homologies . These comparisons suggested a subfamily of mobile elements distinct from but related to the Tn21 group . Based on these results, an evolutionary model is proposed and a pedigree is presented for the genesis of multiresistance beta-lactamase transposons.

J Biol Chem, 1989 Nov 25, 264(33), 20074 - 81
Illicit secretion of a cytoplasmic protein into the periplasm of Escherichia coli requires a signal peptide plus a portion of the cognate secreted protein . Demarcation of the critical region of the mature protein; Summers RG et al.; The beta-lactamase signal peptide alone is not sufficient to direct secretion of chicken muscle triosephosphate isomerase, a normally cytoplasmic protein, into the periplasm of Escherichia coli . The signal peptide and at least the first 3 residues of the mature beta-lactamase are required before any secretion of the isomerase can be observed . At this point the level of secretion is very low, but the addition of further residues of the mature beta-lactamase enhances the secretion of the hybrid protein . The maximum level of secretion is achieved when 12 or more residues of the mature beta-lactamase intervene between the signal peptide and the isomerase . It is the proximity of an arginine residue at position 3 of the isomerase that is responsible for the blockade to secretion of these hybrid proteins (see Summers, R.G., Harris, C.R., and Knowles, J.R . (1989) J . Biol . Chem . 264, 20082-20088) . With 12 residues of the mature beta-lactamase between the signal peptide and the isomerase, the offending arginine now lies at position 15 of the hybrid . The 14 residues that immediately follow the signal peptide therefore define a region of constrained properties that is critical to the secretability of proteins from E . coli.

J Biol Chem, 1989 Nov 25, 264(33), 20082 - 8
A conservative amino acid substitution, arginine for lysine, abolishes export of a hybrid protein in Escherichia coli . Implications for the mechanism of protein secretion; Summers RG et al.; A hybrid protein that comprises the beta-lactamase signal peptide fused precisely to chicken muscle triosephosphate isomerase is not secreted into the periplasm of Escherichia coli . The protein can be secreted, however, if an arginine residue at position 3 of the isomerase is replaced by either a serine or a proline residue . In contrast, replacement of a neighboring lysine residue has no effect on secretion of the protein . Furthermore, if the arginine is removed from position 3 to generate a secreted protein, but is then reintroduced in place of the neighboring lysine, the blockade to secretion is re-established . The singular effect of the arginine residue on secretion does not result from the role this residue plays in the formation or stabilization of the native isomerase structure: mutational alterations remote from the N terminus of the isomerase that prevent the proper folding of the protein do not relieve the block to secretion . The finding that an arginine residue prevents secretion while a lysine residue does not, suggests that basic residues near the mature N terminus of a secreted protein must be deprotonated if orderly export is to occur . This implies that the signal peptide along with the N-terminal portion of the mature protein partitions directly into the lipid bilayer in the course of the secretory process.

Plasmid, 1989 Nov, 22(3), 268 - 70
Lack of expression of RP4-specified beta-lactamase in Azospirillum brasilense; Dutta SH et al.; Plasmid RP4, which normally confers resistance to ampicillin (Apr), tetracycline (Tcr), and kanamycin (Kmr) to its hosts, failed to express enhanced Apr when transferred from Escherichia coli to Azospirillum brasilense which has its own intrinsic beta-lactamase . Even in a beta-lactamase-deficient mutant, A . brasilense RG-D16, no increase in beta-lactamase or significant Apr appeared following transfer of RP4 . However, A . brasilense RG (RP4) and A . brasilense RG-D16 (RP4) did exhibit Tcr Kmr . When RP4 was transferred back from A . brasilense to E . coli all three drug resistances and beta-lactamase activity were fully expressed.

Antimicrob Agents Chemother, 1989 Nov, 33(11), 1958 - 63
TEM-4, a new plasmid-mediated beta-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate of Escherichia coli; Paul GC et al.; A clinical isolate of Escherichia coli, strain CB-134, recovered in 1986 from an abdominal abscess, exhibited resistance to penams, oxyimino-beta-lactams including broad-spectrum cephalosporins (cefotaxime, ceftriaxone, ceftazidime), and aztreonam but remained susceptible to cephamycins (cefoxitin, cefotetan) and to moxalactam and imipenem . Clavulanate (2 micrograms/ml) restored the susceptibility of the strain to broad-spectrum cephalosporins and aztreonam . A beta-lactamase with an isoelectric point (pI) of 5.9 was detected in strain CB-134, and the corresponding gene was transferred by conjugation to E . coli together with the associated aminoglycoside resistance determinant {AAC(3)-II} and tetracycline, trimethoprim, and sulfonamide resistance . The beta-lactamase efficiently hydrolyzed cefotaxime and ceftriaxone but only moderately hydrolyzed ceftazidime and was inhibited by clavulanate and sulbactam (1 microM) and by anti-TEM-1 and anti-TEM-2 sera . This extended-spectrum beta-lactamase, conferring resistance to cefotaxime, ceftriaxone, ceftazidime, and aztreonam, was comparable to CTX-1 (TEM-3) but differed from it by pI . Agarose gel electrophoresis of the plasmid DNA indicated that this new enzyme was coded by pUD16, a plasmid of 220 kilobases which belongs to the Inc6 incompatibility group . Hybridization with an intragenic probe for TEM-1 revealed that this beta-lactamase derives from TEM-type beta-lactamases and hence it was named TEM-4.

J Int Med Res, 1989 Nov-Dec, 17(6), 532 - 8
Oral bioavailability of ampicillin and amoxycillin alone and bound in fixed proportions to sulbactam and clavulanic acid; Desager JP et al.; Oral bioavailability of ampicillin when bound to sulbactam (sultamicillin) compared with ampicillin alone and that of amoxycillin with a ligand of clavulanic acid versus amoxycillin alone were assessed in 16 healthy subjects using an open label, multiple crossover study . After a single administration of the drugs, the bioavailability of ampicillin released from sultamicillin was more than twice (2.17) that of ampicillin administered alone, whereas that of amoxycillin was increased by a factor of 1.64 by the presence of clavulanic acid . Higher peak concentrations and areas under the curves, along with shorter lag times, were observed in both cases . The increased oral bioavailability of these aminopenicillins when associated with a beta-lactamase inhibitor was well demonstrated in this study, the bioavailability of ampicillin being more enhanced by sulbactam than that of amoxycillin by clavulanic acid.

Infection, 1989 Nov-Dec, 17(6), 429 - 33
Biochemical characteristics of extended broad spectrum beta-lactamases; Bush K et al.; Extended broad spectrum beta-lactamases such as TEM-3 (CTX-1), TEM-5 (CAZ-1), TEM-10 and RHH-1 were purified and found to have lower specific activities than the TEM-1 or TEM-2 beta-lactamases . Total hydrolytic activity in crude extracts was also lower for the extended broad spectrum enzymes . These beta-lactamases hydrolyzed not only penicillins such as carbenicillin, cloxacillin and piperacillin, but also cephalosporins and monobactams . The most notable differences in substrate profiles between the extended broad spectrum enzymes and TEM-2 enzymes occurred with oxime-containing antibiotics . Although all the extended broad spectrum enzymes described above hydrolyzed cefotaxime, ceftazidime and aztreonam, the four enzymes could be easily differentiated: TEM-3 hydrolyzed cefotaxime preferentially, TEM-5 and RHH-1 hydrolyzed ceftazidime approximately three times faster than cefotaxime, whereas TEM-10 hydrolyzed ceftazidime 42 times faster than cefotaxime . All the enzymes were inhibited well by clavulanic acid, with I50 values ranging from 4.3 to 12 nM, compared to 130 nM for TEM-2 . Inhibition by sulbactam was also better for the extended broad spectrum than for the TEM-2 beta-lactamases, with I50 values of 12-940 nM for the extended broad spectrum enzymes, compared to 1600 nM for the TEM-2 beta-lactamase.

Antimicrob Agents Chemother, 1989 Nov, 33(11), 1845 - 54
BRO beta-lactamases of Branhamella catarrhalis and Moraxella subgenus Moraxella, including evidence for chromosomal beta-lactamase transfer by conjugation in B . catarrhalis, M . nonliquefaciens, and M . lacunata; Wallace RJ Jr et al.; Two closely related beta-lactamases, BRO-1 and BRO-2 (formerly called Ravasio and 1908), are found in Moraxella (Branhamella) catarrhalis . We screened strains of B . catarrhalis recovered in the United States since 1952 and identified the first beta-lactamase-positive isolate in August 1976 . The prevalence of the enzymes among 394 clinical isolates from one Texas hospital has averaged 75% since testing began in 1983 . Screening of isolates of Moraxella subgenus Moraxella revealed the BRO enzymes in two other human respiratory tract species, M . lacunata and M . nonliquefaciens, beginning in 1978 . A different beta-lactamase with a pI of 6.4 predominated in other species of subgenus Moraxella . BRO-2 had a different isoelectric focusing pattern and was produced in lesser amounts than BRO-1, but the two enzymes were indistinguishable by substrate or inhibitor profile . BRO enzymes from B . catarrhalis, M . nonliquefaciens, and M . lacunata could be transferred by conjugation and, for B . catarrhalis, also by transformation to B . catarrhalis . Plasmid bands were demonstrated in 90% of M . nonliquefaciens and in one previously reported strain of B . catarrhalis, but no change in plasmid profiles was seen in beta-lactamase-positive recombinants, supporting previous studies that suggested the beta-lactamase genes are chromosomal.

Mol Microbiol, 1989 Oct, 3(10), 1361 - 9
Identification of amino acid sequences that can function as translocators of beta-lactamase in Escherichia coli; Zhang Y et al.; A plasmid vector, pYZ1, was constructed which lacks most of the beta-lactamase signal-peptide coding region, but has a unique EcoRI site spanning codons 2 and 3 of the resultant cytoplasmic beta-lactamase derivative . Short quasi-random DNA sequences were cloned into the EcoRI site and Escherichia coli transformants in which some translocation of beta-lactamase across the cytoplasmic membrane was restored were selected by their ability to survive and form colonies on plates containing a low level of ampicillin . About 15-20% of all in-frame inserts restored some beta-lactamase translocation and the salient feature of these sequences was their marked hydrophobicity . These results are discussed in the light of a similar study in which sequences able to function as translocators of invertase in yeast were cloned and analysed (Kaiser et al., 1987).

Biochem J, 1989 Oct 1, 263(1), 309 - 11
Purification of TEM-1 beta-lactamase by immunoaffinity chromatography; Bibi E; A monoclonal antibody prepared against TEM-1 beta-lactamase was found to compete with penicillins and cephalosporins for binding to the enzyme . The purified antibody preparation was linked to Sepharose 4B and used for immunoaffinity-chromatography purification of TEM-1 beta-lactamase . Elution with either benzylpenicillin or cloxacillin yielded a highly purified, concentrated and active enzyme preparation.

J Clin Pharm Ther, 1989 Oct, 14(5), 393 - 401
Kinetics of bacterial growth in simple intravenous solutions and admixtures; Akpan UE et al.; The growth of bacteria in intravenous solutions and admixtures has been studied under stationary conditions of incubation . All the solutions were inoculated with 100 organisms/ml, incubated at room temperature (27 degrees C) or (37 degrees C), with samples withdrawn at specified time intervals, and plated in quadruplicates . The simple intravenous (i.v.) solutions did not support significant growth (P greater than 0.05) of any of the micro-organisms . Growth in i.v . solutions containing 1% blood was very significant (P greater than 0.05), as demonstrated by the high apparent growth rate constants (K) . The ratio of K for beta-lactamase producing bacteria (beta-lac+) over that for non-beta-lactamase producing bacteria (beta-lac-) was significant (P less than 0.05) at 37 degrees C compared to that at 27 degrees C . The higher K values for B . cereus in benzylpenicillin and cefuroxime solutions, respectively, compared to those in antibiotic-free solutions, may be attributable to hydrolysis of the drugs, while the low K values for B . subtilis in the same solutions may be attributed to the inhibitory effects of the drugs . In conclusion, minute quantities of blood in i.v . solution tend to cause bacteria to multiply rapidly . The presence of beta-lactamase producing species might, in addition, hydrolyse susceptible beta-lactam antibiotics which are common additives to i.v . fluids.

Res Microbiol, 1989 Oct, 140(8), 579 - 90
The analysis of five carbenicillin-hydrolysing enzymes by electrophoretic methods; Vedel G et al.; Five carbenicillin-hydrolysing enzymes (carbenicillinases, or CARB), PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3, CARB-4 and CARB-5, and the beta-lactamase PSE-2 were compared by analysing their isoelectric points (pI), electrophoretic mobilities (mR) and titration curves (pH gradient electrophoresis) . The pI determined by isoelectric focusing were 4.3 (CARB-4), 5.3 (PSE-4/CARB-1), 5.7 (PSE-1/CARB-2), 5.75 (CARB-3), 6.1 (PSE-2) and 6.35 (CARB-5) . Their mR were estimated by zone electrophoresis as congruent to 26 for PSE-1 (CARB-2), CARB-3 and CARB-5, congruent to 30 for PSE-2, congruent to 33 for PSE-4 (CARB-1) and congruent to 61 for CARB-4 . Titration curve analyses indicated that (1) PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5 are closely related variants differing by a few amino acid substitutions; (2) the qualitative titration curve of CARB-4 is different from those of PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5, although their patterns are somewhat similar; and (3) PSE-2 has no structural relationship with any of the other carbenicillin-hydrolysing enzymes or carbenicillinases (CARB) studied . Electrophoretic methods, and in particular titration curve determination combined with other physicochemical and enzymatic data, allowed a rapid comparison of the molecular structures of the beta-lactamases, and hence their classification.

J Bacteriol, 1989 Oct, 171(10), 5452 - 7
The translation start signal region of TEM beta-lactamase mRNA is responsible for heat shock-induced repression of amp gene expression in Escherichia coli; Kuriki Y; pBR322 contains the amp gene encoding beta-lactamase . When Escherichia coli carrying this plasmid is exposed to heat shock, beta-lactamase synthesis is repressed transiently at the translational level . To identify the DNA element responsible for this translational repression, DNA segments containing the translation start region of the amp gene were excised from pAT153 and fused in frame with the lacZ reading frame in the open reading frame vector pORF1 . These constructs were introduced into E . coli, and the effect of heat shock of the cells on the synthesis of beta-galactosidase starting from the amp start codon was examined . As is the case for pBR322-encoded synthesis of beta-lactamase, the synthesis of beta-galactosidase encoded by the fused genes also ceased transiently upon heat shock . It is concluded that the heat shock-induced repression of the amp gene occurs at the initiation step of translation . As far as the present study is concerned, the minimum DNA segment responsible for the repression is AT TGA AAA AGG AAG AGT ATG AG, which includes the Shine-Dalgarno sequence (AAGGA) and the initiation codon (ATG).

Biochem J, 1989 Sep 15, 262(3), 849 - 54
Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase of Actinomadura R39; Piron-Fraipont C et al.; By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans . The same 100-200-copy-number plasmid was used to clone in S . lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase . Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).

J Antimicrob Chemother, 1989 Sep, 24 Suppl A, 219 - 23
Interactions of meropenem, with beta-lactamases, including enzymes with extended-spectrum activity against third-generation cephalosporins; Labia R et al.; The interactions of a meropenem were studied with a set of beta-lactamases including the new TEM- and SHV-related plasmid-mediated enzymes that have extended-spectrum activity against third-generation cephalosporins ('cefotaximases' and 'ceftazidimases') . Meropenem and imipenem were highly resistant to the hydrolytic activity of all the TEM and SHV related beta-lactamases, and to the OXA enzymes, as were the cephamycins: cefoxitin and cefotetan . The two carbapenems were also highly stable to Class C beta-lactamases (chromosomal cephalosporinases) whereas third-generation cephalosporins and cephamycins were slowly hydrolyzed . Both carbapenems demonstrated quite similar affinities for all the enzymes studied . In some instances, and particularly with Class A (TEM- and SHV-derived) enzymes, meropenem inactivated the beta-lactamase activity . Imipenem appeared less reactive in this respect.

Plasmid, 1989 Sep, 22(2), 163 - 8
Effect of induction of SOS response on expression of pBR322 genes and on plasmid copy number; Bertrand-Burggraf E et al.; Several lines of evidence are presented that indicate that the level of tetracycline resistance of Esherichia coli strains harboring plasmid pBR322 varies according to whether the SOS system of the host bacteria has been induced . These include use of strains in which the SOS system is expressed constitutively (lexA def.), is thermoinducible (recA441) or noninducible (lexA ind-), or is highly repressed (multiple copies of lexA+) . Similar induction was observed with the product of another plasmid gene, beta-lactamase . The amounts of extractable plasmid DNA were also increased by SOS induction, and we propose that the SOS-induced increases in levels of tetracycline resistance and beta-lactamase activity are due to an increased plasmid copy number.

Mol Microbiol, 1989 Aug, 3(8), 1131 - 40
Translational repression of TEM beta-lactamase synthesis as a response of Escherichia coli to heat shock; Kuriki Y; As a result of a temperature shift-up from 30 degrees C to 42 degrees C, beta-lactamase synthesis in Escherichia coli carrying pBR322 ceased transiently, even though the level of beta-lactamase mRNA was not altered . pBR328-directed pre-beta-lactamase synthesis in an in vitro transcription-translation coupled system was also repressed by incubation at the higher temperature . Translation of the lacZ sequence from the amp translation start signal, inserted into the open reading frame vector pORF1, was also repressed transiently upon the temperature shift-up . Pre-heating of the in vitro coupled system at 45 degrees C specifically reduced its capacity for pre-beta-lactamase synthesis . This capacity was restored by the addition of a 160,000 x g supernatant prepared from E . coli grown at 30 degrees C, but not by the supernatant from the cells incubated at 42 degrees C . These and other results indicate that (i) the 160,000 x g supernatant contains a heat-labile protein(s) that is required for efficient initiation of the translation of pre-beta-lactamase mRNA, and (ii) the heat shock-induced repression of beta-lactamase synthesis is due to inactivation of the protein(s) in the 160,000 x g supernatant.

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