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 b