Penicillinase activity in three strains ofClostridium butyricum

Three strains ofClostridium butyricum exhibited elevated minimal inhibitory concentrations (MICs) to penicillin (64–1,024 g/ml), ampicillin (32–256 g/ml), carbenicillin (128–1,024 g/ml), and oxacillin (32–64 g/ml). Cephalosporin/cephamycin agents were more active than penicillin drugs. All isolates were found to possess a -lactamase. The -lactamases were primarily cell associated during the logarithmic phase of growth. Stationary-phase cells released most of the enzyme into the culture medium. Cephalothin supplementation of broth cultures with concentrations equivalent to one-eighth of the MIC significantly increased the quantity of -lactamase synthesized. The -lactamases produced by these three isolates exhibited greatest activity with penicillin followed by ampicillin>cephaloridine>carbenicillin and oxacillin. No enzymatic activity was observed using cephalothin, cephalexin, cefazolin, cefoxitin, or cephamandole substrates. The -lactamases were inhibited by clavulanic acid and para-chloromurcuribenzoate and not inhibited by cloxacillin. Each enzyme exhibited an isoelectric point of 4.2.     

Characterization ofCardiobacterium hominis: Antibiotic susceptibility,morphological variations,plasmid and membrane analysis

Trypticase soy broth and Mueller Hinton broth lacking yeast extract resulted in morphological alterations (pleomorphism) ofCardiobacterium hominis, as demonstrated by light and transmission electron microscopy. Outer membrane protein profiles were influenced by the composition of growth media, especially in the region of porin proteins. In contrast, the protein composition of inner membranes was not influenced by media composition.C. hominis isolates tested were susceptible to 16 -lactam antibiotics. All isolates exhibited minimal inhibitory concentrations of 1 µg/ml to 13 of the 16 -lactams tested. The changes in outer membrane profiles induced by media composition did not influence susceptibility to -lactam antibiotics. None of the isolates examined contained detectable plasmids, -lactamase, or resistant mutants. Characteristic -lactam-induced morphological defects were observed.     

The purification of TEM-β-lactamase from cell extract using immobilized copper affinity chromatography

Summary TEM--lactamase was purified by immobilized metal affinity chromatography from E. coli cell extracts. It was hypothesized that this protein could be purified in one step due to the abundence of metal binding residues. A pH gradient eluted the cell extract proteins. EDTA treatment released TEM--lactamase. This procedure is much simpler than the current methods employed for TEM--lactamase purification.     

Elimination of the disulphide bond alters the conformation of mature lipo-β-lactamase in yeast

Summary When expressed in Saccharomyces cerevisiae the precursor of the hybrid prokaryotic protein lipo--lactamase is accumulated in reduced form whereas the majority of the mature form contains an intra molecular disulphide bond (oxidized form). We have previously shown that mature mutant lipo--lactamases in which the cysteine residue 131 was changed to either tyrosine or threonine lack the capacity to form a disulphide bond but are nevertheless processed and secreted efficiently in yeast. Here we show that these mutant mature lipo--lactamases, in yeast cell extracts, exhibit a tenfold lower -lactamase-specific activity than the wild-type protein and that the mature mutant proteins are more susceptible to trypsin digestion. Thus, elimination of the disulphide bond alters the conformation of mature lipo--lactamase in yeast.Correspondence to: O. Pines     

Physicochemical characterization of β-crystallins from bovine lenses: Hydrodynamic and aggregation properties

A detailed investigation of the hydrodynamic and aggregation behaviors has been made on the -crystallins of bovine lens. Results from this study indicated that H (high-molecular-weight -crystallin) and L (low-molecular-weight -crystallin) exhibited considerable heterogeneity in their native structures and subunit polypeptides. Low-speed sedimentation equilibrium showed a heterogeneous paucidisperse system in each -crystallin fraction. Viscosity and circular dichroism studies pointed to a compact and globular shape and the presence of -sheet and -turns in these crystallins. Dissociation of H by urea and guanidinium HCl followed by reassociation during gel-filtration chromatography produced an elution pattern with two fractions corresponding to L crystallin and high-molecular-weight aggregates without the formation of native H. By contrast, under similar treatment, about 60% L reassociated into the correct native structure and the rest into high-molecular-weight fractions. Amino acid analyses of H and L and their corresponding subunit polypeptides demonstrated the close similarity of these crystallins. Trace element analyses indicated that both Ca and Mg are present in H and L crystallins and may be involved in maintaining the native quarternary structures of these proteins.     

Factors affecting the synthesis and distribution ofβ-lactamase inMycobacterium smegmatis SN2 cultures

A high level of extracellular -lactamase activity was detected in cultures ofMycobacterium smegmatis SN2. The extracellular distribution of the enzyme varied with growth conditions such as additional carbon source and pH of the medium. Addition of chloramphenicol tothe culture inhibited the increase in the extracellular -lactamase activity. Cell wall damage or autolysis may be responsible for the extracellular -lactamase activity.     

Degradation of β-lactam antibiotics by polyacrylamide-entrapped β-lactamase-producingE. coli cells

Summary Cells of a -lactamase producingE. coli strain were immobilized with acrylamide and lyophilized. The gel particles containing the entrapped cells were used like an immobilized enzyme to study the inactivation of -lactam antibiotics. The substrate profile of the -lactamase was determined and the action of -lactamase inhibitors studied.     

Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

β-Lactamases from Actinopolyspora halophila,an extremely halophilic actinomycete

Actinopolyspora halophila, an extremely halophilic actinomycete, produced both cellular and exocellular -lactamases when grown in liquid media containing 20% (w/v) NaCl. Maximal exocellular -lactamase production occurred at 48 h growth and exceeded cellular enzyme levels 7-fold. Maximal cellular -lactamase was observed at 72 h as cultures achieved full growth.Both -lactamases were purified to molecular homogeneity by a sequence involving gel filtration on Bio-Gel P-100, DEAE Bio-Gel A chromatography, preparative isoelectric focusing, and gel filtration on Sephadex G-75. Cellular -lactamase was purified 99-fold with 12% recovery and had a molecular weight of 42,200, and an isoelectric point of 4.15. Exocellular -lactamase was 12-fold purified with 1.2% recovery of initial activity and had a molecular weight of 38,000 and an isoelectric point of 3.85. Its specific activity was 7-fold greater than that of the cellular enzyme.A variety of penicillin and cephalosporin substrates were degraded by both enzymes. While the cellular -lactamase degraded phenoxymethylpenicillin, methicillin, and cephaloglycin most efficiently, the exocellular enzyme was most active against methicillin, carbenicillin, ampicillin, and cephalosporin C. Both enzymes were stimulated and protected from thermal deactivation by NaCl but not KCl or MgCl₂-Neither enzyme was inhibited by iodine.Abbreviations IEF Isoelectric focusing - SDS-PAGE sodium dodecylsulphate-polyacrylamide electrophoresis - PEG polyethyleneglycol - DTT dithiothreitol - DEAE diethylaminoethyl Issued as NRCC 25164     

Extracellular β-lactamase from Escherichia coli

Summary Escherichia coli strain O 127: K63: (B8): H—was grown in nutrient broth (Difco). Penicillinase activity was found in the culture supernatant after only five hours of incubation, i.e. during the exponential phase of growth. At this phase the levels of typical intracellular markers, did not indicate cell lysis or gross cell damage. The bioautographic revelation of penicillin-splitting enzymes on electropherograms of cell-free liquids confirmed the presence of one basic broad-spectrum -lactamase. This possibly extracellular -lactamase seems to be also present in the cellular extracts where it coexists with several other cell-bound penicillinases.     

Production of glucosamine containing disaccharides of the Lewis-A and -X types employing glycosidases

Disaccharide derivatives of interest for inhibition studies and for synthesis of the blood group determinants Lewis-a and Lewis-x were obtained with glycosidases as catalysts. Thus, Fuc(1–4)(6-OBn)GlcNH₂SEt and Gal1–3(6-OBn)GlcNH₂-SEt were produced employing (6-OBn)GlcNH₂SEt as acceptor and -L-fucosidase and -D-galactosidase, respectively, as catalysts. The phthalimido derivative of lactosamine, Gal1-4GlcNPhthSEt, was prepared from lactose employing GlcNPhthSEt as the acceptor and a yeast -galactosidase as catalyst. The reactions were both regio- and stereospecific, which allowed straightforward production of pure products on a g scale and higher.     

The degradation of amyloid beta as a therapeutic strategy in Alzheimer's disease and cerebrovascular amyloidoses

The deposition of 4-kDa amyloid peptide in the brain is a prominent feature of several human diseases. Such process is heterogeneous in terms of causative factors, biochemical phenotype, localization and clinical manifestations. Amyloid accumulates in the neuropil or within the walls of cerebral vessels, and associates with dementia or stroke, both hereditary and sporadic. Amyloid is normally released by cells as soluble monomeric-dimeric species yet, under pathological conditions, it self-aggregates as soluble oligomers or insoluble fibrils that may be toxic to neurons and vascular cells. Lowering amyloid levels may be achieved by inhibiting its generation from the amyloid -precursor protein or by promoting its clearence by transport or degradation. We will summarize recent findings on brain proteases capable of degrading amyloid with a special focus on those enzymes for which there is genetic, transgenic or biochemical evidence suggesting that they may participate in the proteolysis of amyloid in vivo. We will also put in perspective their possible utilization as therapeutic agents in amyloid diseases.     

Isolation of transposon Tn5 mutant affected in the metabolism of 18β-glycyrrhetinic acid

Transposon Tn5 mutagenesis was carried out to clarify the metabolism of 18-glycyrrhetinic acid (18-GRA) in Sphingomonas paucimobilis G5. A Tn5-induced mutant strain, named TM9638 that was affected in its metabolism of 18-GRA, was isolated. This mutant accumulated three metabolites, designated as M-A, M-B and M-C, from 18-GRA in the culture broth. M-A was accumulated in the culture broth of wild type strain, but M-B and M-C were not accumulated in the culture broth of wild type strain. M-B and M-C were isolated from the culture broth of TM9638 and the chemical structures were elucidated by NMR and GC-MS.     

Factors influencing synthesis and activity of β-galactosidase inLactobacillus acidophilus

Summary In the type-strainLactobacillus acidophilus ATCC 4356 -galactosidase (-gal) was inducible; lactose, galactose, melibiose and probably maltose, but not glucose, fructose, mannose, sucrose and cellobiose, induced -gal synthesis. Glucose partially inhibited -gal-induction by lactose but not by isopropyl--D-thiogalactoside. -gal synthesis during cell growth was maximal at 0.4% lactose, stimulated by Ca²⁺ but inhibited by Mg²⁺ and Mn²⁺. -gal in the cell-free extract had optimum activity at pH 6.5 and at 45°C. The enzyme activity was stimulated by Mg²⁺, inhibited by Ca²⁺, destroyed by oxidizing agents and protected by reducing agents.     

Xylan-hydrolysing enzymes of the yeast Pichia stipitis

Summary Two xylanolytic enzymes, xylanase and -xylosidase from the yeast Pichia stipitis were purified to homogeneity and characterized. Both enzymes are secreted into the culture medium upon growth on xylan. The xylanase is a glycoprotein with an approximate molecular mass of 43 kDa. The N-linked carbohydrate content was estimated to be 26% by endoglycosidase H digestion. The -xylosidase protein has a molecular mass of 37 kDa as determined by sodium dodecyl sulphate gel electrophoresis. Synthesis of xylanase was found to be inducible by xylan and repressible by xylose and glucose. By contrast, -xylosidase is synthesized constitutively to a considerable degree. The purified -xylosidase is able to hydrolyse aryl--D-glucosides with an even higher rate than -xylosides. Thus, this enzyme may not be a specific component of the xylan-degrading system of P. stipitis. Offprint requests to: M. Ciriary     

Conformational transmission in ATP synthase during catalysis: Search for large structural changes

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Defective mutation at Gly-149 was suppressed by the second mutations at the outer surface of the subunit, indicating that the defect by the first mutation was suppressed by the second mutation through long range conformation transmission. Extensive mutant/pseudorevertant studies revealed that / and / subunits interactions are important for the energy coupling between catalysis and H⁺ translocation. In addition, long range interaction between amino and carboxyl terminal regions of the subunit has a critical role(s) for energy coupling. These results suggest that the dynamic conformation change and its transmission are essential for ATP synthase.     

β-Alanine metabolism and high salinity stress in the sea anemone,Bunodosoma cavernata

Summary During high salinity stress, -alanine accumulates to high levels in the sea anemone,Bunodosoma cavernata. Following a salinity increase from 26 to 40 -alanine increased 28-fold from 1.5 to 41.9 moles/g dry weight. Both whole animal studies and experiments with cell free homogenates indicate that under high salinity conditions an increase in the rate of -alanine synthesis from aspartic acid as well as a decrease in the rate of -alanine oxidation are responsible for the observed accumulation of -alanine. The rate of aspartic acid decarboxylation to -alanine is about 3 times greater in anemones acclimated to 40 than for those in normal salinity water (26). -alanine oxidation to CO₂ and acetyl-CoA proceeds 2.5 to 3 times slower in high salinity adaptedB. cavernata than in those acclimated to normal salinity. There is always a rapid degradation of uracil to -alanine, but this does not change with salinity.Abbreviations CASF cold acid soluble fraction - FAA free amino acids - MES 2(N-morpholino) ethane sulfonic acid - NPS ninhydrin positive substances - PCA perchloric acid - TCA trichloroacetic acid     

Metal resistance in Acinetobacter and its relation to β-lactamase production

Thirty nine clinical isolates of Acinetobacter belonging to six species were tested for resistance to 20 metal ions and their ability to produce -lactamase. Fifty two percent of the strains produced -lactamase. -Lactamase producers and non-producers were almost equally distributed in the different species. A. baumannii was the predominant biotype and was found to be most resistant to metals. Resistance to mercury was prevalent in -lactamase-producing A. baumannii only. Silver resistant strains of A. baumannii produced -lactamase. Sensitivity and resistance to copper and cadium was equally distributed between -lactamase producers and non-producers. -Lactamase-producer and -non-producer strains were uniformly sensitive to cadmium except Acinetobacter genospecies 1.     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine