National validation study of a swab protocol for the recovery of Bacillus anthracis spores from surfaces

Twelve Laboratory Response Network (LRN) affiliated laboratories participated in a validation study of a macrofoam swab protocol for the recovery, detection, and quantification of viable B. anthracis (BA) Sterne spores from steel surfaces. CDC personnel inoculated steel coupons (26 cm²) with 1-4 log₁₀ BA spores and recovered them by sampling with pre-moistened macrofoam swabs. Phase 1 (P1) of the study evaluated swabs containing BA only, while dust and background organisms were added to swabs in Phase 2 (P2) to mimic environmental conditions. Laboratories processed swabs and enumerated spores by culturing eluted swab suspensions and counting colonies with morphology consistent with BA. Processed swabs were placed in enrichment broth, incubated 24 h, and cultured by streaking for isolation. Real-time PCR was performed on selected colonies from P2 samples to confirm the identity of BA. Mean percent recovery (%R) of spores from the surface ranged from 15.8 to 31.0% (P1) and from 27.9 to 55.0% (P2). The highest mean percent recovery was 31.0% (sd 10.9%) for P1 (4 log₁₀ inoculum) and 55.0% (sd 27.6%) for P2 (1 log₁₀ inoculum). The overall %R was higher for P2 (44.6%) than P1 (24.1%), but the overall reproducibility (between-lab variability) was lower in P2 than in P1 (25.0 vs 16.5%CV, respectively). The overall precision (within-lab variability) was close to identical for P1 and P2 (44.0 and 44.1, respectively), but varied greatly between inoculum levels. The protocol demonstrated linearity in %R over the three inoculum levels and is able to detect between 26 and 5 × 10⁶ spores/26 cm². Sensitivity as determined by culture was > 98.3% for both phases and all inocula, suggesting that the culture method maintains sensitivity in the presence of contaminants. The enrichment broth method alone was less sensitive for sampled swabs (66.4%) during P2, suggesting that the presence of background organisms inhibited growth or isolation of BA from the broth. The addition of real-time PCR testing to the assay increased specificity from > 85.4% to > 95.0% in P2. Although the precision was low at the 1 log₁₀ inoculum level in both phases (59.0 and 50.2%), this swab processing protocol, was sensitive, specific, precise, and reproducible at 2-4 log₁₀/26 cm² spore concentrations.     

Synthesis and characterization of silver(I) derivatives containing acylpyrazolonate and phosphino ligands: X-ray crystal structures of monomeric [Ag(Q)(PPh3)2] and of dimeric [{Ag(Q)(PiBu3)}2] (Q = 1-phenyl-3-methyl-4-tert-butylacetylpyrazolon-5-ato)

New silver(I) acylpyrazolonate derivatives [Ag(Q)], [Ag(Q)(PR₃)]₂ and [Ag(Q)(PR₃)₂] (HQ = 1-R¹-3-methyl-4-R²(CO)pyrazol-5-one, HQ^Bn = R¹ = C₆H₅, R² = CH₂C₆H₅; HQ^CHPh2 = R¹ = C₆H₅, R² = CH(C₆H₅)₂; HQ^nPe = R¹ = C₆H₅, R² = CH₂C(CH₃)₃; HQ^tBu = R¹ = C₆H₅, R² = C(CH₃)₃; HQ^fMe = R¹ = C₆H₄-p-CF₃, R² = CF₃; HQ^fEt = R¹ = C₆H₅, R² = CF₂CF₃; R = Ph or iBu) have been synthesized and characterized in the solid state and solution. The crystal structure of 1-(4-trifluoromethylphenyl)-3-methyl-5-pyrazolone, the precursor of proligand HQ^fMe and of derivatives [Ag(Q^nPe)(PPh₃)₂] and [Ag(Q^nPe)(PiBu₃)]₂ have been investigated. [Ag(Q^nPe)(PPh₃)₂] is a mononuclear compound with a silver atom in a tetrahedrally distorted AgO₂P₂ environment, whereas [Ag(Q^nPe)(PiBu₃)]₂ is a dinuclear compound with two O₂N-exotridentate bridging acylpyrazolonate ligands connecting both silver atoms, their coordination environment being completed by a phosphine ligand.     

A microtiter plate assay for the selection of 6-thioguanine-resistant mutants in Chinese hamster V79 cells in the presence of phorbol-12-myristate-13-acetate

6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (10³ – 10⁴ cells/0.2 ml growth medium/0.4 cm²) when selected with 30 μM 6-thioguanine and 0.1 μg/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells. Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies. After treatment of the V79 cells with the carcinogen benzo[a]pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes. The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure. The active carcinogen benzo[a]pyrene at 1 μg/ml yielded about 100 mutants per 10⁵ colony-forming cells. The same dose of a less active carcinogen, cyclopenta-[c,d]pyrene, yielded about 20 mutants per 10⁵ colony-forming cells, and benz[a]anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested. Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells.     

Influence of carbon source and inoculum type on anaerobic biomass adhesion on polyurethane foam in reactors fed with acid mine drainage

This paper analyzes the influence of carbon source and inoculum origin on the dynamics of biomass adhesion to an inert support in anaerobic reactors fed with acid mine drainage. Formic acid, lactic acid and ethanol were used as carbon sources. Two different inocula were evaluated: one taken from an UASB reactor and other from the sediment of a uranium mine. The values of average colonization rates and the maximum biomass concentration (C_max) were inversely proportional to the number of carbon atoms in each substrate. The highest C_max value (0.35 g TVS g⁻¹ foam) was observed with formic acid and anaerobic sludge as inoculum. Maximum colonization rates (v_max) were strongly influenced by the type of inoculum when ethanol and lactic acid were used. For both carbon sources, the use of mine sediment as inoculum resulted in a v_max of 0.013 g TVS g⁻¹ foam day⁻¹, whereas 0.024 g TVS g⁻¹ foam day⁻¹ was achieved with anaerobic sludge.     

Palladium(II) and platinum(II) monoselenocarboxylates: Synthesis, spectroscopy, structure and their use as molecular precursors for metal selenides

Treatment of MCl₂(P^∩P) or MCl₂(PⁿPr₃)₂ with two equivalents of ArCOSeK readily yields cis-[M(SeCOAr)₂(P^∩P)] and trans-[M(SeCOAr)₂(PⁿPr₃)₂], respectively (M = Pd or Pt; Ar = Ph or 4-MeC₆H₄; P^∩P = dppm, dppe, dppp). The reaction of Pd(SeCOAr)₂(dppe) with PdCl₂(dppe) in the presence of NaBPh₄ in methanol gave a tri-nuclear ionic complex, [Pd₃(μ₃-Se)₂(dppe)₃][BPh₄]₂. These complexes were characterized by UV-Vis, IR and NMR spectroscopy. The complex [Pt(SeCOPh)₂(dppp)] has been structurally characterized by X-ray crystallography. The coordination environment around square planar platinum atom is defined by chelating dppp ligand and two unidentate selenocarboxylates bonded through selenium atoms. Pyrolysis of [Pd(SeCOAr)₂(PⁿPr₃)₂] either in tri-n-butylphosphate (TBP) (at 200 °C) or in the solid state (furnace heating at 350 °C) gave Pd₁₇Se₁₅.     

The Study of Nanosized Silicate-Substituted Hydroxyapatites Co-Doped with Sr2+ and Zn2+ Ions Related to Their Influence on Biological Activities

Nanosized silicate-substituted hydroxyapatites, characterized by the general formula Ca_9.8−x−nSr_nZn_x(PO₄)_6−y(SiO₄)_y(OH)₂ (where: n = 0.2 [mol%]; x = 0.5–3.5 [mol%]; y = 4–5 [mol%]), co-doped with Zn²⁺ and Sr²⁺ ions, were synthesized with the help of a microwave-assisted hydrothermal technique. The structural properties were determined using XRD (X-ray powder diffraction) and Fourier-transformed infrared spectroscopy (FT-IR). The morphology, size and shape of biomaterials were detected using scanning electron microscopy techniques (SEM). The reference strains of Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa were used to assess bacterial survivability and the impact on biofilm formation in the presence of nanosilicate-substituted strontium-hydroxyapatites. Safety evaluation was also performed using the standard cytotoxicity test (MTT) and hemolysis assay. Moreover, the mutagenic potential of the materials was assessed (Ames test). The obtained results suggest the dose-dependent antibacterial activity of nanomaterials, especially observed for samples doped with 3.5 mol% Zn²⁺ ions. Moreover, the modification with five SiO₄ groups enhanced the antibacterial effect; however, a rise in the toxicity was observed as well. No harmful activity was detected in the hemolysis assay as well as in the mutagenic assay (Ames test).     

Caffeine, pentoxifylline and theophylline form stacking complexes with IQ-type heterocyclic aromatic amines

Methylxanthines (MTX), in particular caffeine (CAF), are known as the most widely consumed alkaloids worldwide. Many accumulated statistical data indicate the protective effect of CAF intake against several types of cancer. One of the possible explanations of this phenomenon is direct non-covalent interaction between CAF and aromatic mutagen/carcinogen molecules through stacking (π-π) complexes formation. Here we demonstrate that CAF and other MTX, pentoxifylline (PTX) and theophylline (TH), form stacking complexes with carcinogenic imidazoquinoline-type (IQ-type) food-borne heterocyclic aromatic amines (HCAs). We estimated neighborhood association constants (K_AC of the order of magnitude of 10² M⁻¹) in neutral and acidic environment and enthalpy changes (ΔH values between −15.1 and −39.8 kJ/mol) for these interactions using UV-Vis spectroscopy, calculations based on thermodynamical model of mixed aggregation and titration microcalorimetry. Moreover, using Ames test with Salmonella typhimurium TA98 strain and recently developed mutagenicity assay based on bioluminescence of Vibrio harveyi A16 strain, we demonstrated a statistically significant reduction in HCAs mutagenic activity in the presence of MTX.     

Mono, di and polynuclear Cu(II)-azido complexes incorporating N,N,N reduced schiff base: syntheses, structure and magnetic behavior

Three kinds of copper(II) azide complexes have been synthesised in excellent yields by reacting Cu(ClO₄)₂ · 6H₂O with N,N-bis(2-pyridylmethyl)amine (L¹); N-(2-pyridylmethyl)-N′,N′-dimethylethylenediamine (L²); and N-(2-pyridylmethyl)-N′,N′-diethylethylenediamine (L³), respectively, in the presence of slight excess of sodium azide. They are the monomeric Cu(L¹)(N₃)(ClO₄) (1), the end-to-end diazido-bridged Cu₂(L²)₂(μ-1,3-N₃)₂(ClO₄)₂ (2) and the single azido-bridged (μ-1,3-) 1D chain [Cu(L³)(μ-1,3-N₃)]_n(ClO₄)_n (3). The crystal and molecular structures of these complexes have been solved. The variable temperature magnetic moments of type 2 and type 3 complexes were studied. Temperature dependent susceptibility for 2 was fitted using the Bleaney-Bowers expression which led to the parameters J = −3.43 cm⁻¹ and R = 1 × 10⁻⁵. The magnetic data for 3 were fitted to Baker’s expression for S = 1/2 and the parameters obtained were J = 1.6 cm⁻¹ and R = 3.2 × 10⁻⁴. Crystal data are as follows. Cu(L¹)(N₃)(ClO₄): Chemical formula, C₁₂H₁₃ClN₆O₄Cu; crystal system, monoclinic; space group, P2₁/c; a = 8.788(12), b = 13.045(15), c = 14.213(15) Å; β = 102.960(10)°; Z = 4. Cu(L²)(μ-N₃)(ClO₄): Chemical formula, C₁₀H₁₇ClN₆O₄Cu: crystal system, monoclinic; space group, P2₁/c; a = 10.790(12), b = 8.568(9), c = 16.651(17) Å; β = 102.360(10)°; Z = 4. [Cu(L³)(μ-N₃)](ClO₄): Chemical formula, C₁₂H₂₁ClN₆O₄Cu; crystal system, monoclinic; space group, P2₁/c; a = 12.331(14), b = 7.804(9), c = 18.64(2) Å; β = 103.405(10)°; Z = 4.     

Mutagenic evaluation of carcinogens and non-carcinogens in the L5178Y/TK assay utilizing postmitochondrial fractions (S9) from normal rat liver

As part of a continuing effort to investigate various metabolic activation procedures in the L5178Y TK^+/− → TK^−/− mutation assay, a series of 18 chemicals including both carcinogens and non-carcinogens selected from 7 chemical classes were tested in the presence of 5% (v/v) 9000 × g postmitochondrial supernatant fraction (S9) prepared from the livers of untreated Sprague-Dawley rats. Excepting ethyl carbamate and thioacetamide, 8 of 10 carcinogens tested produced mutant counts significantly greater than controls when the results were analyzed by a 2-sample log_et-test. Of 8 non-carcinogens assayed, treatment with p-aminophenol and chloroacetic acid yielded mutant counts significantly greater than control levels in at least one test with S9 while styrene oxide, an Ames positive mutagen, produced mutagenic activity in the absence of S9. These results indicate the L5178Y TK^+/− → TK^−/− mutation assay coupled with 5% (v/v) liver homogenate from normal rodent liver and applied to 18 chemicals correctly distinguished the majority of carcinogens from structurally related non-carcinogens on the basis of chemically-induced gene mutations.     

Myometrial LH/hCG receptors during the estrous cycle and pregnancy in pigs

Receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) have been identified in porcine, rabbit, rat, and human myometrium. To determine the estrous cycle and pregnancy related changes in the receptor capacity and affinity, radioreceptor assays were performed with membrane homogenates of porcine uterine tissues. Cycling gilts were divided into four experimental groups: I (n=6), day 1–2; II (n=5), day 6–7; III (n=5), day 11–12; and IV (n=6), day 18–20 of the estrous cycle. Pregnant pigs were divided into three experimental groups: I (n=5), day 35–40; II (n=5), day 65–70; and III (n=4), day 95–105 of pregnancy. The concentrations [femtomoles/mg protein (fmol/mg protein)] and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. Receptor concentrations were highest (P<0.01) in groups II and III (19.3±2.5 and 35.8±2.1 fmol/mg protein, respectively), and was lowest in groups I and IV (5.3±1.4 and 7.5±0.7 fmol/mg protein, respectively). Receptor affinity constants (K_a) were consistent (P>0.05) throughout the estrous cycle [I, (5.1±1.5)×10⁹; II, (3.0±0.8)×10⁹; III, (3.2±0.9)×10⁹; IV, 5.5±0.7×10⁹ lm⁻¹]. Plasma hormone concentrations of progesterone, estrogen and LH were typical of values noted at these times. During pregnancy, receptor concentrations were greatest (P<0.05) in group II (85.4±18.5 fmol/mg protein). In groups I and III receptor numbers were 10.8±2.3 and 26.7±6.6 fmol/mg protein, respectively. The K_a in group I was 10 times greater (P<0.05) than K_a in groups II and III, (I, 3.1±0.9×10¹⁰ lm⁻¹; II, 3.4±0.3×10⁹ lm⁻¹; III, 3.3±1.1×10⁹ lm⁻¹). Plasma hormone concentrations typically found during pregnancy were noted. The function of these LH/hCG binding sites remains unknown; however, changes in receptor capacity during the estrous cycle and pregnancy support a role for modulation of the receptor by hormonal factors.     

Effects of tunnel structures of two termite species on territorial competition and territory size

The foraging territories of 2 subterranean termites, Coptotermes formosanus Shiraki and Reticulitermes flavipes (Kollar), were simulated using a model to explore how territorial intraspecific competition changes with 4 variables characterizing the formation of territory: the number of primary tunnels, N₀; the branching probability, P_branch; the number of territories, N; and the blocking probability, P_block. The blocking probability P_block quantitatively describes the probability that a tunnel will be terminated when another tunnel is encountered; higher P_block values indicate more likely termination. Higher tunnel-tunnel encounters led to denser tunnel networks. We defined a territory as a convex polygon containing a tunnel pattern and explored the effects of competition among termite colonies on territory size distribution at steady state attained after sufficient simulation time. At the beginning of the simulation, N = 10, 20,…, 100 initial territory seeds were randomly distributed within a square area. In our previous study, we introduced an interference coefficient γ to characterize territorial competition. Higher γ values imply higher limitations on network growth. We theoretically derived γ as a function of P_block and N. In this study, we considered the constants in γ as functions of N₀ and P_branch so as to quantitatively examine the effect of tunnel structure on territorial competition. By applying statistical regression to the simulation data, we determined the generalized γ functions for both species. Under competitive conditions, territory size is most strongly affected by N₀, while the outcome of territorial competition is most strongly affected by N, followed by P_block and N₀.     

Synthesis, characterizations, and crystal structures of triorganotin(IV) derivatives with areneseleninic acids

Six new triorganotin(IV) complexes, [R₃Sn(O₂SeC₆H₄Cl)]_n (R = Me 1; Ph 2), [R₃Sn(O₂SeC₆H₄Me)]_n (R = Me 3; Ph 4), [R₃Sn(O₂SeC₆H₄Bu)]_n (R = Me 5; Ph 6) have been synthesized by the reaction of 4-chlorobenzeneseleninic acid, p-Tolueneseleninic acid, and 4-tert-butylbenzeneseleninic acid with triorganotin(IV) chloride in the presence of sodium ethoxide. All of the complexes were characterized by elemental analysis, FT-IR, NMR (¹H, ¹³C, and ¹¹⁹Sn) spectroscopy, and X-ray crystallography. Crystal structures show that all of the complexes exhibit 1D infinite chain structures which are generated by the bidentate oxygen atoms and the five-coordinated tin centers.     

Activity of a “thermostable exotoxin” of Bacillus thuringiensis subsp. morrisoni in the Salmonella/microsomal assay for bacterial mutagenicity

The purpose of this study was to employ the Salmonella/microsomal assay (Ames test) to investigate the mutagenic potential of a thermostable exotoxin of Bacillus thuringiensis subsp. morrisoni. Bacteria are ideal for the detection of infrequently occurring point mutations because the large number of organisms (200 to 400 million bacteria per plate) exposed to the mutagen at any one time increases the possibility of observing a random mutational event. The exotoxin used in this study was produced using the shaker flask fermentation procedure with mineral casein broth. A Petri dish method of bioassay using fresh bovine feces was used to determine the efficacy of the exotoxin against horn flies. The LD₅₀ was found to be 5.35 μl/g of feces. Five bacterial tester strains were identified and characterized for the genetic markers described by Ames et al. (B. N. Ames et al., 1975, Mutat. Res., 31, 347–364). Appropriate doses of the B. thuringiensis supernatant, solvent or positive control were added to agar plates. The supernatant was tested at five dose levels against all five strains of bacteria. Controls of bacteria only were included for spontaneous reversions. All treatments were performed in triplicate. The numbers of revertant colonies from each set of triplicate plates were averaged and the standard deviation calculated and compared to that found with the solvent control. The negative controls, positive controls, and sterility controls all fulfilled requirements for determination of a valid test. No detectable mutagenic activity was found for the thermostable exotoxin of B. thuringiensis morrisoni.     

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells: I. Application to genetic toxicological testing

We have systematically evaluated the mouse lymphoma TK^+/- → TK^-/- mutagenesis assay to determine if this somatic-cell test system would be a useful addition to the routine screening battery already used in our laboratory for the detection of chemical mutagens. During these investigations we observe that, with certain modifications of the basic assay, mutagenicity data could be obtained in as little as 9 days once the relative cytotoxic properties of the test substance were known. By improving the culturing conditions, we were able to reduce the serum requirements by as much as 50–75% without appreciably altering either cell viability or the recovery of chemically-induced mutants. Phenotypic stability of test-derived trifluorothymidine resistant (TFT^R) mutants was confirmed by demonstrating cross-resistance to bromodeoxyuridine and concomitant sensitivity to methotrexate (THMG) in TFT^R cells grown for 20 generations under non-selective conditions. While reduced growth rates resulting from temporary cell-division delay in treated cells is probably not a contributing factor to the observed mutation frequencies, only TFT^R colonies which formed large distinct colonies in the presence of trifluorothymidine were clearly phenotypically stable mutants when spontaneous mutants were isolated and verified. When a non-mutagen, a weak mutagen, and a well-established mutagen were compared at equitoxic doses under these modified conditions, clear quantitative differences were seen in the respective mutation frequencies induced by these 3 types of agents. With these technical modifications, we feel this assay is both reliable and amenable to the screening of diverse chemical compounds for point-mutational activity in a mammalian cell.     

Growth Kinetics of Attached Iron-Oxidizing Bacteria

A model of growth and substrate utilization for ferrous-iron-oxidizing bacteria attached to the disks of a rotating biological contactor was developed and tested. The model describes attached bacterial growth as a saturation function in which the rate of substrate utilization is determined by a maximum substrate oxidation rate constant (P), a half-saturation constant (K_s), and the concentration of substrate within the rotating biological contactor (S₁). The maximum oxidation rate constant was proportional to flow rate, and the substrate concentration in the reactor varied with influent substrate concentration (S₀). The model allowed the prediction of metabolic constants and included terms for both constant and growth-rate-dependent maintenance energies. Estimates for metabolic constants of the attached population of acidophilic, chemolithotrophic, iron-oxidizing bacteria limited by ferrous iron were: maximum specific growth rate (μ_max), 1.14 h⁻¹; half-saturation constant (K_s) for ferrous iron, 54.9 mg/liter; constant maintenance energy coefficient (m₁), 0.154 h⁻¹; growth-rate-dependent maintenance energy coefficient (m′), 0.07 h⁻¹; maximum yield (Y_g), 0.063 mg of organic nitrogen per mg of Fe(II) oxidized.     

Chemical implantation of Group 4 cations on silica via cyclopentadienyl- and N,N-dialkylcarbamato derivatives

Chemical implantation of Group 4 cations [Ti(III), Ti(IV), Zr(IV), Hf(IV)] has been carried out under mild conditions by the reaction of polycyclopentadienyl- (MCp_n; M = Ti, n = 3, 4; M = Zr, Hf, n = 4), mixed cyclopentadienyl/N,N-dialkylcarbamato (ML_x(O₂CNEt₂)_y; M = Ti, L = Cp, C₅Me₅ (Cp*), x = 2, y = 1; M = Hf, L = Cp, x = 1, y = 3), and N,N-dialkylcarbamato (M(O₂CNR₂)_n, M = Ti, n = 3, R = ⁱPr; M = Ti, Hf, n = 4, R = Et; M = Zr, n = 4, R = ⁱPr) derivatives, with the silanol groups of amorphous silica. Cyclopentadiene/pentamethylcyclopentadiene and/or carbon dioxide and the secondary amine are released in the process. The amount of implanted cations depends on the metal and on the ligands, the pentamethylcyclopentadienyl complex being less reactive than the unsubstituted congener. The starting complexes and the final products have been characterized by EPR or by ¹³C CP-MAS NMR spectroscopy.     

Racemic twin crystals containing left- and right-handed polyoxometalate chains induced by the asymmetric coordination of metal-organic units

Two racemic twin crystals containing left- and right-handed polyoxometalates (POMs) chains [Cu(en)₂][Cu(en)₂(H₂O)]₂ [SiW₁₁CuO₃₉] · 5H₂O (D-1 and L-1; en = ethylenediamine) have been synthesized and structurally characterized. Both chiral compounds (D-1 and L-1) were constructed from the achiral building blocks [SiW₁₁CuO₃₉]⁶⁻ and [Cu(en)₂(H₂O)_n]²⁺ (n = 0 or 1). The structural chirality is induced by the [Cu(en)₂(H₂O)_n]²⁺ units asymmetrically coordinated on the chainlike [SiW₁₁CuO₃₉]_n⁶ⁿ⁻ polyoxoanions, leading to the whole molecules crystallized in the chiral P1 space group. The chiroptical activities of D-1 and L-1 were confirmed by the solid-state circular dichroism spectroscopy. Their electrochemical and electrocatalytic properties were also investigated.     

Influence of the surface speciation on biofilm attachment to chalcopyrite by Acidithiobacillus thiooxidans

Surfaces of massive chalcopyrite (CuFeS₂) electrodes were modified by applying variable oxidation potential pulses under growth media in order to induce the formation of different secondary phases (e.g., copper-rich polysulfides, S _n ^2?; elemental sulfur, S⁰; and covellite, CuS). The evolution of reactivity (oxidation capacity) of the resulting chalcopyrite surfaces considers a transition from passive or inactive (containing CuS and S _n ^2?) to active (containing increasing amounts of S⁰) phases. Modified surfaces were incubated with cells of sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans) for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the density of cells attached to chalcopyrite surfaces, the structure of the formed biofilm, and their exopolysaccharides and nucleic acids were analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy coupled to dispersive X-ray analysis (SEM-EDS). Additionally, CuS and S _n ^2?/S⁰ speciation, as well as secondary phase evolution, was carried out on biooxidized and abiotic chalcopyrite surfaces using Raman spectroscopy and SEM-EDS. Our results indicate that oxidized chalcopyrite surfaces initially containing inactive S _n ^2? and S _n ^2?/CuS phases were less colonized by A. thiooxidans as compared with surfaces containing active phases (mainly S⁰). Furthermore, it was observed that cells were partially covered by CuS and S⁰ phases during biooxidation, especially at highly oxidized chalcopyrite surfaces, suggesting the innocuous effect of CuS phases during A. thiooxidans performance. These results may contribute to understanding the effect of the concomitant formation of refractory secondary phases (as CuS and inactive S _n ^2?) during the biooxidation of chalcopyrite by sulfur-oxidizing microorganisms in bioleaching systems.     

Isolation and properties of Escherichia coli ATPase mutants with altered divalent metal specificity for ATP hydrolysis

A method was devised for isolation of large numbers of energy-transducing ATPase (coupling factor) mutants based on a modification of the procedure of Hong and Ames (Hong, J. and Ames, B. N. (1971) Proc. Natl. Acad. Sci. U.S. 68, 3158–3162) for localized mutagenesis of any small region of the bacterial chromosome using transducing phages. The principle of this procedure is to mutate P1-transducing phage particles carrying the ATPase genes (Unc (uncoupled) DNA) using the strong chemical mutagen hydroxylamine. By transducing ilv^? auxotrophs, a marker closely linked to Unc, to prototrophs, mutated Unc DNA can be introduced into the chromosome. We have used this method in conjunction with suitable selection procedures to isolate about 90 Unc^? strains which have been classified by physiological, genetic, and biochemical criteria into three different phenotypes (Unc A, B, D). Mutants of the Unc D phenotype which were studied in detail were found to have the following properties: (1) aerobic growth yields on glucose are considerably lower than the wild type; growth occurs on glucose under anaerobic conditions; (2) Unc D lesions map near the ilv operon; (3) O₂ uptake is comparable to the rate of wild type; (4) vesicles catalyze respiratory-dependent transhydrogenation, but show very low levels of Ca²⁺ ATP-dependent transhydrogenation; Mg²⁺ is ineffective; (5) oxidative phosphorylation is almost completely blocked irrespective of which metal ion is used; (6) the specific activity of ATPase is only about 20% of the wild type; (7) purified ATPase was found to have a marked specificity for Ca²⁺ as a divalent metal for ATP hydrolysis. A summary of properties of the new Unc mutants is discussed.     

A case of mutagen specificity attributable to a plating medium effect

Summary In anadn ^– met ^– di-auxotrophic strain ofSchizosaccharomyces pombe met ⁺ reversions are several hundred times more frequent thanadn ⁺ reversions after treatment with ultra-violet light. They are only slightly more frequent thanadn ⁺ reversions when HNO₂ is used as a mutagen (mutagen specificity). The poor response of theadn-1,199 allele to the mutagenic action of U.V. can be largely overcome by replacing themet-4,D19 allele with its normalmet ⁺ allele (influence of the genetic background). It was shown that both the mutagen specificity and its dependence on the genetic background are due, largely at least, to the inhibition ofadn ⁺ reversions on a plating medium containingl-methionine. This inhibition is very strong for U.V.-induced reversions but only weak for HNO₂-induced ones. It would be wrong to assume that other mutants at theadenine-1 locus behave in the same manner.With 1 Figure in the Text