Catalytic activity of cytochrome oxidase and cytochrome c in apolar solvents containing phospholipids and low amounts of water

Cytochrome c and cytochrome oxidase, in bovine heart submitochondrial particles and in their purified forms, were transferred to a ternary system that contained phospholipids (10 mg/ml toluene), the apolar solvent toluene, and water at concentrations of 13-15 microliters (high water) and 3 microliters (low water) per milliliter of toluene. When the enzymes were transferred back to an all water system, they exhibited full catalytic capacity. In the low water ternary system, cytochrome c could be reduced by ascorbate introduced via inverted micelles. Also in this system, cytochrome oxidase was reduced by ascorbate and cytochrome c but its oxidation was highly impaired. Data on the kinetics of reduction by ascorbate of cytochrome c and cytochrome oxidase under these conditions are presented. Cytochrome oxidase reduced in the organic solvent by ascorbate failed to form a complex with CO, but formed a complex with cyanide introduced via inverted micelles. The oxidized and the ascorbate-reduced cytochrome oxidase-cyanide complex exhibited a trough at 415 nm and a peak at 433 nm. The extent and rate of formation of the cyanide complex were higher with the reduced form of cytochrome oxidase. To achieve protein-protein interactions (cytochrome c-cytochrome oxidase) in the ternary system, it was necessary to extract the two proteins together. There was no functional interaction when they were extracted separately and mixed. In the high water ternary system reduced cytochrome oxidase was not detected, and it oxidized ascorbate at a higher rate than in the low water system; however, this rate was several orders of magnitude lower than in aqueous media.     

The source of the heat resistance of bacterial spores: Study of water in spores by NMR

It has been postulated that the heat stabilization of the essential macromolecules in the core of the spore may be produced by dehydration at two levels: (i) the spore is drier at high relative humidity than the vegetative cell and (ii) the core of the spore may be less hydrated than the cortex and the coat. The latter postulate was subjected to experimental testing by ¹H-NMR studies of the water signal in the five species of spores and coat and (coat + cortex) preparations. The transverse relaxation rate $\text{(}\text{1}\text{T}{}_2\text{)}$ was determined in samples equilibrated at constant relative humidity. To allow for the effect of paramagnetic ions on $\text{1}\text{T}{}_2$ a model system (wool keratin) was studied in the presence of known amounts of Ca(II), Mn(II), Cu(II), Ni(II) and Fe(III). Because of the dominant effect of Mn(II) on $\text{1}\text{T}{}_2$, the effect of small amounts of other metal ions in spores was neglected. The relaxation rate of water at a particular relative humidity and manganese concentration was consistently less for intact spores than for coat or coat + cortex, hence the water in the core is more mobile than in the outer integuments. Sorption isotherm studies have shown that at a particular relative humidity there is about as much water in the core as in the cortex and coat. These two results taken together indicate that the hypothesis that the core is drier than the cortex and coat is incorrect, hence the spore is not heat-stabilized in this way. A theory is proposed in which heat stabilization is attributed to immobilization of essential enzymes and nuclei acids by a solid support, calcium dipicolinate, in a similar fashion to the heat stabilization of enzymes in a charged polymer matrix. It is proposed that stabilization is effected by electrostatic and hydrogen bonds between the calcium dipicolinate and the essential macromolecules. Experiments in progress show that enzymes and DNA are heat-stabilized in vitro by calcium dipicolinate.     

Germination-induced bioluminescence, a route to determine the inhibitory effect of a combination preservation treatment on bacterial spores

In this work, we have used spores of Bacillus subtilis that specifically induce bioluminescence upon initiation of germination as a rapid, real-time monitor of the effects of preservative treatments on germination. Using this tool, we have demonstrated that the combination of mild acidity (pH 5.5 to 5.0), lactic acid (0. 5%), and a pasteurization step (90 degrees C for 5 min) results in enhanced inhibition of spore germination compared with the effects of the individual treatments alone. Inhibition by the combination treatment occurred as a result of both direct but reversible inhibition, entirely dependent on the physical presence of the preservative factors, and permanent, nonreversible damage to the L-alanine germination apparatus of the spore. However, we were able to restore germination of the preservative-damaged spores unable to germinate on L-alanine by supplementing the medium with the nonnutrient germinant calcium dipicolinic acid. The demonstration that simple combinations of preservative factors inhibit spore germination indicates that food preservation systems providing ambient stability could be designed which do not adhere to the strict limits set by commonly accepted processes and which are based on precise understanding of their inhibitory action.     

Fate of bioterrorism-relevant viruses and bacteria, including spores, aerosolized into an indoor air environment

An aerosol physics test facility was used in a series of eight experiments to gather an integrated comprehensive broad base of data on the fate of surrogates of microorganisms that cause smallpox, plague, glanders, anthrax, and viral hemorrhagic fevers. The results are directly relevant to the public health issue of how to protect the occupants of buildings against bioterrorism. The test conditions were directly relevant to the indoor air environment situation, and the results can be generalized to buildings that are now occupied. The reductions in concentrations of relevant viruses and bacteria--including gram-negative fermenters and nonfermenters, gram-positive cocci and bacilli, and spores--were substantial and statistically robust. The data show that the bioterrorism-relevant aerosolized viruses and bacteria, including spores, respond like small particulates to the primary (electrical) forces that control the distribution of small particulates in a room. Further, these relevant microorganisms respond like small particulates to means designed to control airborne particulates. The results could be used to anticipate the effects of a bioterrorist attack on the public health, provide information on means that can be used to minimize such effects, and used to make decisions on how best to protect occupants of specific buildings at minimal cost and with assurance of success.     

Studies on the diversity of inositol-containing yeast phospholipids: incorporation of 2-deoxyglucose into lipid

Seven inositol-containing phospholipids in Saccharomyces cerevisiae can be resolved by two-dimensional chromatography on silicic acid-impregnated paper. Four of these lipids are stable to mild alkaline methanolysis; one of these has already been characterized as a mannosyl di(inositolphosphoryl) ceramide. Addition of labeled 2-deoxy-d-glucose to a growing culture results in label appearing in five lipids, some of which are alkali-stable and have the same R(F) values as the alkali-stable inositol-containing phospholipids. These lipids are labeled rapidly. The deoxyglucose is incorporated intact, probably in a glycosidic link, since the deoxyglucose is released by mild acid treatment.     

Thermal destruction of dried vegetative yeast cells and dried bacterial spores in a convective hot air flow: strong influence of initial water activity

Thermal treatment of Bacillus subtilis spores and Saccharomyces cerevisiae cells dried on glass beads was performed at various initial water activities (in the range 0.10-0.90). Experiments were carried out at 150 degrees C, 200 degrees C and 250 degrees C for 5-120 s. Significant destruction of up to 10(7) vegetative cells and up to 10(5) spores g(-1) was achieved, depending upon treatment conditions. This study demonstrated that the initial water activity (a(w)) value of a sample is very important in the destruction or survival of microorganisms treated with hot air stresses. As described previously, the heat resistance of spores and vegetative cells was strongly enhanced by low initial a(w) values until an optimal a(w) value between 0.30 and 0.50, with maximal viability at 0.35 for both S. cerevisiae and B. subtilis. However, our results highlighted for the first time that very low initial a(w) values (close to 0.10) greatly improved the destruction of spores and vegetative cells. Factors and possible mechanisms involved in the death of vegetative cells and spores are discussed.     

Sterilization of bacteria, yeast, and bacterial endospores by atmospheric-pressure cold plasma using helium and oxygen

Atmospheric-pressure cold plasma (APCP) using helium/oxygen was developed and tested as a suitable sterilization method in a clinical environment. The sterilizing effect of this method is not due to UV light, which is known to be the major sterilization factor of APCP, but instead results from the action of reactive oxygen radicals. Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae deposited on a nitrocellulose filter membrane or Bacillus subtilis spores deposited on polypropylene plates were exposed to helium/oxygen plasma generated with AC input power at 10 kHz, 6 kV. After plasma treatment, nitrocellulose filter membranes were overlaid on fresh solid media and CFUs were counted after incubation overnight. D-values were 18 sec for E. coli, 19 sec for S. aureus, 1 min 55 sec for S. cerevisiae, and 14 min for B. subtilis spores. D-values of bacteria and yeast were dependent on the initial inoculation concentration, while the D-value of B. subtilis spores showed no correlation. When treated cells were observed with a scanning electron microscope, E. coli was more heavily damaged than S. aureus, S. cerevisiae exhibited peeling, and B. subtilis spores exhibited shrunken morphology. Results showed that APCP using helium/oxygen has many advantages as a sterilization method, especially in a clinical environment with conditions such as stable temperature, unlimited sample size, and no harmful gas production.     

Microbiological treatment of industrial wastes containing toxic chromium involving successive use of bacteria,yeast and algae

Bacteria, yeasts, protozoa and algae, observed in industrial effluents from tanneries, were checked for their possible use in the detoxification of polluted water. Two bacterial strains were found to be highly resistant to Cr^VI. Several bacteria, yeasts and algae were observed to be capable of reducing Cr^VI to Cr^III.     

A carboxyl-terminal hydrophobic region of yeast cytochrome c1 is necessary for functional assembly into complex III of the respiratory chain

Cytochrome c1 is an amphiphilic protein which binds to the mitochondrial inner membrane, presumably through a hydrophobic region near the carboxyl (C)-terminus. In the preceding study (Hase, T., et al. (1987) J. Biochem. 102, 401-410), two cytochrome c1 mutations were constructed: delta 1 and delta 2 cytochromes c1, in which the C-terminal segments of 17 and 71 residues were replaced by foreign sequences of 20 and 15 residues, respectively. delta 2 cytochrome c1 had lost the putative membrane anchor. The two cytochrome c1 mutants were localized in mitochondria, but succinate-cytochrome c1 reductase activity was detected only in the mitochondria containing delta 1 cytochrome c1. The membrane association of the two mutant molecules as well as that of authentic cytochrome c1 was investigated. These three molecules were firmly attached to mitochondrial membranes and not solubilized on either sonication or sodium carbonate (pH 11) treatment. However, when the membranes were solubilized with Triton X-100, both the delta 1 and authentic cytochromes c1 were extracted from the membranes more easily than delta 2 cytochrome c1. By fractionating cholate extracts of mitochondrial membranes with ammonium sulfate, delta 1 cytochrome c1 was cofractionated with the enzymatic activity of complex III, but delta 2 cytochrome c1 was clearly separated from the complex III fraction. Trypsin treatment of mitochondria and mitoplasts showed that delta 2 cytochrome c1 was exposed to the intermembrane space, with such a topology that its trypsin susceptibility became much higher than that of the authentic molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of yeast growth limitation on the respiratory chain, the economic coefficient and the critical oxygen concentration for respiration

The effect of the yeast growth limitation by oxygen on the economical coefficient (EC), the operation of the cyanide resistant electron transport pathway (CrETP), and the critical for respiration oxygen concentration concentration ([O2]cr) was studied. The operation of CrETP was found to differ among various yeasts growing on glucose: it could function during both the exponential phase and limitation of growth (Torulopsis candida), or only in the conditions of growth limitation (Candida tropicalis, C. mycoderma, C. lipolytica), sometimes for a very long period (Endomyces geotrichum); in certain cases (C. utilis), it cannot be detected at all. If the main respiratory chain is inhibited by cyanide (i. e. if only CrETP operates), the value of [O2]cr sharply increases; such an increase can be also found in the absence of cyanide but in the conditions of active operation of CrETP. Apparently, the value of [O2]cr is higher for cyanide resistant oxydase of the studied organisms than for cytochrome oxydase. A decrease in EC observed upon the limitation of yeast growth by oxygen (Lozinov et al., 1974) correlates with the appearance or intensification of CrETP. Therefore, the decrease of EC can be attributed to the operation of non-phosphorylating CrETP which occurs in all the studied yeasts (with an exception of C. utilis) when their growth is limited by oxygen.     

Assessing the activity of microbicides against bacterial spores: knowledge and pitfalls

Bacterial endospores (spores) have a higher intrinsic resistance to microbicides as compared to other microbial forms, most likely due to their impermeable outer layers and low water content. Though structural differences between the spores of various bacterial species may account for observed variations in their resistance to microbicides, flaws in methods for testing the sporicidal activity of microbicides often exaggerate the differences. This has major implications when considering the selection of one or more surrogates to assess microbicides against clinically relevant spore‐formers such as Clostridium difficile. The mounting significance of Cl. difficile as a pathogen is leading to a corresponding increase in the number of commercially available microbicidal formulations claiming activity against its spores without proper differentiation between the product's sporistatic and sporicidal actions. In this review we critically assess the situation and the implications of product claims on the field use of microbicidal products.     

Structure of the DNA-SspC complex: implications for DNA packaging, protection, and repair in bacterial spores

Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the alpha/beta-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the alpha/beta-type SASP.     

Transfer of phospholipid and protein into the envelope of gram-negative bacteria by liposome fusion

A liposome-bacterial fusion system was developed in order to introduce preformed terminal complement complexes, C5b-9, into the outer membrane of Gram-negative bacteria. Liposomes were prepared from a total phospholipid extract of Salmonella minnesota Re595. Fusion between liposomes and Salmonella sp. or Escherichia coli 17 was dependent on time, temperature, pH, and Ca2+ and PO4- concentration. Only Salmonella sp. with attenuated LPS core regions were able to fuse efficiently with liposomes. It was demonstrated that fusion of liposomes with S. minnesota Re595 or E. coli 17 under optimum conditions resulted in (i) quantitative transfer of the self-quenching fluorescent membrane probe octadecyl rhodamine B chloride from the liposomal bilayer to the bacterial envelope, (ii) transfer of radiolabeled liposomal phospholipid to the bacterial outer membrane and its subsequent translocation to the cytoplasmic membrane, demonstrated by isolation of the bacterial membranes following fusion, and (iii) delivery of liposome-entrapped horseradish peroxidase (HRP) to the periplasmic space, confirmed by a chemiluminescent assay. Following fusion of liposomes incorporating C5b-9 complexes with S. minnesota Re595 or E. coli 17, immunological analysis of the isolated membranes revealed C5b-9 complexes located exclusively in the outer membrane.     

Effects of algal concentration, bacterial size and water chemistry on the ingestion of natural bacteria by cladocerans

Journal of Plankton Research, 11, 1273–1295, 1989. The values of P/U⁰ (Table I) and fluid velocity used to calculatethe energy required for sieving (pp. 1289–1290) and severalequations (footnote b of Table I; p. 1290, lines 3–4)are incorrect. The corrected table appears below: Table I. Filter setule measurements (mean and within specimenstandard deviation) of the gnathobases for the cladocerans studied^aGnathobaseof trunklimb number. ^bP = 8µU₀/(b(1 – 21nt + 1/6(t²) - 1/144(t⁴))), whereP = pressure drop in dyn cm^–2, =3.1416, U₀ = fluid velocityin cm s^–1, b = distance between setule centres in cm,t = ( x setule diameter)/b and µ = 0.0101 dyn s^–1cm^–2. Formula from Jørgensen (1983). The text (p. 1289, line 19 to p. 1290, line 10) should read: organism. Using a similar argument, a 0.5 mm Ceriodaphnia witha filter area of 0.025 mm² (Ganf and Shiel, 1985) and pressuredrop P = 2757 dyn cm^–2 (with fluid velocity of 0.07 cms^–1) allocates only 2171 ergs h^–1 to filtrationof a total energy expenditure of 10⁴ ergs h^–1 [filtrationenergy (ergs h^–1) = area (cm²) x pressure drop (dyn cm^–2)x 3600 (s h^–1) x 1/0.2 (efficiency of conversion of biochemicalinto mechanical work); total energy (ergs h^–1) = respiration(0.05 µl O₂ ind^–1 h^–1 consumed; Gophen, 1976)x conversion factor (2 x 10⁵ ergs µl^–1 O₂). Withan estimated 0.034 mm² in filter area, fluid velocity of 0.041cm s^–1 and respiration of 1.8 x 10⁴ ergs h^–1 (calculatedfrom Porter and McDonough, 1984), a 0.5 mm Bosmina uses <4%of its metabolism to overcome filter resistance. The velocities used in the original examples (0.4 cm s^–1for Ceriodaphnia, 0.2 cm s^–1 for Bosmina) were derivedfrom literature values of appendage beat rate and estimatesof the distance travelled by the appendages during each beatcycle. This approach unnecessarily assumes that all water movedpasses through the filter. In the new calculations, the flowacross the filter needed for food to be collected by sieving(0.07 cm s^–1 for Ceriodaphnia and 0.041 cm s^–1 forBosmina) was determined from the maximum clearance rate/filterarea. The amended energy expenditures, although higher, do notrefute the sieve model of particle collection.