Effect of alkylbenzenes on malting processes

It has been shown that one of alkyl hydroxybenzenes, C7-AHB, can be used in malting for regulating barley growth. Depending on concentration (0.01-1.0%) and duration (10 min to 6 h), treatment of barley with a C7-AHB solution stimulates embryo development (0.01-0.02%) or suppresses the growth of vegetative organs (> 0.5%) and modulates enzyme activities in germinating grains. Stimulation of the activities of the amylolytic and protein-proteinase complexes in barley depending on C7-AHB concentration improves malt quality by increasing both the degree of its saccharification and protein dissolution.     

Protection of Saccharomyces cerevisiae against Oxidative and Radiation-Caused Damage by Alkylhydroxybenzenes

The effects of C₇-alkylhydroxybenzene (₇-AHB) and p-hydroxyethylphenol (tyrosol), chemical analogs of microbial anabiosis autoregulators, on the viability of yeast cells under oxidative stress were investigated. The stress was caused by reactive oxygen species (ROS) produced under irradiation of cell suspensions using doses of 10–150 krad at an intensity of 194 rad/s or by singlet oxygen generated in cells photosensitized with chlorin e ₆ (10 g/l). C₇-AHB was found to exert a protective effect. The addition of 0.05–0.16 vol % of C₇-AHB to cell suspensions 30 min before irradiation protected yeast cells from radiation (50 krad). The protective effect of C₇-AHB manifested itself both in the preservation of cell viability during irradiation and in the recovery of their capacity to proliferate after irradiation. In our studies on photodynamic cell inactivation, the fact that the phenolic antioxidant C₇-AHB protects cells from intracellular singlet oxygen was revealed for the first time. The analysis of difference absorption spectra of oxidized derivatives of C₇-AHB demonstrated that the protective mechanism of ₇-AHB involves the scavenging of ROS resulting from oxidative stress. The fact that tyrosol failed to perform a photoprotective function suggests that the antioxidant properties of microbial ₇-AHB are not related to its chaperon functions. The results obtained make an important addition to the spectrum of known antioxidant and antistress effects of phenolic compounds.     

Enzyme Modification by Natural Chemical Chaperons of Microorganisms

We demonstrated for the first time that alkyl hydroxybenzenes (the d₁ microbial autoregulatory factors involved in stress responses of cells) are capable of stabilizing enzymes in aqueous media and increasing their catalytic activity. The stabilizing effect of a chemical analogue of alkyl hydroxybenzenes, C₇-AHB, was established in in vitro studies with enzymes of microbial origin: a protease produced by Bacillus licheniformis, cellulase produced by Trichoderma viride, and -amylase produced by Bacillus subtilis. This effect manifested itself in considerable extension of the temperature and pH ranges of the enzymatic activity. The modulation of the catalytic activities of the stabilized enzymes depended on the C₇-AHB concentration and on the time of preincubation of the complexes obtained. We demonstrated that not only enzymes but also their polymeric substrates formed complexes with C₇-AHB and this significantly influenced the efficiency of hydrolytic reactions. We also conducted comparative studies on the efficiency of hydrolytic reactions in systems in which the structure of enzymes and/or substrates was modified with C₇-AHB.     

Effect of alkylhydroxybenzenes,microbial anabiosis inducers,on the structural organization of <Emphasis Type="Italic">Pseudomonas aurantiaca</Emphasis> DNA and on the induction of phenotypic dissociation

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas aurantiaca and the phenotypic dissociation of this bacterium. The addition of 0.1–0.3 mM hexylresorcinol (C₆-AHB), a chemical analogue of microbial anabiosis autoinducers, caused the formation of cystlike refractile cells (CRC) in these gram-negative, nonsporulating bacteria. Inoculating pseudomonad CRC on solid nutrient media resulted in phenotypic dissociation of the microbial population that yielded several variants with different colony structure and morphology. This manifested itself in the conversion of the original S-colony-forming phenotype into the R form and in the formation of less pigmented colonies. These transitions were possibly linked to AHB-induced structural changes in the DNA. In vitro studies revealed that AHB could interact with DNA SC, resulting in their structural modification that manifested itself in changes in their viscoelasticity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their viscoelasticity and capacity to interact with AHB homologues with different hydrophobicity, such as hexylresorcinol and methylresorcinol (C₁-AHB). The DNA SC from actively proliferating cells were characterized by smaller viscoelasticity compared with those from stationary-phase and anabiotic cells, due to the difference in the DNA superspiralization degree and the physiological age of the bacteria involved. C₆-AHB produced a pronounced relaxing effect on the DNA SC from exponential-phase P. aurantiaca cells. The less hydrophobic C₁-AHB produced a similar relaxing effect on the DNA SC from stationary-phase cells. The curve of the dose-effect dependence of C₆-AHB had a breaking point within the submillimolar (10^–4 M) concentration range. These concentrations induce the formation of cystlike anabiotic pseudomonad cells that are characterized by an unstable phenotype and dissociate into distinct variants upon inoculation on solid media.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 157–165.Original Russian Text Copyright © 2005 by Mulyukin, Vakhrushev, Strazhevskaya, Shmyrina, Zhdanov, Suzina, Duda, Kozlova, El-Registan.     

Dormant state and phenotypic variability of Staphylococcus aureus and Corynebacterium pseudodiphtheriticum

Ability to produce dormant forms (DF) was demonstrated for non-spore-forming bacteria Staphylococcus aureus (a nonpathogenic strain) and Corynebacterium pseudodiphtheriticum (an organism of the normal oropharyngeal flora). The salient features of the sthaphylococcal and corynebacterial DF were (1) prolonged (4 months) preservation of viability; (2) resistance to damaging factors (heat treatment); and (3) specific morphology and ultrastructure. The optimal conditions for DF formation were (1) transfer of stationary-phase cultures into saline solution with CaCl₂ (10–300 mM) (for S. aureus); (2) growth in SR1 synthetic medium with fivefold nitrogen limitation (for C. pseudodiphtheriticum); and (3) incubation with (1–5) × 10^?4 M of C₁₂-AHB, an alkylhydroxybenzene akin to microbial anabiosis autoinducers. Increase of C₁₂-AHB concentration to 7 × 10^?4–2 × 10^?3 M resulted in “mummification” of cells with irreversible loss of viability without autolytic processes. Germination of dormant forms was followed by increasing of phenotypic variability, as seen from (1) diversity of colony types and (2) emergence of antibiotic-resistant clones on selective media. The share of kanamycin-resistant S. aureus variants was most numerous (0.002–0.01%) in 4-month DF suspensions in SALINE with CaCl₂. In the C. pseudodiphtheriticum DF produced under the effect of C₁₂-AHB, the share of kanamycin-resistant variants was also found to increase. These data point to an association between the emergence of antibiotic-resistant variants of bacteria and their persistence in dormant state mediated by starvation stress and regulated by AHB.     

Occurrence of (+)-7-iso-jasmonic acid inVicia faba L. and its biological activity

The plant growth regulator (–)-jasmonic acid (JA) and its stereoisomer (+)-7-iso-jasmonic acid (7-iso-JA) have been isolated from young fruits ofVicia faba L. and identified by TLC, GC, GC-MS, and chemical transformation. Different isolation procedures gave different ratios of JA/7-iso-JA because of partial isomerization of (+)-7-iso-JA to (–)-JA. Optimal conditions, excluding artificial isomerization, were checked by the addition of [U-¹⁴C]-7-iso-JA. The naturally occurring isomer ratio was determined to be 65% (–)-JA: 35% (+)-7-iso-JA in immature broad bean fruits. The biological activities of both isomers of JA have been studied using several bioassays. Growth of wheat and GA₃-stimulated dwarf rice seedlings is inhibited more effectively by (+)-7-iso-JA than by (–)-JA. In growing seedlings of barley and oat, both isomers caused a senescence-like bleaching effect characterized by chlorophyll and carotenoid decrease. The free acids are more active than their methyl esters in intact barley plants in contrast to results obtained in leaf segment tests. Highest activity was obtained with (+)-7-iso-JA.     

The Effect of Anabiosis Autoinducers on the Bacterial Genome

The mutagenic activity of chemical analogues of microbial anabiosis autoinducers (the autoregulatory d₁ factors of cell differentiation), which act to inhibit cell proliferation, to enhance cell tolerance, and to induce the transition of cells to an anabiotic state, was studied using the Ames test. In the range of concentrations studied (0.1 to 100 g/ml), alkyl-substituted hydroxybenzenes (AHBs) differing in hydrophobicity, i.e., methylresorcinol (C₁-AHB) and hexylresorcinol (C₆-AHB), as well as unsubstituted resorcinol, showed different growth-inhibiting and mutagenic effects. C₆-AHB was found to inhibit the growth of Salmonella typhimurium TA100 and to induce its mutagenesis at a rate of 1.8 revertants/nmol. C₁-AHB taken at low concentrations not only failed to inhibit bacterial growth but even stimulated it and exerted an antimutagenic effect. Unsubstituted resorcinol virtually did not influence bacterial growth and showed weak mutagenic activity. The growth-inhibiting effect of elevated C₆-AHB concentrations correlated with the degree of the transition of the original phenotype producing S-type colonies to a phenotype producing R-type colonies. The role of AHB homologues, as microbial autoregulators with mutagenic activity, in the regulation and correlation of two processes: the phenotypic dissociation of microbial populations and the formation of resting microbial forms, is discussed. The accumulation of AHBs in senescent microbial cultures may induce adaptive mutations, change the expression of genes, and promote the development of minor cell subpopulations (phenotypes), thus providing for the adaptation of these cultures to varying environmental conditions.     

Properties of the phenotypic variants of Pseudomonas aurantiaca and P. fluorescens

Different capacity for phenotypic variation of Pseudomonas aurantiaca and P. fluorescens in populations of cyst-like resting cells (CRC) during their germination on solid media, was shown to be a characteristic trait of biodiversity for the dormant forms of these bacteria. This biodiversity manifests itself as qualitative and quantitative differences in the spectra and emergence frequency of phenotype variants, obtained by plating of CRC, and depends on the conditions of CRC formation and storage time. In P. aurantiaca, the variation was associated with transition of the wild-type S-colonial phenotype into the R-type or the more pigmented P-type. These transitions were most pronounced for the CRC obtained under nitrogen depletion (a twofold N limitation), as well as under the influence of a chemical analogue of microbial anabiosis autoinducers, C₁₂-AHB. In the latter case, the frequency of S?R and S?P transitions (up to 70% and 80%, respectively) depended on the C₁₂-AHB concentration (1.0 × 10^?4 M and 2.5 × 10^?4 M) and on the storage time of CRC suspensions (from 3 days to 1.3 months). In the CRC populations grown in nitrogen-deficient media, R-type appeared with a frequency of up to 45% after at least four months of storage. In the case of P. fluorescens, S?R transitions depended not only on the storage time of CRC and C₁₂-AHB concentrations, but also on the composition of the solid medium used for plating. Differences were shown between the R-, P-, and S-variants of P. aurantiaca in such morphological, physiological, and biochemical characteristics as the growth rate (μ_max) in a poor medium, biomass yield (Y _max), resistance to streptomycin and tetracycline (LD₅₀), and the productivity in extracellular proteases. The R-and S-variants of P. fluorescens differed in their growth characteristics, resistance to high salinity and oxidative stress, as well as in their sensitivity to exogenous introduction of chemical analogues of microbial autoregulators (C₁₂-AHB and C₇-AHB). Hence, both the formation of dormant forms of the various morphological types [1] and intrapopulation phenotypic variability observed during their germination are important for the survival strategy of pseudomonads under unfavorable environmental conditions.     

The Role of Bacterial Growth Autoregulators (Alkyl Hydroxybenzenes) in the Response of Staphylococci to Stresses

The investigation of the response of a batch culture of Staphylococcus aureus to exogenous alkyl-substituted hydroxybenzenes (AHBs), chemical analogues of anabiosis autoinducers, showed that C₁-AHB at concentrations from 5 M to 1.5 mM did not influence the culture growth, whereas the more hydrophobic C₆-AHB inhibited it at concentrations of 0.5 mM and higher. Either of the AHBs drastically enhanced the phenotypic dissociation of staphylococcal cultures, which manifested itself in an increase in the fraction of cells producing small nonhemolyzing colonies of G type when plated on solid media with erythrocytes. In a submerged staphylococcal culture, the relative number of cells producing G-type colonies varied from 10 to 90%, depending on the concentration of the AHB added. The growth of S. aureus in the presence of AHBs also enhanced cell tolerance to heat shock (heating at 45 or 60°C for 10 min). The role of AHBs, which are structural modifiers of membranes and possess chaperone activity, in the mechanisms responsible for cell tolerance and phenotypic dissociation of microbial populations is discussed.     

Experimental and mathematical simulation of plant growth promoting rhizobacteria and plant interaction under cadmium stress

Bacterial inoculants of the commercially available plant growth promoting rhizobacteria (PGPR) Arthrobacter mysorens 7, Flavobacterium sp. L30, and Klebsiella mobilis CIAM 880 were selected to obtain ecologically safe barley crop production on cadmium (Cd) polluted soils. All the PGPR immobilized 24–68% soluble cadmium from soil suspension. A. mysorens 7 and K. mobilis CIAM 880 were highly resistant to Cd and grew in up to 1 and 3 mmol CdCl₂ on DAS medium respectively. All PGPR were able to fix nitrogen (276–1014 nmol mg^–1 bacterial DW) and to produce indole acetic acid (IAA) (126–330 nmol mg^–1 bacterial DW) or ethylene (4.6–13.5 nmol bacterial DW). All the PGPR actively colonized barley root system and rhizosphere and significantly stimulated root elongation of barley seedlings (up to 25%), growing on soil containing 5 or 15 mg Cd kg^–1 of soil. Created in the simulation mathematical model confirms our hypothesis that PGPR beneficial effect on barley growing under Cd-stress is a complex process. One of mechanisms underlying this effect might be increase of bacterial migration from rhizoplane to rhizosphere, where PGPR bind soluble free Cd ions in biologically unavailable complex forms. Among the studied PGPR K. mobilis CIAM 880 was the most effective inoculant. Inoculation with K. mobilis CIAM 880 of barley plants growing on Cd contaminated soil (5 mg Cd kg^–1 of soil) under field conditions increased by 120% grain yield and 2-fold decreased Cd content in barley grain. The results suggest that the using K. mobilis CIAM 880 is an effective way to increase the plant yield on poor and polluted areas.     

Halophilic n-alkane utilizing yeast from the Arabian gulf

Summary A facultative halophilic yeastTorulopsis candida (Saito) Lodder, isolated from the Arabian Gulf, was oua to grow on n-alkanes (C₁₃–C₁₉) at 37°C and pH 3–7 in media containing 1.4- 3M NaCl or 30–90% seawater. The influence of pH, temperature, growth factors, salt and pure n-alkanes on its growth were studied. Its amino acid profile and crude protein were compatible with those of commercial n-alkane yeast products.     

The effects of alkylhydroxybenzenes on homoserine lactone-induced manifestations of quorum sensing in bacteria

The effects of four alkylhydroxybenzene (AHB) homologs with different hydrocarbon chain lengths on the synthesis of violacein pigment induced by C₆-homoserine lactone (HSL) and biofilm formation by Chromobacterium violaceum NCTC 13274 and on Escherichia coli pAL103 bioluminescence in the presence of C₆-oxo-HSL were studied. Alkylhydroxybenzenes inhibit the growth of C. violaceum increased in the C₅-AHB → C₁₂-AHB series in the absence of this activity in C₁-AHB. Subinhibitory AHB concentrations reduced violacein production and suppressed biofilm formation. These effects were presented as individual and group regression dependencies between the analysed parameters. Using the bioluminescent model, the regulatory effects of AHBs were not associated with their direct competition with HSL and that they develop as a result of changes in the sensitivity of bacterial cells to the respective quorum sensing inducer.     

The fate of15N-labelled organic nitrogen in sheep manure applied to soils of different texture under field conditions

The fate of nitrogen from¹⁵N-labelled sheep manure and ammonium sulfate in small lysimeters and plots in the field was studied during two growth seasons. In April 1991,¹⁵N-labelled sheep faeces (87 kg N ha^–1) plus unlabelled (NH₄)₂SO₄ (90 kg N ha^–1), and (¹⁵NH₄)₂SO₄ (90 kg N ha^–1) were each applied to three soils; soil 1 (100% soil + 0% quartz sand), soil 2 (50% soil + 50% quartz sand) and soil 3 (25% soil + 75% quartz sand). The lysimeters were cropped with spring barley (Hordeum vulgare L.) and undersown ryegrass (Lolium perenne L.). The barley crop recovered 16–17% of the labelled manure N and 56% of the labelled (NH₄)₂SO₄-N. After 18 months 30% of the labelled manure N and 65% of the labelled (NH₄)₂SO₄-N were accumulated in barley, the succeeding ryegrass crop and in leachate collected below 45 cm of soil, irrespective of the soil-sand mixture. Calculating the barley uptake of manure N by difference of N uptake between manured and unmanured soils, indicated that 4%, 10% and 14% of the applied manure N was recovered in barley grown on soil-sand mixtures with 16%, 8% and 4% clay, respectively. The results indicated that the mineralization of labelled manure N was similar in the three soil-sand mixtures, but that the manure caused a higher immobilization of unlabelled ammonium-N in the soil with the highest clay content. Some of the immobilized N apparently was remineralized during the autumn and the subsequent growth season. After 18 months, 11–19% of the labelled manure N was found in the subsoil (10–45 cm) of the lysimeters, most of this labelled N probably transported to depth as organic forms by leaching or through the activities of soil fauna. In unplanted soils 67–74% of the labelled manure N was recovered in organic form in the 0–10 cm soil layer after 4 months, declining to 55–64% after 18 months. The lowest recovery of labelled N in top-soil was found in the soil-sand mixture with the lowest clay content. The mass balance of¹⁵N showed that the total recovery of labelled N was close to 100%. Thus, no significant gaseous losses of labelled N occurred during the experiment.     

Responses of alfalfa and Barley to foliar application of N and P on a coal mine spoil

Summary To ensure adequate growth of plants on the highly impoverished and erodable surface mined lands, the application of N and P fertilizers by suitable methods is essential. In the present study, five growth chamber experiments were conducted to evaluate the relative efficacy of foliar and spoil application of N and P using alfalfa (Medicago sativa L. var. Erand) and barley (Hordeum vulgare L. var. Manker) as test crops on a freshly exposed coal mine spoil collected from western North Dakota. In general, barley responded to both N and P, but alfalfa mainly to P. Growth responses of barley to foliar or spoil-applied N+P were substantial and similar in magnitude. However, the yields were much higher when the plants received 3–4 sprays of 1.5–2.2% urea, with P supplied through the spoil. Increasing the number of 2.2% urea sprays from 1 to 3 increased the growth response from 40 to 243%. In another study, increasing the concentration of foliar-applied urea from 0 through 1% resulted in further increases in the dry weights of barley at all the levels of spoil-applied (0, 25, 75, 225 g/g) N.Foliar sprays of 0.5–1.0% NaH₂PO₄ increased the dry weights of alfalfa and barley by an average of 366% and 86%, respectively. However, the yield response of alfalfa to spoil-applied P (100 g/g) was as high as 782% compared to only 117% for barley. Alfalfa responded significantly to increasing concentrations of H₃PO₄ (0–0.3%) in foliar sprays only in the absence of spoil-applied P. With increasing rates of spoil-applied P, alfalfa yields increased steadily, but additional supply of P sprays caused leaf burning which intensified as the P concentration in sprays increased.The results of chemical analyses indicated that foliar applications were more effective than soil applications in increasing the concentration of N or P in the plants. Moreover, urea sprays increased the uptake of K, Zn, and Fe in barley, whereas spraying alfalfa with P compounds caused increases in its K and Fe content and decreases in those of Zn and Na. The results of these experiments indicated that the nutritional requirements of plants grown on coal mine spoils can be met through foliar fertilization as effectively as, or better than, through conventional soil fertilization methods.Presented at the Annual Meeting, American Society of Agronomy, Chicago, Illinois, Dec. 3–8, 1978.     

Protein production from whey usingPenicillium cyclopium; growth parameters and cellular composition

Summary The growth parameters ofPenicillium cyclopium have been evaluated in a continuous culture system for the production of fungal protein from whey. Dilution rates varied from 0.05 to 0.20 h^–1 under constant conditions of temperature (28°C) and pH (3.5). The saturation coefficients in the Monod equation were 0.74 g l^–1 for lactose and 0.14 mg l^–1 for oxygen, respectively. For a wide range of dilution rates, the yield was 0.68 g g^–1 biomass per lactose and the maintenance coefficient 0.005 g g^–1 h^–1 lactose per biomass, respectively. The maximum biomass productivity achieved was 2 g l^–1 h^–1 biomass at dilution rates of 0.16–0.17 h^–1 with a lactose concentration of 20 g l^–1 in the feed. The crude protein and total nucleic acid contents increased with a dilution rate, crude protein content varied from 43% to 54% and total nucleic acids from 6 to 9% in the range of dilution rates from 0.05 to 0.2 h^–1, while the Lowry protein content was almost constant at approximately 37.5% of dry matter.Nomenclature (mg l^–1) C_o initial concentration of dissolved oxygen - (h^–1) D dilution rate - (mg l^–1) K₀₂ saturation coefficient for oxygen - (g l^–1) K_s saturation coefficient for substrate - (g g^–1 h^–1) lactose per biomass) m maintenance energy coefficient - (mM g^–1 h^–1O₂ per biomass) Q₀₂ specific oxygen uptake rate - (g l^–1) S residual substrate concentration at steady state - (g l^–1) S_o initial substrate concentration in feed - (min) t_1/2 time when C_o is equal to C_o/2 - (g l^–1) X biomass concentration - (g l^–1) X biomass concentration at steady state - (g g^–1 biomass per lactose) Y_G yield coefficient for cell growth - (g g^–1 biomass per lactose) Y_x/s overall yield coefficient - (h^–1) specific growth rate     

The fate of carbon in pulse-labelled crops of barley and wheat

Wheat (cv. Gutha) and barley (cv. O'Connor) were grown as field crops on a shallow duplex soil (sand over clay) in Western Australia with their root systems contained within pvc columns. At four stages during growth, the shoots were pulse-labelled for 1.5h with¹⁴CO₂; immediately prior to labelling, the soil was isolated from the shoot atmosphere by pvc sheets. After labelling, the soil atmosphere was pumped through NaOH to trap respired CO₂ and after 2.5, 5, 7.5 and 24 h from the start of labelling, columns were destructively sampled to recover¹⁴C from the roots, soil and shoot.Both species showed similar patterns of¹⁴C distribution and changes in distribution through the growing season. During early tillering, 15–25% of the¹⁴C recovered after 24 h had been respired by the roots and rhizosphere, 17–27% was retained in the roots, 0.4–1.8% was recovered as water-soluble¹⁴C in the soil and the remainder (45–67%) was present in the shoot. These percentages changed during growth so that during grain filling only 2–3% of the¹⁴C recovered after 24 h was as respired CO₂, 2–6% was in the roots, 0.2% was in the soil and over 90% was in the shoot.The distribution of¹⁴C in components of the soil-plant system changed during the 24 h after labelling with the most rapid changes occurring generally during the first 7.5 h after labelling.Using growth measurements from adjacent plots, the amounts of C added to the soil were estimated for the whole season. Carbon input to the soil was about 48 gC m^–2 for wheat and 58 gC m^–2 for barley; the crops produced total shoot dry matter of 494 (wheat) and 735 g m^–2 (barley). Of the C input to the soil, 27.8% (wheat) and 40.3% (barley) was as respired C and only 3.3 (wheat) and 4.1% (barley) was collected as exudate (water-soluble material).     

Production of Lactobacillus kefir cells for asymmetric synthesis of a 3,5-dihydroxycarboxylate

An efficient fedbatch process for the production of Lactobacillus kefir DSM 20587 cells was developed. An improvement in space time yield of 270% (3.7 g_DCW l^–1 day^–1) and in final enzyme activity of 440% (9.1 U/ml) was achieved on a 150 l scale by controlling the oxygen transfer rate to 7–9 mmol l^–1 h^–1. The cells exhibited good and highly stereoselective reducing activities against tert-butyl 6-chloro-3,5-dioxohexanoate. tert-Butyl (3R,5S)-6-chloro-dihydroxyhexanoate, a chiral building block for HMG-CoA reductase inhibitor synthesis, was produced with 47.5% yield and >99% ee at C₃3 and C₅5 in a simple batch biotransformation process.     

Mapping of the Synaptosome-Binding Regions on the Heavy Chain of Botulinum Neurotoxin A By Synthetic Overlapping Peptides Encompassing the Entire Chain

The purpose of this work was to map, on the heavy (H) chain of botulinum neurotoxin A (BoNT/A), the regions that bind to mouse brain synaptosomes (snps). We prepared 60 synthetic overlapping peptides that had uniform size and overlaps and encompassed the entire H chain (residues 499 to 1296) of BoNT/A. The ability of each peptide to inhibit the binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was studied. The binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was completely inhibited by free unlabeled BoNT/A, but not by unrelated proteins, indicating that the binding of BoNT/A to mouse brain snps was a specific event. Inhibition studies with the individual peptides showed that, on the H_N domain, inhibitory activities greater than 10% were exhibited, in decreasing order, by peptides 799–817, 659–677, 729–747, 533–551, 701–719, and 757–775. Lower inhibitory activities (between 5.6% and 8.7%) were exhibited by five other peptides, 463–481, 505–523, 519–537, 603–621 and 645–663. The remaining 18 H_N peptides had little or no inhibitory activity. In the H_C domain, peptides 1065–1083, 1163–1181 and 1275–1296 had the highest inhibitory activities (between 25% and 29%), followed (10–12% inhibitory activity) by peptides 1107–1125, 1191–1209 and 1233–1251. Two other peptides, 1079–1097 and 1177–1195, had very low (5.8% and 4.9 %) inhibitory activities. The remaining 23 H_C peptides had no inhibitory activity. Inhibition with mixtures of equimolar quantities of the most active 6 peptides of H_N, 5 of H_C or all 11 of H_N and H_C revealed that the peptides contain independent non-competing binding regions. Comparison of the locations of the snp-binding regions on the H-subunit with the regions that bind blocking mouse anti-BoNT/A Abs helped explain the protecting ability of these Abs. In the three-dimensional structure of BoNT/A, the snp-binding regions that completely coincide or significantly overlap with the antigenic regions occupy surface locations and most of them reside in the last half of the H_C domain. But some of the regions reside in the H_N domain and might play a role in the translocation event.     

Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria

Recently, there has been a resurgence of interest in bioorganic fertilizers as part of sustainable agricultural practices to alleviate drawbacks of intensive farming practices. N₂-fixing and P-solubilizing bacteria are important in plant nutrition increasing N and P uptake by the plants, and playing a significant role as plant growth-promoting rhizobacteria in the biofertilization of crops. A study was conducted in order to investigate the effects of two N₂-fixing (OSU-140 and OSU-142) and a strain of P-solubilizing bacteria (M-13) in single, dual and three strains combinations on sugar beet and barley yields under field conditions in 2001 and 2002. The treatments included: (1) Control (no inoculation and fertilizer), (2) Bacillus OSU-140, (3) Bacillus OSU-142, (4) Bacillus M-13, (5) OSU-140 + OSU-142, (6) OSU-140 + M-13, (7) OSU-142 + M-13, (8) OSU-140 + OSU-142 + M-13, (9) N, (10) NP. N and NP plots were fertilized with 120 kg N ha^–1 and 120 kg N ha^–1 + 90 kg P ha^- for sugar beet and 80 kg N ha^–1 and 80 kg N ha^–1 + 60 kg P ha^–1 for barley. The experiments were conducted in a randomized block design with five replicates. All inoculations and fertilizer applications significantly increased leaf, root and sugar yield of sugar beet and grain and biomass yields of barley over the control. Single inoculations with N₂-fixing bacteria increased sugar beet root and barley yields by 5.6–11.0% depending on the species while P-solubilizing bacteria alone gave yield increases by 5.5–7.5% compared to control. Dual inoculation and mixture of three bacteria gave increases by 7.7–12.7% over control as compared with 20.7–25.9% yield increases by NP application. Mixture of all three strains, dual inoculation of N₂-fixing OSU-142 and P-solubilizing M-13, and/or dual inoculation N₂-fixing bacteria significantly increased root and sugar yields of sugar beet, compared with single inoculations with OSU-140 or M-13. Dual inoculation of N₂-fixing Bacillus OSU-140 and OSU-142, and/or mixed inoculations with three bacteria significantly increased grain yield of barley compared with single inoculations of OSU-142 and M-13. In contrast with other combinations, dual inoculation of N₂-fixing OSU-140 and P-solubilizing M-13 did not always significantly increase leaf, root and sugar yield of sugar beet, grain and biomass yield of barley compared to single applications both with N₂-fixing bacteria. The beneficial effects of the bacteria on plant growth varied significantly depending on environmental conditions, bacterial strains, and plant and soil conditions.