Hydrophobic characteristics ofBacillus spores

Spores from severalBacillus species displayed a strong affinity for hexadecane and other hydrophobic solvents. The binding ofBacillus subtilis spore suspensions to octyl-Sepharose was enhanced by ammonium sulfate and other salts, but was inhibited by detergents. Treatment of spore suspensions with strong denaturants promoted their adherence to hexadecane, presumably by exposing hydrophobic residues in coat proteins. The hydrophobic characteristics of spores may be important in the ecological adaptation of certain bacteria.     

Copper retention by a strain ofBacillus

Summary A survey has been made of the copper accumulation by resting cells of bacteria selected as copper-resistant, isolated from activated sludges. The best selected strain, classified asBacillus, retained copper at up to 3.8% of its cell dry weight. These values were lower in the presence of glucose, unlike a type culture ofBacillus cereus, in which the retention of copper was higher when glucose was present. Possible reasons for these changes in uptake of both strains are suggested.     

Selective regeneration ofBacillus subtilis protoplasts transformed to kanamycin resistance

An HTY medium osmotically stabilized with 0.5 M D-glucitol was used for regeneration ofBacillus subtilis protoplasts. The application of glucitol as osmotic stabilizer allows simultaneous selection of cells resistant to kanamycin to be made since this antibiotic is not inactivated by glucitol when added to the regeneration medium.     

Protoplast formation and regeneration in <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.     

Cell growth and α-amylase production characteristics ofBacillus subtilis

Growth, differential rate of α-amylase synthesis and production characteristics ofBacillus subtilis DP 1 (isolate from starch materials) in comparison with 10Bacillus strains were examined in batch fermentation. The effect of the carbon and nitrogen source was evaluated with regard to cell growth and enzyme production. The pH optimum of enzyme activity was 6.5 and temperature optimum 60°C.     

Efficient protoplast regeneration ofBacillus thuringiensis andAgrobacterium tumefaciens

Summary Treatment ofBacillus thuringiensis andAgrobacterium tumefaciens taken from the early growth phase (8 h) with lysozyme at 1 mg/ml gave 90–99% protoplast formation and 10–12% protoplast regeneration on the minimal medium in absence of plasma expander (Bovine serum albumin). Enhanced fusion frequency was obtained when protoplasts from 8 h grown cells were used for fusion experiments.     

Nontranslation of foreign genetic information for fraction 1 protein under circumstances favorable for direct transfer ofNicotiana gossei isolated chloroplasts intoN. Tabacum protoplasts

Summary The nuclei and cytoplasm ofN. gossei andN. tabacum are compatible to the extent that reciprocal, interspecific F₁ hybrids can be produced by conventional breeding techniques. Conditions were established in which manyN. gossei isolated chloroplasts could be seen by phase and fluorescence microscopy to adhere to 40% of the population of protoplasts obtained from white tissue of variegatedN. tabacum plants and to remain attached after washing the protoplasts. Chloroplasts also could be seen to enter the interior of the protoplasts. After treating albino protoplasts withN. gossei chloroplasts, the protoplasts were subjected to further conditions whereby 65 calluses containing shoots developed. TwentyN. tabacum protoplasts not treated with foreign chloroplasts also produced calluses with shoots to serve as a control. All calluses developed chlorophyll irrespective of whether or not the albino protoplasts had been treated with isolatedN. gossei chloroplasts. The Fraction 1 protein ofN. tabacum has a different electrophoretic mobility from the protein ofN. gossei or anN. gossei xN. tabacum F₁ hybrid. The Fraction 1 protein large subunit is coded by chloroplast DNA, whereas the small subunit is coded by nuclear DNA. Fraction 1 protein was isolated from the variegated shoots of the 65 calluses obtained after treating albino protoplasts with foreign chloroplasts. Immunoelectrophoresis demonstrated the protein from each callus to have a mobility identical toN. tabacum protein. Therefore, under circumstances highly favorable for the direct transfer ofN. gossei isolated chloroplasts (and possibly nuclei also) intoN. tabacum protoplasts, no evidence was obtained to suggest that genetic information contained in the isolated foreign organelles was being translated into the polypeptides of either the large or small subunits of Fraction 1 protein contained in newly differentiated leaves derived from the protoplasts. Supported by Research Grant PCM-75-07368 from the National Science Foundation.     

Instability of mosquito larvicidal activity ofBacillus sphaericus (ISPC-6=WHO-2377)

Rapid loss of mosquito larvicidal potency ofBacillus sphaericus (ISPC-6) during subculturing is correlated to the appearance of non toxic variants. Simultaneous loss of streptomycin resistance, urease and exoprotease is also observed in majority of the non toxic variants. The loss is however reversible because of the reappearance of toxic phenotype resembling wild type in the non toxic variants.     

Transfer of kanamycin resistance fromNicotiana tabacum toNicotiana plumbaginifolia by fusion of x-irradiated protoplasts

Summary Nuclear hybrids have been obtained by fusion of mesophyll protoplasts ofNicotiana plumbaginifolia and x-irradiated or iodoacetate-treated mesophyll protoplasts of a kanamycin-resistant line ofN. tabacum. The effect of irradiation on the recovery of asymmetric hybrids was evaluated by analysis of their morphology, fertility, chromosome number, isozyme patterns, restriction patterns in their organelle DNAs, and presence of the kanamycin-resistance gene. The results presented in this paper show that x-ray irradiation leads to a significant reduction in the amount ofN. tabacum genome present in the hybrids and demonstrates, once more, the power of this technique to induce directional loss of genomic traits of the irradiated parent.     

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respeot to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3 – 5 h of incubation. After 7 h 20 – 50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg mg^-1 h^-1. The intracellular proteolytic activity was inhibited to 65 – 88% by EDTA, to 23 – 76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50 – 65% of the activity were localized in protoplasts. Another strain ofBacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

Glucanases and chitinases ofBacillus circulans WL-12

Summary Lysis of cell walls of various yeast species by -1,3- and -1,6-glucanases ofBacillus circulans WL-12 was investigated. Selective enzymolysis of cell walls ofPyricularia oryzae by single and combined actions of -1,3-, -1,6-glucanases and chitinase was followed. Chemical structure of the cell wall glucan ofP. oryzae was determined by chemical and enzymatic methods. Multiple component nature of glucanases ofB. circulans WL-12, their induction and lytic actions on cell walls of various yeasts were studied. Genes specifying glucanases and chitinases ofB. circulans WL- 12 were cloned inE. coli, and their nucleotide sequences were determined. Fibronectin type III modules were found in the chitinases. Functions of the domains of the deduced structures of the glucanases and the chitinases were studied by various methods including molecular genetic techniques.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Characteization of bacterial spores from high-temperature growth transformants ofBacillus subtilis

Spores from high-temperature growth transformants ofBacillus subtilis were examined for a number of sporal characteristics. Analyses showed a dramatic increase in both the calcium (Ca) and dipicolinic acid (DPA) contents, a slight increase in the Ca/DPA mole ratio, but a reduction in magnesium (Mg) content and the Mg/Ca mole ratio. The spore wet density increased, whereas the core/core+cortex volume ratio and protoplast water content decreased. Spore heat resistance increased but not to levels normally observed for thermophilicBacillus species. It is concluded that the biophysical and biochemical changes within the spores of the transformants are influenced by both the inherent mesophilic genotype and the transformant's inherited ability to grow at elevated temperatures.Florida Agricultural Experiment Station, Journal Series No. 8189     

Characteristics of intracellular proteolytic activities ofBacillus megaterium

Intracellular proteolytic activities ofB. megaterium KM occur soluble in the cytoplasm and periplasm and insoluble in the membrane. Two proteolytic enzymes were found in the cytoplasmic fraction by gel filtration on Sephadex G 150 and by polyacrylamide gel electrophoresis. The first enzyme called C_I was stable, had a relative molecular mass ofM _r=105000 (M=105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated C_II, was labile and had a relative molecular mass ofM _r=46000 (M=46 kg/mol). Because of its lability it could not be characterized in detail. In the “periplasm” only a single proteolytic enzyme P (M _r=28000;M=28 kg/mol) inhibited by EDTA could be demonstrated. The extracellular enzyme exhibited similar properties. The membrane proteolytic activity was sensitive to PMSF and EDTA. The membrane enzymes have not yet been solubilized. In cells of the mutant KM 12 that does not produce the extracellular proteinase, only one type of proteinase, in all its properties identical with the cytoplasmic proteinase C_I, could be demonstrated.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

Quantification of<Emphasis Type="Italic">Bacillus</Emphasis> species in a wastewater treatment system by the molecular analyses

Bacillus species were observed and quantified by molecular approaches, using the 165 rDNA primers/probes, in a wastewater treatment plant designed for the purpose of stimulating the growth ofBacillus species. The plant has been operating as a test plant since 1997 in the city of Ina, Japan, with excellent treatment performance. Observations byin situ hybridization, usingBacillus-specific probes, indicated thatBacillus strains were inhabited in the plant and their numbers decreased during the treatment process. Similar results were obtained from a quantitative PCR analysis using aBacillus-specific primer set, and the amount of DNA originating from variousBacillus species was maximally 1.91% of the total DNA in the wastewater treatment tank. Clone library analysis using theBacillus-specific primers suggested that, while the population was noticeably increased, the phylogenetic diversity of the increasingBacillus species was very low.     

Protoplast Formation and Regeneration of Streptococcus bovis Cells

The simultaneous use of hen egg lysozyme and mutanolysin, N-acetylmuramidase SG, showed synergistic effects on the lytic action and more effectiveness for protoplast formation of Streptococcus bovis IFO 12058. The frequency of regeneration reached 100% after 7 days of incubation under the improved conditions and by a method in which protoplasts were overlaid with the agar medium layer.     

Behavior,activities, and effects of bacteria on synthetic quartz monocrystal surfaces

Two strains ofBacillus sp. and a strain ofBrevibacterium sp., originally isolated from a natural quartzite surface, were characterized and employed as test strains with several methods: acridine orange fluorochromation and epifluorescence microscopy were used for detection of individual cells; scanning and transmission microscopy for studying attachment behavior; replica techniques in combination with electron microscopy for following surface interaction effects; and chemical analysis of SiO₂ for detecting possible silica leaching activities. The experimental results clearly showed that the three test strains were able to attach to and grow on the precleaned quartz surfaces. Attachment modes were either by direct sorption mechanisms (Brevibacterium sp. S) or the production of adhesive polymers (Bacillus sp. U andBacillus sp. W). In short-term contact incubation experiments with rich media, neither quartz crystal surface structures nor bacterial cell surfaces appeared to be changed. Likewise, significant biochemical dissolution and mechanical dislocation of SiO₂ (which would have indicated rapid bacterial weathering activities) could not be detected. The importance of quartz purity and crystalline structure for the initiation of weathering processes is discussed.     

Wall ultrastructure in regenerating protoplasts ofAspergillus nidulans

Summary The ultrastructure of the wall formed by regenerating protoplasts ofAspergillus nidulans was investigated by electron microscopy using shadowing and freeze-etching techniques. The first evidence of wall formation by the protoplasts was seen in the development of a microfibrillar network composed of chitin. As the protoplasts develop further amorphous material was deposited on the outer surface of the skeletal net.     

Application of pattern recognition to monitoring fermentations ofBacillus amyloliquefaciens

Summary Pattern recognition techniques were applied to analytical data to distinguish abnormal from normal microbial fermentations usingBacillus amyloliquefaciens as a model system. Patterns of fermentation end products during growth ofB. amyloliquefaciens were obtained from HPLC analysis of broth samples. Data were also obtained from fermentations using other bacterial species, strains, and environmental conditions, and were compared with the model data set. The bacterial species cultured includedB. subtilus, B. licheniformis, andEscherichia coli. Environmental variables included acration and temperature. The chromatographic patterns were compared by using hierarchical cluster and principal component analysis to obtain a quantitative measure of their similarity and to establish the normal variability within a model data set. Statistical analysis of the data indicated that individual fermentations can be assigned to distinct clusters on the basis of their divergence from the model system. Altered environments and other species can be identified as outliers from the model set. These results show that pattern recognition analysis has direct applicability to monitoring fermentation processes.     

Bacteriophages ofBacillus subtilis: Comparison of different isolation techniques and possible use for classification ofBacillus subtilis strains

When different techniques are used for the isolation of bacteriophages ofBacillus subtilis a number of different phages may be obtained. Furthermore defective phages are found in old cultures of all strains ofB. subtilis tested so far. The possible use of the phages and the defective phages for classifyingB. subtilis strains into a number of groups according to their susceptibility to different phages and according to the presence of certain defective phages in the cells is discussed.