“Spore Plate Method” for Transformation of Steroids by Fungal Spores Entrapped in Silica Gel G

A new technique for investigating steroid biotransformations involving the use of glucose-treated Silica Gel G thin-layer chromatography plates spotted with fungal spores and steroid substrates is described. The conversion is followed by the detection and identification of steroid metabolites and is carried out on single plates by using the spores of different fungi. During the entire process, the spores remain on the original spots and microscopical examination revealed no germination. The method was successfully applied to as little as 30 μg of substrates, and a single plate could be used to detect the steroid metabolizing activity of spores of as many as 15 different cultures.     

Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.     

Toxicity of terpenes to spores and mycelium of Penicillium digitatum

Spores, although often considered metabolically inert, catalyze a variety of reactions. The use of spores instead of mycelium for bioconversions has several advantages. In this paper, we describe the difference in susceptibility of mycelium and spores against toxic substrates and products. A higher resistance of spores toward the toxic effects of bioconversion substrates and products is an advantage that has not been studied in detail until now. This paper shows that spores of Penicillium digitatum ATCC 201167 are on average over 2.5 times more resistant than mycelium toward the toxicity of substrates, intermediates, and products of the geraniol bioconversion pathway. Furthermore, the higher resistance of spores to citral was shown as an advantage in its biotransformation by P. digitatum. Using three different approaches the toxicity of the compounds were tested. The order of toxicity toward P. digitatum was, starting with the most toxic, citral > nerol/geraniol > geranic acid > methylheptenone > acetaldehyde.     

Enhancement of sporulation in species of Bipolaris, Curvularia, Drechslera, and Exserohilum by growth on cellulose-containing substrates

Nine species of Bipolaris, Curvularia, Drechslera, and Exserohilum were compared for sporulation on agar media and for enhancement of sporulation by growth on four cellulose-containing substrates (index card, filter paper, cheesecloth, cotton fabric). On two natural and one synthetic agar media, sporulation varied from profuse to nonexistent among three isolates of each species. Growth of all species on cellulose substrates resulted in large and significant increases in sporulation. Growth on index card pieces often provided the greatest increases, but no single substrate was superior for all species, and significant substrate × isolate interactions were observed within species. Overlay of filter paper onto whole colonies in agar plates resulted in 2 to 18-fold increases in sporulation for eight of nine species and production of spores in sufficient quantity for most experimental purposes. Overlay of soil dilution plates with filter paper to promote sporulation of colonies enabled detection of B. spicifera, B. hawaiiensis, C. lunata, and E. rostratum at relatively low population levels (≤1.3 × 10³ colony-forming units per gram of soil) in samples of a naturally infested soil. Results indicate that enhancement of sporulation by growth of species of Bipolaris, Curvularia, Drechslera, and Exserohilum on cellulose substrates may facilitate (i) their identification in culture, (ii) production of spores at relatively high concentrations, and (iii) detection and enumeration of these fungi in soil.     

Simple Method for the Separation of Ascospores

A simple method for the separation of ascospores is described. To isolate single spores from adhesive ascospores and the mycelium, the suspension was sucked through a combination of sintered-glass plates with different pore sizes.     

LIGHT BOUNDARIES AND THE COUPLED EFFECTS OF SURFACE HYDROPHOBICITY AND LIGHT ON SPORE SETTLEMENT IN THE BROWN ALGA HINCKSIA IRREGULARIS (PHAEOPHYCEAE) 1

We examined the effects of light and surface hydrophobicity individually and in tandem on Hincksia irregularis (Kützing) Amsler spore settlement. Hincksia irregularis spores were determined to be negatively phototactic by the use of computer‐assisted motion analysis. Spore settlement was significantly influenced by surface hydrophobicity and by light, individually and in tandem. Experiments conducted using culture plates modified to reduce well edge artifacts revealed significantly higher settlement on hydrophobic surfaces and in dark environments when compared with negatively charged surfaces and lighted environments. Experimental light/dark boundaries elicited distinct spore settlement responses, with spores displaying dissimilar settlement patterns on plates with different surface hydrophobicities. The results of this study indicate H. irregularis spores possess the capacity for complex responses to their environment. These complex responses may influence dispersal and aid spores in the detection of suitable settlement locations in marine microenvironments.     

The resistance of Bacillus atrophaeus spores to the bactericidal activity of peracetic acid is influenced by both the nature of the solid substrates and the mode of contamination

Aims: To evaluate the impact of the mode of contamination in relation with the nature of solid substrates on the resistance of spores of Bacillus atrophaeus ‐selected as surrogates of Bacillus anthracis‐ to a disinfectant, peracetic acid. Methods and Results: Six materials confronted in urban and military environments were selected for their different structural and physicochemical properties. In parallel, two modes of contamination were examined, i.e. deposition and immersion. Deposition was used to simulate contamination by an aerosol and immersion by an extended contact with liquids. A pronounced difference in the biocontamination levels and spatial organization of spores was observed depending on the mode of contamination and the nature of the solid substrate considered, with consequences on decontamination. Contamination by immersion led to lower efficiency of peracetic acid decontamination than contamination by deposition. Infiltration of spores into porous materials after immersion is one reason. In contrast, the deposition mode aggregates cells at the surface of materials, explaining the similar disinfecting behaviour of porous and nonporous substrates when considering this inoculation route. Conclusions: The inoculation route was shown to be as influential a parameter as material characteristics (porosity and wettability) for decontamination efficacy. Significance and Impact of the Study: These results provide comparative information for the decontamination of B. atrophaeus spores in function of the mode of contamination and the nature of solid substrates.     

Analysis of Airborne Actinomycete Spores with Fluorogenic Substrates

The reactions between seven fluorogenic substrates and different groups of enzymes, esterases, lipases, phosphatases, and dehydrogenases, were studied in a search for a new method for the detection of actinomycete spores. Fluorescence measurement was chosen as a fast and sensitive method for microbial analysis. The focus of the research was on the spores of important air contaminants: Streptomyces albus and Thermoactinomyces vulgaris. For the measurement of the enzymatic activity, the chosen fluorogenic substrate was added to a mixture of spores and nutrient media, and the resulting fluorescence was measured with a spectrofluorometer. Fluorogenic substrates were found to show enzymatic activities even for dormant spores. Comparison of the enzymatic activities of dormant spores with those of vegetative cells showed similarity of the enzymatic profiles but higher activity for vegetative cells. The increase of enzymatic activity from dormant spores to vegetative cells was not linear but fluctuating. The largest fluctuations were found after 4 to 5 h of incubation. The enzymatic activities of S. albus were 10 to 50 times lower than those of T. vulgaris, except for the dehydrogenase activity, which was seven times higher. These results indicate that analysis with fluorogenic substrates has the potential for becoming a fast and sensitive method for the enumeration and identification of airborne actinomycete spores.     

An optimized method for Aspergillus niger spore production on natural carrier substrates

Aspergillus niger spores have wide ranging applications in the fermentation industry as well as in wastewater treatment. We present an optimized method for production of A. niger spores on natural substrates such as rice, split pea, and millet. The specific productivity (number of spores per gram of dry substrate) was 31-fold greater and volumetric productivity was 750-fold greater compared to agar slopes. The important process variables were incubation temperature, moisture content, and inoculum quantity. We find that the optimal condition for total spore count is different from the viable spore count for millet. The optimum lies in a narrow region defined by the process parameters. Of the three substrates tested split pea gave the highest specific spore productivity of 3.1 x 10(10) spores per gram of dry substrate. This is the first report of systematic study on the effect of process parameters on spore viability. The method of A. niger spore production on natural substrate appears advantageous as compared to the currently practiced method in terms of scale-up, cost, and ease of operation.     

Morphological Observations of Diplodia maydis on Synthetic and Natural Substrates as Revealed by Scanning Electron Microscopy

Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion.     

Actinoplanetes in soil and on plant litter from freshwater habitats

An isolation method for actinoplanetes is described which uses the ability of the sporangium to withstand desiccation and release motile spores when subsequently rehydrated. The desiccation stage reduces the number of associated Gram negative bacteria which cause problems when attempts are made to isolate species on agar plates from natural substrates. The method has enabled the isolation of actinoplanetes directly from soil and plant litter and indirectly from soil with the aid of baits such as pollen and hair. Actinoplanetes appear to be common in dry soils collected from arid areas and sand dune systems. They are normally present on leaves submerged for a time in rivers, streams and lakes and subsequently deposited in heaps on shores or as debris on overhanging vegetation. An actinomycete resembling the 'spore-dome' organism recovered from leaves and twigs by Willoughby (1969) was frequently isolated during the course of these studies. Preliminary work suggests that different species can be recognized and could constitute a new genus within the family Streptomycetaceae.     

Transformation of steroids by mixed cultures

Some results of our studios on transformation of steroids by mixed culture fermentation are presented in this paper. Arthrobacter simplex was paired in turn with each of the following: Streptomyces roseochromogenes, Curvularia lunata, Absidia coerulea, and Aspergillus ochraceus. The steroid substrates examined for multiple transformation were 16α-hydroxy-cortexolone, 16α-hydroxy-cortexolone 16,17-acetonide, 9α-fluorohydrocortisone, 9α-fluorohydrocortisone 21-acetate, and 9α-fluorohydrocortisone 21-hemisuccinate. The effects of media, steroid substrate, and microbial interaction in a mixed culture on the induction and repression of steroid transforming enzymes were unique to each case studied. The reaction mechanism of the multiple steroid transformation was also found to vary from one mixed culture system to another. Two different reaction mechanisms were observed, namely, consecutive and parallel. In the former, one of the two enzymatic reactions always preceded the other, while in the latter, two different enzyme reactions occurred simultaneously, thereby giving rise to two different intermediates. Multiple transformation of steroids by a single step mixed culture fermentation has potential economic advantages.     

Certain aspects of transformation of 21-acetate of the Reichstein substance "S" by the culture of Tieghemella orchidis]

The transformation of acetate from the Reichstein substance "S" by the culture of Tieghemella orchidis was studied upon a single and fractional addition of the substrate. The effect of streptomycin sulphate, Tween-80, ethanol (steroid solvent) and steroid solution on the respiratory activity of the culture was investigated upon a single and fractional addition of the steroid. During transformation the respiratory activity of the culture decreased--by the end of the process (18-20 hours) it fell 2-2.5-fold upon a single addition of the steroid (1 g/l) and by 20% upon a fractional addition of the steroid (2 g/l). In the latter case the respiratory activity dropped step-by-step if the concentration of the steroid was 1.4-1.6 g/l; simultaneously the transformation activity declined.     

The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-density,homogeneous endospore monolayer deposition on test surfaces

Bacillus subtilis spores were deposited in high-density single layers on metal, glass, and polymer substrates using vacuum filtration followed by a wetted filter transfer step. Quantitative analysis of spore transfer was performed using culture-based and germinability assays, and spore distributions were observed with electron microscopy.     

Bacillus cereus cell and spore properties as influenced by the micro-structure of the medium

Aim:  To investigate the effect of different growth conditions on Bacillus cereus cell and spore properties.
Methods and Results:  Bacillus cereus was grown on agar plates with different surface water conditions (wet and dry) or viscosity. Cell populations displayed different types of behaviour, and heterogeneity was manifested in cell motility and dimension. Spore populations were heterogeneous regarding their properties, namely size and thermal resistance. The smallest spores were produced from flagellated cells, which also displayed jet-motility, growing on the wettest agar. Cytometric analysis also revealed within the smallest spores a sub-population labelled by propidium iodide (PI), indicating that spore populations were partly damaged. Nonmotile cells grown on diffusion-limiting media were elongated and produced the least thermal-resistant spores.
Conclusions:  The micro-structural properties of the media were found to influence cell and spore properties. Abundant surface water enabled flagellar motility and resulted in a heterogeneous cell and spore population, the latter including small and damaged spores. High viscosity gave rise to filamentous cells and more heat-sensitive spores.
Significance and Impact of the Study:  This study provides useful information on conditions resulting in heterogeneous populations of damaged and heat-sensitive spores.     

Relations between phenotypic changes of spores and biofilm production by Bacillus atrophaeus ATCC 9372 growing in solid-state fermentation

Bacillus spp. spores are usually obtained from strains cultivated in artificial media. However, in natural habitats, spores are predominantly formed from bacteria present in highly surface-associated communities of cells. Solid-state fermentation (SSF) is the culture method that best mimetizes the natural environment of many microorganisms that grow attached to the surface of solid particles. This study aims to confirm that sporulation through SSF of Bacillus atrophaeus occurs by biofilm formation and that this model of fermentation promotes important phenotypic changes in the spores. Sporulation on standard agar and by SSF with sand and sugarcane bagasse as support was followed by a comparative study of the formed spores. Growth characteristics, metabolic and enzymatic profiles confirmed that sporulation through SSF occurs by biofilm formation promoting important phenotypic changes. It was possible to demonstrate that spores coat had different structure and the presence of ridges only on SSF spores' surface. The sporulation conditions did not affect the dry-heat spore resistance. The type of support evaluated also influenced in the phenotypic alterations; however, the used substrates did not cause interference. This work provides novel information about B. atrophaeus response when submitted to different sporulation conditions and proposes a new concept about bacterial biofilm formation by SSF.     

Individual and Coupled Effects of Echinoderm Extracts and Surface Hydrophobicity on Spore Settlement and Germination in the Brown Alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30?min were >?400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were >?300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30?min and the settled spores allowed to subsequently germinate for 24?h. Spore germling numbers were consistently >?25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24?h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30?min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24?h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.     

Individual and coupled effects of echinoderm extracts and surface hydrophobicity on spore settlement and germination in the brown alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30 min were > 400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were > 300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30 min and the settled spores allowed to subsequently germinate for 24 h. Spore germling numbers were consistently > 25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24 h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30 min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24 h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.     

Protease and peptidase activities in growing and sporulating cells and dormant spores of Bacillus megaterium.

Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.