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.     

Application of a simple yeast extract from spent brewer's yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a Physiological Study

The suitability of using a simple brewer's yeast extract (BYE), prepared by autolysis of complete beer slurry, for growth and sporulation of Bacillus thuringiensis kurstaki was studied in baffled shake flasks. In a standard buffered medium with 2.5% (w/v) glucose and 1% (w/v) brewer's yeast extract, growth of B. t. kurstaki resulted in a low biomass production with considerable byproduct formation, including organic acids and a concomitant low medium pH, incomplete glucose utilization and marginal sporulation, whereas growth in the same medium with a commercial laboratory-grade yeast extract (Difco) resulted in a high biomass concentration, complete glucose utilization, relatively low levels of byproducts and complete sporulation (2.6 × 10⁹ spores/ml). When glucose was left out of the medium, however, growth parameters and sporulation were comparable for BYE and commercial yeast extract, but absolute biomass levels and spore counts were low. Iron was subsequently identified as a limiting factor in BYE. After addition of 3 mg iron sulphate/l, biomass formation in BYE-medium more than doubled, low byproduct formation was observed, and complete sporulation occurred (2.8 × 10⁹spores/ml). These data were slightly lower than those obtained in media with commercial yeast extract (3.6 × 10⁹spores/ml), which also benefited, but to a smaller extent, from addition of iron.     

Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties

Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores’ inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50–150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l‐PG) synthesis exhibited a 30–50% decrease. Spore sensitivity to H₂O₂ and tert‐butylhydroperoxide was increased 30–60% in the absence of the major CL synthase, but these spores’ sensitivity to NaOCl or Oxone^? was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l‐PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10‐fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore’s inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.     

Sporulation of Streptomyces griseus in submerged culture.

A wild-type strain of Streptomyces griseus forms spores both on solid media (aerial spores) and in liquid culture (submerged spores). Both spore types are highly resistant to sonication, but only aerial spores are resistant to lysozyme digestion. Electron micrographs suggest that lysozyme sensitivity may result from the thinner walls of the submerged spores. Studies of the life cycle indicate that neither streptomycin excretion nor extracellular protease activity is required for sporulation: the analysis of mutants, however, suggests that antibiotic production may be correlated with the ability to sporulate. A method was devised to induce the rapid sporulation of S. griseus in a submerged culture. This method, which depends on nutrient deprivation, was used to determine that either ammonia or phosphate starvation can trigger sporulation and that the enzyme glutamine synthetase may be useful as a sporulation marker after phosphate deprivation.     

脱落酸对丛枝菌根真菌侵染和产孢的调控效应研究

【目的】揭示脱落酸(ABA)对丛枝菌根(AM)真菌侵染和产孢的影响,建立利用外源ABA促进孢子产量的高效菌剂扩繁方法。【方法】利用番茄毛状根和AM真菌Rhizophagus irregularis DAOM 197198建立双重培养体系,通过外源施用ABA、赤霉素(GA)或者使用ABA、GA的缺陷突变体,染色观察菌根侵染,荧光定量PCR测定丛枝发育和脂质合成运输相关基因的表达,统计丛枝和孢子的数量,从而揭示ABA对AM真菌侵染和产孢的影响。【结果】ABA缺陷突变体not中的F%(侵染频率)、a%(丛枝丰度)、丛枝数量,以及丛枝发育特异性相关基因EXO70A1-like (LOC101253481)、脂质合成运输相关基因RAM2和STR2的表达均显著低于其野生型MT;外源施用ABA显著促进了F%、M%(侵染强度)、丛枝数量、孢子产量,以及脂质合成运输相关基因RAM2和STR2的表达,外源添加ABA处理的孢子产量约为不添加处理的4.5倍;外源GA处理极显著抑制了菌根侵染的所有指标和孢子产量;GA缺陷突变体gib3与其野生型MM的AM真菌侵染之间没有显著差异,但gib3的孢子产量显著高于MM...     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.     

MonitoringAspergillus fumigatus aerosols from a composting facility

The large, outdoor Islip Yard Waste Composting Facility on Long Island, New York was investigated as a source of airborne fungus spores. The Burkard-Hirst volumetric spore trap was used for the first extensive sampling of small mold spores for this application. Samplers were operated continuously from 21 August to 30 November 1992 in the facility and in a suburban community about 540 m from the facility. A control site approximately 10 000 m from the facility was also sampled to establish background levels of fungus spores. The facility site had higher average readings ofAspergillus fumigatus spores than did the community and both were higher than the control.A. fumigatus was the only fungus among 30 categories tracked that differed significantly between the facility and control sites. It was also isolated repeatedly from the compost. Higher average levels ofA. fumigatus were measured in the community when winds blew from the facility, and also during times when the compost was moved or mixed at the facility. No correlation was found between wind direction or work times andA. fumigatus conidia at the control site. The study shows that this compost facility can produce a measurable increase in the number of airborneA. fumigatus conidia both at the edge of the facility and at 540 m downwind. It also demonstrates that the Burkard spore trap can be used for monitoring small, airborne mold spores, but it is a difficult and labor intensive task.     

Chitinase and protease activities in germinating zoospore cysts of a parasitic fungus,Aphanomyces astaci,Oomycetes

In germinating spores of the parasitic fungus, Aphanomyces astaci, chitinase was first demonstrated shortly before the germ-tube began to branch, in contrast to protease which was present in both ungerminated and germinated spores. The time at which chitinase would be required when this fungus penetrates the crayfish cuticle is correlated with that of the in vitro production of chitinase.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

Bicarbonate is a stimulus in the inter-species induced sporulation of strict anaerobic Syntrophomonas erecta subsp. sporosyntropha

Previously Syntrophomonas species had been described as the bacteria those did not form spores, however, in our previous studies, a newly isolated S. erecta subsp. sporosyntropha JCM13344^T was found to form spores in the co-culture with methanogens, while not in mono-culture or in co-culture with sulfate reducer. In this study, we examined the sporulation stimulus conferred by methanogens in the co-culture. By reducing bicarbonate in mono-culture and sulfate-reducing co-culture, we could induce S. erecta subsp. sporosyntropha JCM13344^T to form spores, so that bicarbonate at lower concentration was determined as another stimulus for sporulation. Based on the substrate degradation by strain JCM13344^T in different concentration of bicarbonate vs at different pHs, it was suggested that bicarbonate could stimulate the sporulation by mediating a nutrient deprivation but not pH drop. To further confirm the sporulation potential of this group of bacteria, spo0A fragments were amplified from strain JCM13344^T as well as all the recognized Syntrophomonas species, confirming that they were members of the spore-forming group. Since sporulation is a kind of response of spore-forming bacteria to environmental stresses, the observation in this work implies that stresses can be created even between the mutual beneficial partners, in this case, inducing sporulation.     

Genetic instability of sporulation-associated characters in a Bacillus subtilis mutant: relationship between sporulation, segregation and the synthesis of extracellular enzymes (kinetic studies).

In the genetically unstable, protease-overproducing 'medusa (M) strains of Bacillus subtilis, segregation of stable, wild-type-like B cells occurred mainly during sporulation. After the end of the exponential growth phase, a small fraction of M cells sporulated quickly and formed M spores, while the majority of the cells, after a 'critical period', gave rise to B segregants which sporulated after a delay. Segregation occurred without cell division. Delayed sporulation, segregation and protease overproduction are related. Similar but more complex results were obtained with the highly unstable TD strains. Sporulation and the kinetics of protease overproduction were also followed in several stable segregants. Depending on the strain, either the rate of protease production or both the rate and time course were affected. The results are interpreted in terms of sequential activation and de-activation of sporulation genes. The production of the alkaline and the neutral proteases was, in general, under common genetic control. In some strains alpha-amylase was also overproduced.     

Comparative immunological study of catalases in the genus Micrococcus

Intracellular proteases from sporulating Bacillus cereus have been purified by ammonium sulfate fractionation, heat treatment and DEAE cellulose column chromatography. After the last purification step, two protease activities, with an activity ratio of about thirty to one are resolved. Both proteases are resistant to o-phenanthroline but sensitive to phenyl methyl sulfonyl fluoride. Their separation by polyacrylamide gel electrophoresis and DEAE cellulose column chromatography, their difference in heat sensitivity and a mutation affecting only the major intracellular protease (IP1) suggest that the two are products of distinct genes. An IP1 mutant previously shown to produce coat defective spores (4) also turnsover protein with a reduced rate during late sporulation stages. Correlated with the slower turnover rate in this mutant is the more rapid disappearance of IP1. A partial revertant of this mutant has a protein turnover rate intermediate between that of the original mutant and wild type. These correlations imply that IP1 has an important role in protein turnover during sporulation.     

Germination initiation and outgrowth of spores ofBacillus brevis strain Nagano and its gramicidin S-negative mutant

Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to germination of their spores produced in several media. Germination initiation occurred in the presence of nutrient broth orL-alanine but not with inosine, glucose, glycerol or fructose; the process was activated by heat. Parental and mutant spores behaved similarly in these experiments. During outgrowth, parental spores remained in this phase of germination much longer than did mutant spores, but only when the parental spores had been harvested from a sporulation medium where significant gramicidin S synthesis had occurred. When parental spores were extracted or treated with an enzyme that hydrolyzes gramicidin S, rapid outgrowth occurred. Adding exogenous gramicidin S or the extract from parental spores to mutant spores lengthened the outgrowth in a dose-dependent manner. The uptake of labeledL-alanine by parental spores was delayed compared to mutant spores in the presence or absence of chloramphenicol. These data suggest a mechanism of action for gramicidin S whereby it interferes in membrane function, such as transport or energy metabolism, in outgrowing spores.Abbreviations GS Gramicidin S - CFU colony-forming units     

Does Methyl Salicylate, A Component of Herbivore-induced Plant Odour, Promote Sporulation of the Mite-pathogenic Fungus Neozygites tanajoae?

Blends of volatile chemicals emanating from cassava leaves infested by the cassava green mite were found to promote conidiation of Neozygites tanajoae, an entomopathogenic fungus specific to this mite. Methyl salicylate (MeSA) is one compound frequently present in blends of herbivore-induced plant volatiles (HIPV) as well as that of mite-infested cassava. Here, we investigated the effect of methyl salicylate in its pure form on the production of pre-infective spores (conidia), and the germination of these spores into infective spores (capilliconidia), by a Brazilian isolate and a Beninese isolate of N. tanajoae. Mummified mites previously infected by the fungal isolates were screened under optimal abiotic conditions for sporulation inside tightly closed boxes with or without methyl salicylate diffusing from a capillary tube. Production of conidia was consistently higher (37%) when the Beninese isolate was exposed to MeSA than when not exposed to it (305.5 ± 52.62 and 223.2 ± 38.13 conidia per mummy with and without MeSA, respectively). MeSA, however, did not promote conidia production by the Brazilian isolate (387.4 ± 44.74 and 415.8 ± 57.95 conidia per mummy with and without MeSA, respectively). Germination of the conidia into capilliconidia was not affected by MeSA for either isolate (0.2%, 252.6 ± 31.80 vs. 253.0 ± 36.65 for the Beninese isolate and 4.2%, 268.5 ± 37.90 vs. 280.2 ± 29.43 for the Brazilian isolate). The effects of MeSA on the production of conidia were similar to those obtained under exposure to the complete blends of HIPV for the case of the Beninese isolate, but dissimilar (no promoting effect of MeSA) for the case of the Brazilian isolate. This shows that MeSA, being one compound out of many HIPV, can be a factor promoting sporulation of N. tanajoae, but it may not be the only factor as its effect varies with the fungal isolate under study.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

The response to a specific germinant by <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spores in primary mouse macrophages is modulated by a protein encoded on the pXO1 plasmid

A Bacillus anthracis Sterne pXO1 plasmid-encoded protein designated Cot43 was found in coat extracts of purified spores. Cot43 is a tetratricopeptide repeat domain protein related to those which function as phosphatases in the sporulation phosphorelay and as regulators of competence and pathogenic factors. The synthesis of Cot43 began in the late exponential phase downstream from a sigmaA promoter (as mapped by RACE) and it was present at least until the formation of phase white endospores. There was specificity in the association of Cot43 with B. anthracis spores since Bacillus cereus producing Cot43 from a cloned gene had very little of this protein in spore coat extracts. In addition, Cot43 was synthesized by B. anthracis cells to the same extent in glucose-yeast extract and nutrient sporulation media, but was essentially absent from spores formed in the former. l-histidine is an important germinant for B. anthracis spores in macrophages, Spores produced by a mutant with a disruption of cot43 germinated in response to l-histidine both in vitro and within primary mouse macrophages earlier and more extensively than Sterne strain spores. The germination delay due to the presence of Cot43 would enhance spore survival and thus increase the chances for a successful infection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The role of fungal proteinases in pathophysiology of <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis>

The adverse health effects of Stachybotrys chartarum have often been linked to exposure to the trichothecene mycotoxins. Recent studies have shown that in addition to mycotoxins this fungus is capable of producing and secreting in vivo proteins such as hemolysins and proteinases. Spore extracts obtained from a high trichothecene producing isolate JS 58-17 exhibited a significantly lower proteolytic activity compared to the low trichothecene producer, JS 58-06. Growing isolates on rice or potato dextrose agar results in higher proteolytic activity of the spores compared to those grown on drywall. Proteinases in the spore extracts can hydrolyze gelatin and collagen I and IV. Analysis of zymograms shows the presence of several proteins with proteolytic activity in the spores of S. chartarum. Human tracheal epithelial cells exposed to spore extracts produced significantly higher levels of IL-6, IL-8, and TNF-α than control cells. This stimulation of cytokine production was completely abolished by Pefabloc, a serine protease inhibitor. Neutrophil numbers and proinflammatory cytokine (IL1-β and TNF-α) concentrations were highly elevated in the lungs of 7 day old rat pups exposed intratracheally to 4 × 10⁴ spores/gm body weight compared to control. No significant differences in those inflammatory indices in vivo were noted between the treatments with the high trichothecene producer, isolate JS 58-17 and JS 58-06, which does not produce macrocyclic trichothecenes. Immunohistochemistry revealed reduced collagen IV labeling in spore-induced lung granulomas in rat pups exposed to both isolates. These results suggest that proteinases from S. chartarum spores significantly contribute to lung inflammation and injury.     

A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe Streptomyces coelicolor A3(2)

The Gram‐positive aerobe Streptomyces coelicolor undergoes a complex life cycle including growth as vegetative hyphae and the production of aerial hyphae and spores. Little is known about how spores retain viability in the presence of oxygen; however, nothing is known about this process during anaerobiosis. Here, we demonstrate that one of the three respiratory nitrate reductases, Nar‐1, synthesized by S. coelicolor is functional exclusively in spores. A tight coupling between nitrite production and the activity of the cytoplasmically oriented Nar‐1 enzyme was demonstrated. No exogenous electron donor was required to drive nitrate reduction, which indicates that spore storage compounds are used as electron donors. Oxygen reversibly inhibited nitrate reduction by spores but not by spore extracts, suggesting that nitrate transport might be the target of oxygen inhibition. Nar‐1 activity required no de novo protein synthesis indicating that Nar‐1 is synthesized during sporulation and remains in a latently active state throughout the lifetime of the spore. Remarkably, the rates of oxygen and of nitrate reduction by wetted spores were comparable. Together, these findings suggest that S. coelicolor spores have the potential to maintain a membrane potential using nitrate as an alternative electron acceptor.     

不同基质多代培养对蚧虫病原真菌蜡蚧霉致病力的影响

【目的】研究昆虫病原真菌蜡蚧霉Lecanicilliurn lecanii(Zimmermann.)菌株No.V3.4504在不同培养基上继代培养,对菌种的菌落生长特性、胞外酶活力和对蚧虫致病力的影响。【方法】试验菌种蜡蚧霉菌株No.V3.4504是从染病蚧虫上分离的。试验蚧虫是沙里院褐球蚧Rhodococcus sariuoni Borchsenius和日本龟蜡蚧Ceroplastes japonicus Green。采用7种培养基继代培养多代。观察菌落形态特征、测定生长速率、产孢量、胞外蛋白酶和几丁质酶活性及对蚧虫的致死率。【结果】在PDA培养基上,菌落生长速率最快,但产孢量最低,胞外蛋白酶和几丁质酶的活性均呈逐代下降趋势,对两种蚧虫致死率也最低;增加蛋白胨对改善菌种致病力没有明显效果;在增加蚧虫尸体的D、E、F培养基上,菌落生长速率虽然较慢,但产孢量上升为8.83×106-9.13×106孢子/cm2。蛋白酶和几丁质酶的活性平均达到2.16-2.13 U/g和1.01-1.03 U/g,对两种蚧虫的致死率分别在55%-58%和39%-42%;在活蚧虫上连续培养3代,蛋白酶和几丁质酶的活性最高,为3.08-2.92 U/g和1.45-1.42 U/g,是PDA培养基上的1.6倍。对两种蚧虫的致死率也最高,分别达到71.30%和58.89%。蛋白酶和几丁质酶的活性与蚧虫死亡率呈正直线相关关系。【结论】采用PDA培养基连续多代培养会引起菌株No.V3.4504明显退化;在培养基中加入蚧虫尸体,对于保持菌种活力有明显效果;在活蚧虫体上继代培养对复壮菌种,提高菌种毒力的效果最佳。     

In vitro spore formation and completion of the asexual life cycle of Neozygites parvispora, an obligate biotrophic pathogen of thrips

Capilliconidia, the asexual secondary spores of Neozygites parvispora (Zygomycetes, Entomophthorales) were produced in vitro either by entrapment of vegetative cells (hyphal bodies) in alginate pellets or after plating them onto water agar. Cultivation of the fungus for 3 days in a medium lacking hemolymph increased spore production 30 to 40-fold, and about 10% of the cells produced capilliconidia. The in vitro produced capilliconidia were infectious to Thrips tabaci and the fungus was reisolated from infected insects, thus completing its asexual life cycle under laboratory conditions. A decrease in capilliconidia production and a modification of the number of nuclei per spore were observed for isolates cultivated in vitro for more than 2 months, but subsequent host passages restored and increased sporulation efficiency without influencing the number of nuclei. Fungal cultures were stored at —80 °C for up to 7 months, and the capability to sporulate and infect T. tabaci was preserved. A bioassay procedure for infecting T. tabaci with N. parvispora is described, the first mycosed insects dying usually after 8 d of incubation.