The effect of solid medium composition on growth and sporulation ofStreptomyces clavuligerus; spore viability during storage at +4°C

Summary The effect of solid medium composition and pH on growth and sporulation ofStreptomyces clavuligerus was studied in Petri dish and slant cultures at 30 °C. The extent of sporulation was observed under the microscope and the number of viable spores per slant was counted by serial dilution. Abundant aerial mycelium and sporulation were achieved with some of the media tested in this work. In Tris/HCl buffer (0.05 M, pH 7.2), containing 0.1% Tween 80, spores retained 28%, 17% and 2% viability at +4 °C after 1,9 and 17.5 weeks, respectively.     

Inducing sporulation in different strains of alternaria solani

Summary Ultraviolet light induced abundant sporulation in two, out of the three strains ofAlternaria solani studied. Scraped cultures produced larger number of spores than unscraped ones. Ten seconds' exposure was found optimum for maximum sporulation. The optimum temperature of incubation subsequent to irradiation was 20°C. Young cultures were more responsive to ultraviolet light than the older ones. However, old cultures were more tolerant to a greater time of exposure than the younger ones. Intense ultraviolet light greatly reduced or even completely inhibited sporulation whereas low intensity of ultraviolet light was less effective in inducing sporulation. More irradiations than one greatly enhanced sporulation which reached its maximum with four irradiations. Spore length was considerably influenced by the age of the mycelium, temperature of incubation and the intensity and number of irradiations.     

The effect of sporulation temperature on sporal characteristics ofBacillus subtilis A

Spores ofBacillus subtilis A were produced at different temperatures (23°–49°C) and examined for a number of sporal characteristics. Spore heat resistance increased with sporulation temperature to 45°C, with spores grown at 49°C showing a dramatic reduction in resistance. Spore crops showed biphasic thermal death curves whether enumerated on germination medium with or without calcium dipicolinate. This strain produces both rough and smooth variants. Of the spores produced at 23°C, 99% were rough, had a density of 1.305, and an average core/core + cortex volume ratio of 0.1838. At 49°C, 99% were smooth, had a density of 1.275, and an average volume ratio of 0.3098. Between these temperatures both spore types were produced. There appeared to be no direct correlation with sporulation temperature, heat resistance, and dipicolinate content. There was an increase in both the magnesium and calcium contents to 45°C with a dramatic reduction at 49°C. The 1.305 density spores had higher calcium and dipicolinate contents than the 1.275 spores, although both spore types showed biphasic thermal death curves. The mechanisms involved in determining which spore type (rough/smooth) is produced at a specific growth temperature is unknown.Florida Agricultural Experiment Station Journal Series Number R-00312.     

The effect of the pH of the sporulation environment on the heat resistance ofClostridium perfringens spores

The inactivation ofClostridium perfringens NCTC 8239 spores at 95° and 105° C, as determined by colony formation on an agar base containing lysozyme (BASE + lysozyme), was influenced by the initial pH of the sporulation medium. In the pH range of 7.0–8.5, established by the addition of each of several biological buffers or carbonate buffer to Duncan-Strong (DS) medium, increased pH resulted in formation of spores with greater resistance to inactivation at elevated temperatures. An increase of pH from 8.5 to 9.0 resulted in increased resistance of spores formed in DS-carbonate but not DS-TAPS (N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid) medium. Resistance to spore injury, as determined by reduced recovery on BASE compared with BASE + lysozyme, was not increased for spores formed in media with higher pH's. As the pH of the medium increased, cell growth and number of spores formed were decreased, but the percentage of sporulation was apparently not affected.     

Analysis of Metabolism in Dormant Spores of Bacillus Species by 31P Nuclear Magnetic Resonance Analysis of Low-Molecular-Weight Compounds

This work was undertaken to obtain information on levels of metabolism in dormant spores of Bacillus species incubated for weeks at physiological temperatures. Spores of Bacillus megaterium and Bacillus subtilis strains were harvested shortly after release from sporangia and incubated under various conditions, and dormant spore metabolism was monitored by ³¹P nuclear magnetic resonance (NMR) analysis of molecules including 3-phosphoglyceric acid (3PGA) and ribonucleotides. Incubation for up to 30 days at 4, 37, or 50°C in water, at 37 or 50°C in buffer to raise the spore core pH from ∼ 6.3 to 7.8, or at 4°C in spent sporulation medium caused no significant changes in ribonucleotide or 3PGA levels. Stage I germinated spores of Bacillus megaterium that had slightly increased core water content and a core pH of 7.8 also did not degrade 3PGA and accumulated no ribonucleotides, including ATP, during incubation for 8 days at 37°C in buffered saline. In contrast, spores incubated for up to 30 days at 37 or 50°C in spent sporulation medium degraded significant amounts of 3PGA and accumulated ribonucleotides, indicative of RNA degradation, and these processes were increased in B. megaterium spores with a core pH of ∼7.8. However, no ATP was accumulated in these spores. These data indicate that spores of Bacillus species stored in water or buffer at low or high temperatures exhibited minimal, if any, metabolism of endogenous compounds, even when the spore core pH was 7.8 and core water content was increased somewhat. However, there was some metabolism in spores stored in spent sporulation medium.     

Altered heat resistance in spores and vegetative cells of a mutant fromBacillus subtilis

The relationship between sporulation temperature and spore killing temperature is described.Bacillus subtilis YB886, grown and sporulated at 25°, 30°, 37°, and 45°C, produced spores having D₉₀ values of 63.5, 76.3, 89.0, and 106 min respectively. In addition, the vegetative cells of this strain also demonstrated resistance to heat killing when grown at elevated temperatures (D₅₀ of 26.6, 32.5, 39.0, and >50 min for cells grown at 25°, 30°, 37°, and 45°C). A transposon-generated mutant of strain YB886, designated as BUL786, which is missing a heat shock-induced protein (97 kDa) (Qoronfleh MW and Streips UN, BBRC, 138:526–532, 1986 and FEMS 1987), was tested for thermotolerance under similar conditions. The cells failed to respond to growth at high temperature by producing heat-resistant spores or vegetative cells. For strain BUL786 the D₉₀ of spores generated at 20°, 25°, 30°, 37°, and 45°C was 9.4, 11.3, 12.8, 14.1, and 20 min, respectively. Similarly, the D₅₀ of vegetative cells was 15, 16.8, 17.8, 19.0, and 22.3 min when the cells were grown at 20°, 25°, 30°, 37°, and 45°C. Also, sporulation of YB886 cells in the presence of cadmium chloride increased the D₉₀ values for the resulting spores (5µM CdCl₂ resulted in a D₉₀ of 160 min). Strain BUL786 failed to produce spores with any elevated D₉₀ when grown in the presence of CdCl₂.     

Rapid sporulation of Bacillus anthracis in a high iron, glucose-free medium

Spores are the infectious form of Bacillus anthracis (BA), causing cutaneous, inhalation and gastrointestinal anthrax. Because of the possible use of BA spores in a bioterrorism attack, there is considerable interest in studying spore biology. In the laboratory, however, it takes a number of days to prepare spores. Standard sporulation protocols, such as the use of ‘PA broth’, allow sporulation of BA to occur in 3 to 5 days. Another method employs growth of BA on plates in the dark for several days until they have efficiently sporulated. In efforts to determine the effect of iron on gene expression in BA, we grew BA Sterne strain 7702 in a minimal defined medium (CDM; Koppisch et al., 2005) with various concentrations of iron and glucose. As part of our initial observations, we monitored BA sporulation in CDM via light microscopy. In glucose-free CDM containing 1.5 mM Fe(NO₃)₃ (CDM-Fe), > 95% of the BA sporulated by 30 h; a far shorter time period than expected. We pursued this observation and we further characterized spores derived from PA and CDM-Fe media. Purified spores derived from PA or CDM-Fe had similar morphologies when viewed by light or electron microscopy, and were equally resistant to harsh conditions including heat (65 °C), ice and fresh 30% H₂O₂. Spore viability in long term cold storage in water was similar for the two spore preparations. Extracted spore coat proteins were evaluated by SDS-PAGE and silver staining, which revealed distinct protein profiles for PA and CDM-Fe spore coat extracts. ELISA assays were done to compare the interaction of the two spore preparations with rabbit antiserum raised against UV-killed Sterne strain 7702 spores prepared in PA medium. Spores from both media reacted identically with this antiserum. Finally, the interaction and fate of spores incubated with macrophages in vitro was very similar. In summary, BA spores induced in CDM-Fe or in PA medium are similar by several criteria, but show distinct extractable coat proteins. CDM-Fe liquid medium can be used for rapid production of BA spores, and could save considerable time in spore research studies.     

Production of Vesicular-Arbuscular Mycorrhizal Fungus Inoculum in Aeroponic Culture

Bahia grass (Paspalum notatum) and industrial sweet potato (Ipomoea batatas) colonized by Glomus deserticola, G. etunicatum, and G. intraradices were grown in aeroponic cultures. After 12 to 14 weeks, all roots were colonized by the inoculated vesicular-arbuscular mycorrhizal fungi. Abundant vesicles and arbuscules formed in the roots, and profuse sporulation was detected intra-and extraradically. Within each fungal species, industrial sweet potato contained significantly more roots and spores per plant than bahia grass did, although the percent root colonization was similar for both hosts. Mean percent root colonization and sporulation per centimeter of colonized root generally increased with time, although with some treatments colonization declined by week 14. Spore production ranged from 4 spores per cm of colonized root for G. etunicatum to 51 spores per cm for G. intraradices. Infectivity trials with root inocula resulted in a mean of 38, 45, and 28% of bahia grass roots colonized by G. deserticola, G. etunicatum, and G. intraradices, respectively. The germination rate of G. etunicatum spores produced in soil was significantly higher than that produced in aeroponic cultures (64% versus 46%) after a 2-week incubation at 28°C. However, infectivity studies comparing G. etunicatum spores from soil and aeroponic culture indicated no biological differences between the spore sources. Aeroponically produced G. deserticola and G. etunicatum inocula retained their infectivity after cold storage (4°C) in either sterile water or moist vermiculite for at least 4 and 9 months, respectively.     

Temperature sensitivity of flocculation induction,conjugation and sporulation in fission yeast

Homothallic cultures of Schizosaccharomyces pombe, anaerobically grown to stationary phase in broth at 32°C, were induced by aeration to flocculate. Flocculation was followed by copulation, conjugation, zygote formation, meiosis and sporulation. Cultures grown to stationary phase at 32°C and then aerated at 37°C did not sporulate. Grown to stationary phase at 37°C, cultures were not immediately inducible when aerated at 32°C. To identify which events in the developmental sequence were thermosensitive, we grew and induced cultures at 32°C and then shifted them at various times to 37°C. We observed the following events to be thermosensitive: development of respiratory sufficiency, readiness (inducibility of a culture within 1 h), flocculation induction, copulation, conjugation and early sporulation (including meiosis). Respiration, flocculation and spore maturation were thermoresistant. Conjugation-induced lysis and post-developmental deflocculation were enhanced at 37°C.NRCC no. 17775     

Behaviour of proteolytic Clostridium botulinum types A and B near the lower temperature limits of growth

Summary The behaviour of spores of Clostridium botulinum type A and proteolytic C. botulinum type B has been studied in cooked meat medium at 10°C, 12°C, 15°C, and 20°C, using mixed cultures (9 groups of in total 41 strains) and pure cultures (41 strains).At 10°C a decrease of 1–1.5 log cycles for type B and of 2–4 log cycles for type A Clostridia was observed. Neither growth nor toxin formation could be demonstrated.At 12°C spores of some strains developed and formed toxin with 3–4 weeks, whereas other strains did not develop within 7 weeks.At 15°C growth and toxin formation could be observed within 1 week, whereas at 20°C toxin was formed mostly within 2 or 3 days. Incubation at 10°C prior to incubation at 20°C seemed to have some effect on the lag time.     

Hormonal growth-promotant effects on grain-fed cattle maintained under different environments

Six steers (3/4 Charolais×1/4 Brahman) (mean body weight 314±27 kg) and six spayed heifers (3/5 Shorthorn×2/5 Red Angus) (mean body weight 478±30 kg) were used to determine the effects of climatic conditions and hormone growth promotants (HGP) on respiration rate (RR; breaths/min), pulse rate (beats/min), rectal temperature (RT; °C), and heat production (HP; kJ). Cattle were exposed to the following climatic conditions prior to implantation with a HGP and then again 12 days after implantation: 2 days of thermoneutral conditions (TNL) [21.9±0.9°C ambient temperature (T_A) and 61.7±22.1% relative humidity (RH)] then 2 days of hot conditions [HOT; 29.2±4°C (T_A) and 78.3±13.2% (RH)], then TNL for 3 days and then 2 days of cold conditions [COLD; 17.6±0.9°C (T_A) and 63.4±1.8% (RH); cattle were wet during this treatment]. The HGP implants used were: estrogenic implant (E), trenbolone acetate implant (TBA), or both (ET). Both prior to and following administration of HGP, RRs were lower (P<0.05) on cold days and greater (P<0.05) on hot days compared to TNL. On hot days, RTs, were 0.62°C higher after compared to before implanting. Across all conditions, RTs were >0.5°C greater (P<0.05) for E cattle than for TBA or ET cattle. On cold days, RTs of steers were >0.8°C higher than for the heifers, while under TNL and HOT, RTs of steers were 0.2–0.35°C higher than those of heifers. Prior to implantation, HP per hour and per unit of metabolic body weight was higher (P<0.05) for cattle exposed to hot conditions, when compared to HP on cold days. After implantation, HP was greater (P<0.05) on hot days than on cold days. Under TNL, ET cattle had the lowest HP and greatest feed intake. On hot days, E cattle had the lowest HP, and the highest RT; therefore, if the potential exists for cattle death from heat episodes, the use of either TBA or ET may be preferred. Under cold conditions HP was similar among implant groups.     

Studies on anaerobic proteolytic bacteria of Leh,India

Five anaerobic proteolytic bacteria were isolated from water bodies of Leh, India, where the ambient temperature varies from –25 to 25 °C. Isolates showed growth at all temperatures ranging from 5 to 37 °C except SPL-4 and SPL-5 which showed no growth at 5 °C. The cultures could grow and produce proteases on various protein substrates and the yield varied with the substrates. Two of the cultures showed the presence of spores. Acetate was the dominant VFA during hydrolysis of protein substrates.     

Water availability affects the growth,accumulation of compatible solutes and the viability of the biocontrol agent Epicoccum nigrum

Growth of the biocontrol fungus Epicoccum nigrum was more sensitive to ionic solute water stress (NaCl) than non-ionic (glycerol) on potato dextrose-based media at –0.5, –3.0 and –5.5 MPa water potentials. Subsequent physiological manipulation of growth of E. nigrum in glycerol-modified media to –3.0 MPa water potential resulted in a significant increase in the accumulation of compatible solutes in both mycelial liquid cultures and spores, but no enhanced accumulation of the desiccation protectant trehalose, when compared to unmodified media (–0.5MPa). The main solute accumulated was glycerol, followed by arabitol. In temporal studies over 20 days maximum accumulation of glycerol occurred in 5-d old cultures with water stressed cultures having 250× greater amounts than those from unmodified medium. The arabitol content was also higher in mycelium and spores produced under water stress. The difference was maximum after 15 days growth. Glucose content decreased over time in mycelial colonies but increased in spores. The germination of conidia from the two treatments was similar, regardless of compatible solute content, even at –9.25 MPa water potential stress. However, germ tube extension was significantly increased at this water potential level. The production of E. nigrum spores at –3.0 MPa water potential resulted in improved survival when stored fresh at 4 and 25 °C. However, freeze-drying severely affected the viability of spores produced on both media (–0.5 or 3.0 MPa). Accumulation of compatible solutes may assist the fungus in better ecological competence and establishment in the phyllosphere, where water availability is often limited.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effects of L-proline and post-plating temperature treatment on Maize (Zea mays L.) anther culture

Summary Comparison of different post-plating temperature regimes with a control treatment (27° C) revealed that a short-term cold (8/14°C:2/2 days or 14°C:4 days) as well as a heat treatment (30°C:14 days) increased the production of embryro-like-structures (ELS) from cultured maize anthers. The beneficial effects of short-term cold treatments were magnified 2–3 times when L-proline (PROL) was added to the induction medium (125–500 mg/L). In the best treatment (14°C:4 days, 125 mg/L L-proline) one genotype produced 143.5 ELS/100 anthers. Anthers subjected to high temperature (30°C:4 days, 30°C:7 days, 30°C:14 days) generally showed a lower response than did cold treated anthers, although genotypic differences were observed. Regeneration frequency did not appear to be affected by the presence of L-proline in the induction medium.Abbreviations ELS Embryo-like-structures - PROL L-proline     

Inducing sporulation in Alternaria solani II. Effect of light

Among the three different light sources viz. incandescent electric light, infra-red light and sunlight, only sunlight was effective in inducing sporulation in petri dish cultures ofA. solani. The intensity of sporulation depended upon the age and growth stage of the cultures, duration and number of exposures to light and the presence or absence of a sporulating zone in the culture. Maximum sporulation was obtained in the case of 6 days old partially grown cultures by inducing the formation of sporulating zone which appeared in 24 hours after every exposure of 60 minutes to sunlight.     

Sporulation of Streptomyces venezuelae in submerged cultures

Shaken cultures of Streptomyces venezuelae ISP5230 in minimal medium with galactose and ammonium sulphate as carbon and nitrogen sources, respectively, showed extensive sporulation after 72 h incubation at 37 degrees C. The spores formed in these cultures resembled aerial spores in their characteristics. The ability of the spores to withstand lysozyme treatment was used to monitor the progress of sporulation in cultures and to determine the physiological requirements for sporulation. In media containing ammonium sulphate as the nitrogen source, galactose was the best of six carbon sources tested. With galactose S. venezuelae ISP5230 sporulated when supplied with any of several nitrogen sources; however, an excess of nitrogen source was inhibitory. In cultures containing galactose and ammonium sulphate, sporulation was suppressed by a peptone supplement. The onset of sporulation was accompanied by a drop in intracellular GTP content. When decoyinine, an inhibitor of GMP synthase, was added to a medium containing starch and ammonium sulphate, a slight increase in sporulation was seen after 2 d. The suppression of sporulation by peptone in liquid or agar cultures was not reversed by addition of decoyinine. A hypersporulating mutant of S. venezuelae ISP5230 was altered in its ability to assimilate sugars. In cultures containing glucose the mutant sporulated more profusely than did the wild-type and did not acidify the medium to the same extent. However, the suppressive effect of glucose on sporulation was not merely a secondary result of acid accumulation.     

The effects of warming by active and passive means on the subsequent responses to cold water immersion

Two experiments were undertaken to investigate the effects of warming the body upon the responses during a subsequent cold water immersion (CWI). In both experiments the subjects, wearing swimming costumes, undertook two 45-min CWIs in water at 15° C. In experiment 1, 12 subjects exercised on a cycle ergometer until their rectal temperatures (T _re) rose by an average of 0.73°C. They were then immediately immersed in the cold water. Before their other CWI they rested seated on a cycle ergometer (control condition). In experiment 2, 16 different subjects were immersed in a hot bath (40° C) until their T _re rose by an average of 0.9° C; they were then immediately immersed in the cold water. Before their other CWI they were immersed in thermoneutral water (35° C; control condition). Heart rate in both experiments and respiratory frequency in experiment 1 were significantly (P < 0.05) higher during the first 30 s of CWI following active warming. In experiment 1, the rate of fall of T _re during the final 15 min of CWI was significantly (P < 0.01) faster when CWI followed active warming (2.46° C · h^–1) compared with the control condition (1.68°C · h^–1). However, this rate was observed when absolute T _re was still above that seen in the control CWIs. It is possible, therefore, that if longer CWIs had been undertaken, the two temperature curves may have converged and thereafter fallen at similar rates; this was the case with the aural temperature (T _au) seen in experiment 1 and the T _au and T _re in experiment 2. It is concluded that pre-warming is neither beneficial nor detrimental to survival prospects during a subsequent CWI.     

Activation ofStreptomyces viridochromogenes spores by detergents

Constitutively dormant spores ofStreptomyces viridochromogenes germinate rapidly following treatment with 1.0% of the detergents Tween 80, sodium dodecyl sulfate (SDS), or sodium heptadodecyl sulfate. Six other detergents did not activate the spores. Activation by SDS was studied further. The spores were not activated following treatment with 0.09% or less of SDS for 60 min at 37°C. Activation was complete within 1 to 2 min of treatment with 1.0% SDS. the SDS-activated spores became deactivated during incubation in buffer. Deactivation was slow at 4°C and complete after incubation for 12 h at 25°C or 6 h at 37°C. The endogenous respiratory rate of the spores was increased 3-fold by SDS activation.     

Net CO2 output by CAM plants in the light: the role of leaf conductance

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-l-(l,2,4-triazol-l-yl)-2-butanone] is a triazoie fungicide which protects bean ( Phaseolus vulgaris L. cv. Spring Green) plants from heat and chilling injury. When the plants were exposed to heat shock by dipping the shoots in hot (50°C) distilled water for 2 min or exposing the plants to cold (1°C) for 8 h the primary leaves showed visual symptoms of injury 2 days after treatment and thereafter there was a progressive decline in chlorophyll and an increase in electrolyte leakage indicative of a loss of membrane integrity. There was a loss of metabolic (respiratory) activity in the root meristems when the roots were dipped in hot (48°C) water. All these symptoms of heat and chilling injury in the controls were either delayed or prevented by root application of triadimefon.     

Heat resistance in Rhizobium

Summary Seven of eight Rhizobium strains survived 70° C but not 80° C for half an hour in liquid media. The proportion of cells surviving heat treatment varied with the composition of the media and the age of the cultures, but did never exceed 0.7%. It appears that this heat resistance is not an effect of sporulation, according to the classical definition of a spore. Spore staining gave always negative results. However, spore-like structures could be seen in cells of heat resistant cultures. But it was not possible to correlate the number of these cells with the number of heat resistant cells. There was no difference between cells surviving heat treatment and untreated cells concerning infectivity and effectivity.