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.     

Screening of filamentous fungi for lipase production: Hypocrea pseudokoningii a new producer with a high biotechnological potential

Abstract

Filamentous fungi isolated from soil samples were screened for extracellular lipase production. The best producer was Hypocrea pseudokoningii identified by taxonomical criteria, and by rDNA sequencing of the variable internal transcribed spacers (ITS I and II) and the intervening 5.8S gene. The fungus was grown in a complex medium supplemented with 1% Tween 80 and 0.2% yeast extract, for 4 days. The optimum pH for extracellular and intracellular lipases was 7.0 and 8.0, respectively. Both enzymes exhibited maximum activity at 40°C. Extracellular and intracellular lipase activities were highly stable in the pH range 3.0–8.0 at room temperature. The intracellular lipase was thermostable up to 60°C, for 15 min and the extracellular, for 107 min, at the same temperature. The intracellular lipase was stimulated by silver ions. Extracellular lipase was stable in organic solvents, such as DMSO, alcohols, acetone, and acetonitrile, for 24 hours. Lipase activity increased around 80% when detergents were added to the enzymatic assay, such as Tween 80, Triton X-100, and SDS.     

Germination and Survival of Fusarium graminearum Macroconidia as Affected by Environmental Factors

The effects of temperature (4–20°C), relative humidity (RH, 0–100%), pH (3–7), availability of nutrients (0–5 g/l sucrose) and artificial light (0–494 μmol/m²/s) on macroconidial germination of Fusarium graminearum were studied. Germ tubes emerged between 2 and 6 h after inoculation at 100% RH and 20°C. Incubation in light (205 ± 14 μmol/m/s) retarded the germination for approximately 0.5 h in comparison with incubation in darkness. The times required for 50% of the macroconidia to germinate were 3.5 h at 20°C, 5.4 h at 14°C and 26.3 h at 4°C. No germination was observed after an incubation period of 18 h at 20°C in darkness at RH less than 80%. At RH greater than 80%, germination increased with humidity. Germination was observed when macroconidia were incubated in glucose (5 g/l) or sucrose (concentration range from 2.5 × 10^?4 to 5 g/l) whereas no germination was observed when macroconidia were incubated in sterile deionized water up to 22 h. Macroconidia germinated quantitatively within 18 h at pH 3–7. Repeated freezing (?15°C) and thawing (20°C) water agar plates with either germinated or non‐germinated macroconidia for up to five times did not prevent fungal growth after thawing. However, the fungal growth rate of mycelium was negatively related to the number of freezing events the non‐germinated macroconidia experienced. The fungal growth rate of mycelium was not significantly affected by the number of freezing events the germinated spores experienced. Incubation of macroconidia at low humidity (0–53% RH) suppressed germination and decreased the viability of the spores.     

Effect of Temperature on Germination of Spores from Some Bacillus and Clostridium Species

The effect of temperature on germination of spores of Bacillus subyilis, B. megaterium. B. cereus, Clostridium sporogenes, Cl. butyricum and Cl. bifermentans was studied. At lower temperatures (+5°C to +10°C) the three Glostridium species germinated to a less extent than the three Bacillus. species. The optimum temperature for germination of the six species varied between +35°C and +45°C. The Clostridium species were more tolerant to heat than the Bacillus species.     

An effective method based on wet-heat treatment for the selective isolation of Micromonospora from estuarine sediments

Several methods for the isolation of Micromonospora from soil samples have been developed; however, it is unclear whether these methods are optimal for estuarine samples. In this study, we optimized the conditions of a wet-heat method for the selective isolation of Micromonospora from estuarine sediments. Sediments were collected from the Arakawa River (estuarine sediments) and Tokyo Bay (marine sediments). Sediment samples were wet-heated at 45, 55, or 65 °C for 30 min and then incubated at 27 °C for 3 weeks. After incubation, most of the actinomycete colonies were macroscopically determined to be of the genus Micromonospora or Streptomyces. In contrast to the treatment at 55 °C, treatment at 65 °C drastically reduced the number of Streptomyces colonies but increased the number of Micromonospora colonies from the estuarine sediments. This procedure allowed us to grow cultures that were composed of more than 90 % Micromonospora. In addition, treatment at 65 °C did not affect the diversity of Micromonospora species compared with treatment at 55 °C. These results indicate that the wet-heat method, which involves pre-treating the sediment at 65 °C for 30 min, is a very simple and effective method for the selective enrichment of a large number of diverse Micromonospora from estuarine sediments. Our results may lead to the isolation of new Micromonospora species, which produce novel bioactive compounds, from different estuarine sediments.     

Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis

Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90°C and between pH 6.5 and 10; the half-life of the enzymes at 75°C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53–2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.     

Cloning and characterization of a novel GH44 family endoglucanase from mangrove soil metagenomic library

A novel endoglucanase gene, mgcel44, was isolated from a mangrove soil metagenomic library by functional-based screening. It encodes a 648-aa peptide with a catalytic domain of glycosyl hydrolase family 44. The deduced amino acid sequence of mgcel44 shares less than 50 % identity with endoglucanases in GenBank database. mgcel44 was cloned and overexpressed in Escherichia coli. The recombinant enzyme, MgCel44, has a molecular mass of 70.8 kDa as determined by SDS-PAGE. Its optimal pH and temperature for activity were 6 and 45 °C, respectively. It was highly active at 25–45 °C and pH 5–8. Its activity was enhanced in 0.5 M NaCl by >1.6-fold and stable up to 1.5 M NaCl. MgCel44 was resistant to several organic solvents and had high activity at 15 % (v/v) solvent after incubating for 24 h at 25 °C.     

Effect of Variation of Sporulation Time and Temperature on Thermostability of Bacillus cereus Spores

The optimum temperature for sporulation of a strain of Bacillus cereus was estimated at 30°–35°C, where the maximum yield of spores was obtained between 18 and 24 hours’ incubation. Sporulation was more rapid, but less extensive at 40°C and did not occur at all at 45°C. The heat resistance of the spores increased with the sporulation temperature from 20° to 40°C. The spores appear to be more susceptible to heat destruction in the early stage of spore production than after further incubation.     

Properties and Substrate Specificities of Four Esterases from Aspergillus niger NRRL 337

Properties and substrate specificities of four esterases (Esterase-I, -II, -III, -IV) from Aspergillus niger were studied. Esterase-I and Esterase-II were found to be markedly stable to heat. When Esterase-I was assayed at 35°C using methylacetylsalicylate as a substrate, even after heating at 100°C for 15 min 60% of its activity remained. However, Esterase-I scarcely hydrolyzed the substrate at 70°C or over, because of a reversible change in conformation by heating as found by CD measurement. The maximum activity of Esterase-I was found at 55°C at 20 min of reaction time. Esterase-II was stable up to 80°C and had an optimum temperature for reaction at 80°C, but was irreversively inactivated by heating for 15 min at 90°C.

The four esterases hydrolyzed aliphatic esters of short chain fatty acids and acetyl esters of phenols, but neither methyl esters of aromatic carboxylic acids nor acetyl esters of aromatic alcohols.     

Organic solvent tolerance of an alkaline protease from salt-tolerant alkaliphilic <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> strain Mit-1

A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol, benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol; the enzyme was highly active at 60–80°C and displayed optimum activity at 70°C. The protease was significantly stable and catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis of the enzyme adversely. Crude enzyme preparation was more stable at 37°C in solvents as compared to partially purified and purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant protease from salt-tolerant alkaliphilic actinomycetes.     

Bacillus subtilis builds structurally and functionally different spores in response to the temperature of growth

Bacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH-dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat-labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.     

Proceedings: Preservation of rust fungi in liquid nitrogen

Spores of rust fungi can be expected to retain viability without loss of infectivity for at least several years when stored in liquid nitrogen (?196 °C). Addition of liquid suspending media is harmful and not necessary. Some rust fungi experience cold-induced dormancy when exposed to less than 0 °C for a minute or longer but germinability is dependably restored on applying a heat shock by heating the spores to 40 °C for at least 15 sec during or after thawing. Most rust fungi are not sensitive to moisture content at the time of freezing but Puccinia striiformis must be vacuum dried before freezimg. The need for heat shock may not show up until several days after thawing. All of the rust strains tested to date have retained their properties to the extent tested and for the duration of storage. Data are available for up to 11 years. Preliminary experiments to preserve saprophytic mycelial cultures of P. graminis have so far failed, with and without use of 10% glycerol and 5% DMSO. The successful preservation of rust spores has made feasible the development of a collection of living rust fungi at ATCC beginning in 1965 and which now has over 80 strains in 20 species in 7 genera.     

Predicting patterns of post-fire germination in 35 eastern Australian Fabaceae

Germination in 35 species from 15 legume genera of southeastern Australia was promoted by a heat treatment which broke the seed coatcaused dormancy. Once the critical temperature was reached, most seeds had their dormancy broken, independent of the duration of heating. Species fell into three classes according to whether their dormancy was broken by a temperature of 40, 60 or 80°C. Highest germination in all species was achieved by heating in the temperature range 80–100°C, although long durations (120 min) at 100°C caused seed death in several species. At 120°C, seeds of most species were killed at all but one minute's duration. A proportion of seeds from 7 species (Acacia myrtifolia, Pultenaea daphnoides, P. incurvata, P. linophylla, P. polifolia, Dillwynia floribunda and Sphaerolobium vimineurn) was not killed at 120°C and had their dormancy broken. This proportion varied markedly and resultant germination levels were significantly less than those at 80 and 100°C, except in S. vimineum. Between-site variations in the 4 species tested (A. myrtifolia, A. suaveolens, A. terminalis and A. ulicifolia) were small. These variations concerned: (i) the minimum temperature required to break seed dormancy in 2 species: 60°C in one population of A. myrtifolia and A. suaveolens, and 80°C in the other; and (ii) the intensity of the germination response. Duration of heating was less important than temperature as a determinant of germination. Ordination techniques revealed that results from one duration across temperatures were comparable with data from multiple durations. This has significant applications in studying rare species, where seed may be in short supply. Predicted germination levels after a moderate intensity fire should far exceed those after a low intensity fire. Little germination was predicted for many species after a low intensity fire and for one species, A. elongata, no germination was predicted. The potential role of indicator species in relation to the maintenance of species in a community is suggested.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP?g^?1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^?1 µg^?1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

CHANGES IN THE HEAT-RESISTANCE OF ASCOSPORES OF NEUROSPORA UPON GERMINATION

Lingappa , Yamuna , and A. S. Sussman . (U. Michigan, Ann Arbor.) Changes in the heat-resistance of ascospores of Neurospora upon germination. Amer. Jour. Bot. 46 (9): 671–678. Illus. 1959.—A rapid loss in heat-resistance accompanies activation of ascospores of Neurospora tetrasperma after incubation at 27°C. When activated spores are given a 5-min. “heat-flash” at 65°C. after only 5 min. at 27°C., fully % fail to germinate. Such treatment, if administered 25 min. after activation, results in the complete destruction of the spores. By contrast, when incubation at 27°C. is not interposed, more than ½ of the spores will germinate, even when they have been exposed to 65°C. for 30 min. Similar results were obtained with “heat-flashes” at 50 and 60°C., although exposures of longer duration were required to affect the spores. Conidia respond very differently to “heat-flashes” in that germination is stimulated if they are provided after an incubation period at 27°C. On the other hand, conidia are killed by short exposures to 60°C., so that they are far more susceptible to such treatment than are ascospores. A study of the cardinal temperatures of germination revealed that the maximum is about 44°C. for both conidia and ascospores. The maximum for the growth of two strains of N. tetrasperma and for one of N. crassa is between 40–45°C.; however, another strain of the latter species grows at 45°C. Dry heat was shown to be less effective than wet in activating ascospores. Removal of the exospore of ascospores results in the loss of considerable heat-resistance. In addition, the requirement for heat-activation is considerably mitigated in such spores, suggesting that the exospore, or an associated layer is the locus of the ascospore's heat-resistance.     

Laboratory experiments on sugar-beet downy mildew (Peronospora farinosa)

The optimum conditions for Peronospora farinosa betae to produce spores were temperature 8–10 °C and relative humidity 90 % or more, but many spores were produced between 5 and 20 °C and between 80 and 90 % R.H. Most spores were formed in darkness after leaves were exposed to light for 6–8 h. Spores survived exposure to 60 % R.H. for up to 5 days, but were soon killed by temperatures above 20 °C. The germination capacity of spores collected from the field was often very small, but this could not be related to the weather. Most seedlings were infected when inoculated at the growing point and incubated in a saturated atmosphere between 3 and 15 °C for at least 8 h.     

Biochemical Properties of A Thermophilic Alkalophile

A thermophilic alkalophile (IC strain) which can grow well in an alkaline medium at over 55°C was isolated from soil samples, and identified as Bacillus licheniformis; its growth on a neutral medium was, however, very poor. This strain was able to grow at 37°C as well as at 55°C, but the specific growth rate at 55°C was about twice as high as that at 37°C under alkaliné conditions.

The intracellular pH remained below 9.5 when Na⁺ was present in the medium. Na ⁺ stimulated the alanine uptake by cells or membrane vesicles, but was not required ATP synthesis.

Intracellular enzymes were stable on heat treatment up to 60°C. The residual activity of enolase after heating at 60°C for 10 min was about 80%. Cytochrome oxidase in membrane vesicles was completely stable up to 58°C for 30 min. These enzymes were also resistant to SDS treatment, more than 50% of their activities remaining at 5% SDS.     

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35?kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60?°C. It was determined that the enzyme had remained stable at the range of pH 7.0–10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20–80?°C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. K_M and V_max values were calculated as 0.197?mg/mL and 7.29?μmol.mL^?1.min^?1, respectively.     

Life from the ashes: survival of dry bacterial spores after very high temperature exposure

We found that spores of Bacillus amyloliquefaciens rank amongst the most resistant to high temperatures with a maximum dry heat tolerance determined at 420 °C. We found that this extreme heat resistance was also maintained after several generations suggesting that the DNA was able to replicate after exposure to these temperatures. Nonetheless, amplifying the bacterial DNA using BOXA1R and (GTG)₅ primers was unsuccessful immediately after extreme heating, but was successful after incubation of the heated then cooled spores. Moreover, enzymes such as amylases and proteases were active directly after heating and spore regeneration, indicating that DNA coding for these enzymes were not degraded at these temperatures. Our results suggest that extensive DNA damage may occur in spores of B. amyloliquefaciens directly after an extreme heat shock. However, the successful germination of spores after inoculation and incubation indicates that these spores could have a very effective DNA repair mechanism, most likely protein-based, able to function after exposure to temperatures up to 420 °C. Therefore, we propose that B. amyloliquefaciens is one of the most heat resistant life forms known to science and can be used as a model organism for studying heat resistance and DNA repair. Furthermore, the extremely high temperature resistivity of these spores has exceptional consequences for general methodology, such as the use of dry heat sterilization and, therefore, virtually all studies in the broad area of high temperature biology.

     

A cotyledon double inoculation technique for evaluating resistance to anthracnose (Colletotrichum orbiculare) and scab (Cladosporium cucumerinum) in cucumber

A technique for simultaneous inoculation of cucumber cotyledons with Colletotrichum orbiculare race 1 and Cladosporium cucumerinum has been developed. The procedure permitted both resistant and susceptible plants to be recovered. Seedlings were grown at 20°C and inoculated 24 h after emergence with Colletotrichum orbiculare (200 spores in 2 μ1 of water) and Cladosporium cucumerinum (1000 spores in 5 μ1 of water) followed by 48 h of incubation in the dark at 20°C and 100% r.h., and 48 h in a 20°C lighted growth chamber. Seedlings were then moved to a growth chamber at 21°C at night and at 26°C during the day for 4 days and plants were rated as resistant or susceptible 8 days after inoculation. No interference in the expression of resistance or susceptibility of cultivars to either pathogen was detected in simultaneous inoculations.