Beginning Of Sporulation Research Articles

Paleoparasitological analysis of a coprolite assigned to a carnivoran mammal from the Upper Pleistocene Touro Passo Formation, Rio Grande do Sul, Brazil.

A paleoparasitological analysis was carried out on a large coprolite assigned to a carnivoran mammal, recovered from the Municipality of Uruguaiana, in the western region of the State of Rio Grande do Sul, Brazil, where the Upper Pleistocene Touro Passo Formation crops out. For this, an individual sample was extracted from the specimen using an electric drill, dissociated with 10% hydrochloric acid solution, washed with distilled water, and sifted through a 500 mesh Tyler sieve. After laboratory processing, the sediment retained on the sieve was mixed with glycerin and examined by optical microscopy, which revealed the presence of 14 protozoan oocysts and three nematode eggs. The morphological characteristics of the oocysts (i.e., spherical shape, thick-walled, internal zygote apparently at the beginning of sporulation, as well as their size) and of the eggs (i.e., ovoidal shape, rounded ends, smooth surface, thin-shelled, embryo in their interior, along with their morphometry) suggest that these specimens belong respectively to the orders Eucoccidiorida and Strongylida (Family Ancylostomatidae) represented by several parasitic species of the alimentary tract of modern carnivore. This is the first record of paleoparasites discovered in a vertebrate host from the Touro Passo Formation.

Quantification of incubation, latent and infection periods of Phakopsora pachyrhizi in soybean, according to chronological time and degree-days

ABSTRACT In experiments conducted in a growth chamber, the chronological time and the accumulated degree-days were determined for the duration of incubation, latent and infectious periods of Phakopsora pachyrhizi cultivars BRSGO 7560 and BRS 246 RR. Detached soybean leaflets were placed in gerbox-type acrylic boxes and inoculated with 20 x 103 uredospores/mL. The study was conducted at 12-h photoperiod and temperatures of 10ºC, 15ºC, 22ºC, 25ºC and 30°C for 30 days. Lesions and uredia/cm2were evaluated and the number of uredia per lesion was quantified after the beginning of sporulation. The sporulation potential was also quantified for cultivars BRSGO 7560 and BRS 246 RR. The steps of the infection process can be quantified based on both the chronological time and the accumulated heat. The cultivar BRSGO 7560 produced 4,012.8 spores/cm2 and BRS 246 RR, 7,348.4 uredospores/cm2. The largest number of uredia was produced at 25ºC in both cultivars; however, BRS 246 RR presented 372.7 uredia/cm2 and BRSGO 7560, 231.6 uredia/cm2. At 10ºC and 30°C, leaf infection did not occur in both cultivars.

Aeration effects on metabolic events during sporulation of Bacillus thuringiensis.

The metabolism of Bacillus thuringiensis during its sporulation process was investigated under different concentrations of oxygen. At the beginning of sporulation, the aeration conditions were regulated to obtain different oxygen transfer rates (OTR) in four separate fermentations, representing interrupted, limited, non-limited, and saturated oxygenation, respectively. A higher OTR resulted in a higher pH, up to about 9 in the case of saturated oxygenation, while the interrupted oxygenation resulted in a significantly acidic culture. In contrast, the absence of oxygen resulted in rapid sporangia lysis and caused acidification of the medium, indicating a distinctly different sporangia composition and different metabolism. The bacterium also showed different CO2 production rates during sporulation, although a maximum point was observed in every case.With a higher OTR, the maximal value was observed after a longer time and at a lower value (40, 26, and 13 mmol/L/h for limited, non-limited, and saturated cases, respectively). Despite the exhaustion of glucose prior to the sporulation phase, the interrupted oxygenation resulted in acetate, lactate, and citrate in the medium with a maximum concentration of 4.8, 1.3, and 5.0 g/L, respectively. Notwithstanding, while the metabolic events differed visibly in the absence of oxygen, once sporulation was triggered, it was completed, even in the case of an interrupted oxygen supply.

Novel antilisterial bacteriocin licheniocin 50.2 from Bacillus licheniformis VPS50.2 isolated from soil sample

To isolate and characterize bacteriocin, licheniocin 50.2, from soil bacteria identified as Bacillus licheniformis. The strain B.licheniformis VPS50.2 was identified as bacteriocin producer, effective against Gram-positive bacteria, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and β-haemolytic streptococci. The start of bacteriocin production coincides with the beginning of sporulation. Ammonium sulfate precipitation, chloroform extraction and ultrafiltration were used for bacteriocin purification. MALDI TOF/TOF mass spectrometry of purified sample detected the protein with molecular mass of 3253·209Da. N-terminal sequencing recognized first 15 amino acids with the sequence: W E E Y N I I X Q L G N K G Q. We named the newly characterized bacteriocin as subclass II.3 bacteriocin, licheniocin 50·2. The bacteriocin activity was insensitive to lysozyme and proteinase K, heat stable after incubation at 100°C for 30min and over wide range of pH (2-12). MICs of crude bacteriocin extract were determined for L.monocytogenes and MRSA. Time-kill study showed that licheniocin had bactericidal effect to L.monocytogenes. A novel, thermostable, pH-tolerant bacteriocin active against Gram-positive bacteria was isolated. Attributes of new, stable licheniocin 50.2 make it a promising agent for application as biopreservative in food industry.

Production and Release of Asexual Sporangia in Plasmopara viticola

To study the influence of environmental conditions on sporulation of Plasmopara viticola lesions under vineyard's conditions, unsprayed vines were inspected every second or third day and the numbers of sporulating and nonsporulating lesions were counted in two North Italy vineyards in 2008 to 2010. Infected leaves were removed so that only fresh lesions were assessed at each field assessment. Sporulation was studied at two scales, across field assessments and across the seasonal population of lesions. Frequencies of sporulating lesions were positively correlated with the numbers of moist hours in the preceding dark period (i.e., the number of hours between 8:00 p.m. and 7:00 a.m. with relative humidity ≥80%, rainfall >0 mm, or wetness duration >30 min). In a receiver operating characteristic analysis, predicted sporulation based on the occurrence of ≥3 moist hours at night provided overall accuracy of 0.85. To study the time course of sporulation on lesions which were not washed by rainfall, numbers of sporangia produced per square millimeter of lesion were estimated on individual cohorts of lesions over the whole infectious period. The numbers of sporangia per square millimeter of lesion increased rapidly during the first 4 days after the beginning of sporulation and then tapered off prior to a halt; the time course of cumulative sporangia production by a lesion followed a monomolecular growth model (R(2) = 0.97). The total number of sporangia produced by a square millimeter of lesion increased as the maximum temperature decreased and moist hours in the dark increased. To study the release pattern of the sporangia, spore samplers were placed near grapevines with sporulating lesions. Airborne sporangia were caught in 91.2% of the days over a wide range of weather conditions, including rainless periods. The results of this study provide quantitative information on production of P. viticola sporangia that may help refine epidemiological models used as decision aids in grape disease management programs.

Some Genetic, Biochemical and Morphological Analysis of Selected Powdery Mildew Strains at the Beginning of Sporulation on Barley

The present work analyzes some characteristics of four powdery mildew pathotypes, RU-3, Sk-5/11, Sk-12/1 and A-4/0, selected from the wild strains of BGH from Central European regions. Our results showed that the studied BGH strains differ in the virulence and avirulence genes in their genomes, in prolongation of their asexual phase of the growth and also in morphological and biochemical characteristics. Protein analysis confirmed the genetic differences between the studied powdery mildew pathotypes. Abundant acid glucanases in all studied BHG pathotypes were found between molecular weights Mr ? 25-35 kDa and 11-22kDa. Races RU-3 and A-4/0 also contained low molecular weight glucanases with Mr ? 9-14kDa. Immunological analyses showed higher specificity of pathogen chitinases to plant antibody compared to barley cultivars carrying different dominant/semidominant resistance genes. Rabbit antibody prepared against the plant interacellular acid chitinase Chi 14.4 (PR-4) gave the positive signal for two powdery mildew races, Sk-5/11 and A-4/0. These pathotypes were more aggressive compared to races Sk-12/1 and RU-3. Their genomes contained more virulence genes and asexual phase of the growth was shorter. Ultrastructural analyses of BGH body in the sensitive barley cultivar cells, showed presence of virus like particles, which probably play role by the synthesis of some PR-proteins with hydrolytic function. Genetic and biochemical analyses indicate that some powdery mildew pathotypes contain genes in their genome which are orthological to those in their hosts, which makes them suitable subjects for the future as a source of new resistance genes for plant breeding.

Evaluation of whole cells of Bacillus subtilis as substrate for measurement of autolysin activity

Autolysins are a group of endogenous bacteriolytic enzymes that hydrolyze specific bonds in the cell wall peptidoglycan. The conventional method for determining autolysin activity employs purified autolysinless cell walls as substrate. In this study, preparation of autolysinless whole cells of Bacillus subtilis 168 as an alternative to cell walls is described. The protocol developed is a simple, single step procedure and gives improved yields. The enzyme, autolysin was extracted from B. subtilis 168 using 4 M lithium chloride (LiCl). Substrate was prepared by treating vegetative whole cells of B. subtilis 168 with 10 M LiCl. Autolysin activity was measured as the reduction in optical density of autolysinless whole cells at 600 nm. This method of analysis was applied to estimate the autolysin levels at different growth stages of B. subtilis 168. The autolysin levels were found to be maximum in cells at three stages of growth; start of cell separation, beginning of sporulation and during mother cell lysis. This confirms the established role of autolysins in these cellular processes.

MP030 Characterization of the adhesive properties of a novel autolysin/adhesin from Staphylococcus epidermidis

Autolysins are a group of endogenous bacteriolytic enzymes that hydrolyze specific bonds in the cell wall peptidoglycan. The conventional method for determining autolysin activity employs purified autolysinless cell walls as substrate. In this study, preparation of autolysinless whole cells of Bacillus subtilis 168 as an alternative to cell walls is described. The protocol developed is a simple, single step procedure and gives improved yields. The enzyme, autolysin was extracted from B. subtilis 168 using 4 M lithium chloride (LiCl). Substrate was prepared by treating vegetative whole cells of B. subtilis 168 with 10 M LiCl. Autolysin activity was measured as the reduction in optical density of autolysinless whole cells at 600 nm. This method of analysis was applied to estimate the autolysin levels at different growth stages of B. subtilis 168. The autolysin levels were found to be maximum in cells at three stages of growth; start of cell separation, beginning of sporulation and during mother cell lysis. This confirms the established role of autolysins in these cellular processes.

Purification, kinetic properties, and intracellular concentration of SpoIIE, an integral membrane protein that regulates sporulation in Bacillus subtilis.

SpoIIE is a bifunctional protein which controls sigmaF activation and formation of the asymmetric septum in sporulating Bacillus subtilis. The spoIIE gene of B. subtilis has now been overexpressed in Escherichia coli, and SpoIIE has been purified by anion-exchange chromatography and affinity chromatography. Kinetic studies showed that the rate of dephosphorylation of SpoIIAA-P by purified SpoIIE in vitro was 100 times greater, on a molar basis, than the rate of phosphorylation of SpoIIAA by SpoIIAB. The intracellular concentrations of SpoIIE and SpoIIAB were measured by quantitative immunoblotting between 0 and 4 h after the beginning of sporulation. The facts that these concentrations were very similar at hour 2 and that SpoIIE could be readily detected before asymmetric septation suggest that SpoIIE activity may be strongly regulated.

Secretion, localization, and antibacterial activity of TasA, a Bacillus subtilis spore-associated protein.

The synthesis and subcellular localization of the proteins that comprise the Bacillus subtilis spore are under a variety of complex controls. To better understand these controls, we have identified and characterized a 31-kDa sporulation protein, called TasA, which is secreted into the culture medium early in sporulation and is also incorporated into the spore. TasA synthesis begins approximately 30 min after the onset of sporulation and requires the sporulation transcription factor genes spo0H and spo0A. The first 81 nucleotides of tasA encode a 27-amino-acid sequence that resembles a signal peptide and which is missing from TasA isolated from a sporulating cell lysate. In B. subtilis cells unable to synthesize the signal peptidase SipW, TasA is not secreted, nor is it incorporated into spores. Cells unable to produce SipW produce a 34-kDa form of TasA, consistent with a failure to remove the N-terminal 27 amino acids. In cells engineered to express sipW and tasA during exponential growth, TasA migrates as a 31-kDa species and is secreted into the culture medium. These results indicate that SipW plays a crucial role in the export of TasA out of the cell and its incorporation into spores. Although TasA is dispensable for sporulation under laboratory conditions, we find that TasA has a broad-spectrum antibacterial activity. We discuss the possibility that during the beginning of sporulation as well as later, during germination, TasA inhibits other organisms in the environment, thus conferring a competitive advantage to the spore.

Contribution of partner switching and SpoIIAA cycling to regulation of sigmaF activity in sporulating Bacillus subtilis.

sigmaF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-sigma factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or sigmaF depends on the nature of the adenine nucleotide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, sigmaF-SpoIIAB-ADP, and sigmaF-SpoIIAB-ATP. The results suggested that SpoIIAB's choice of binding partner is unlikely to depend on the ATP/ADP ratio in the cell. The intracellular concentrations of sigmaF, SpoIIAB, SpoIIAA, and SpoIIAA-phosphate (SpoIIAA-P) were measured by quantitative immunoblotting between 0 and 3 h after the beginning of sporulation (t0 to t3). sigmaF and SpoIIAB were barely detectable at t0, but their concentrations increased in parallel to reach maxima at about t1.5. SpoIIAA-P increased steadily to a maximum at t3, but nonphosphorylated SpoIIAA was detectable only from t1.5, reached a maximum at t2.5, and then declined. Kinetic studies of the phosphorylation of SpoIIAA catalyzed by SpoIIAB suggested that the reaction was limited by a very slow release of one of the products (SpoIIAA-P or ADP) from SpoIIAB, with a turnover of about once per 20 min. This remarkable kinetic property provides an unexpected mechanism for the regulation of sigmaF. We propose that when SpoIIE (which dephosphorylates SpoIIAA-P) is active at the same time as SpoIIAB, SpoIIAA cycles repeatedly between the phosphorylated and nonphosphorylated forms. This cycling sequesters SpoIIAB in a long-lived complex and prevents it from inhibiting sigmaF.

Transcriptional regulation of the cryIVD gene operon from Bacillus thuringiensis subsp. israelensis.

The CryIVD protein is involved in the overall toxicity of the Bacillus thuringiensis subsp. israelensis parasporal inclusions and is one of the four major components of the crystals. Determination of the DNA sequence indicated that the cryIVD gene is the second gene of an operon which includes three genes. The first one encodes a 19-kDa polypeptide and has sequence homology with the orf1 gene of the Bacillus thuringiensis cryIIA and cryIIC operons. The second and third genes have already been identified and encode the CryIVD crystal protein and the P20 polypeptide, respectively. The promoter region was located by deletion analysis, and the 5' end of the mRNA was determined by primer extension mapping. Transcription of the cryIVD gene in B. thuringiensis subsp. israelensis strains is induced 9 h after the beginning of sporulation. Sequence analysis indicated two potential promoters, a strong one and a weak one, recognized respectively by the RNA polymerase associated with the sigma 35 or the sigma 28 factor of B. thuringiensis (sigma E and sigma K of Bacillus subtilis, respectively). Transcriptional lacZ fusion integrated in single copy into the chromosome of various B. subtilis sporulation mutants confirmed the sigma E dependence of cryIVD gene transcription.

Studies on Antagonistic Effects of Trichoderma Isolates Against Phytophthora cactorum

AbstractBioassays were used to demonstrate the antibiotic effect of Trichoderma isolates on P. cactorum. When both fungi were grown on benomyl‐containing PDA medium, the mycelial growth of Trichoderma was suppressed. However, the production of antibiotics by this fungus remained active, leading to inhibition of the mycelial growth of P. cactorum. The antibiotic effect of Trichoderma on zoospores and cysts was tested on a PDA substrate precultured with Trichoderma on cellophane sheets. On the substrate of some Trichoderma isolates, lysis of zoospores, formation of extracellular vesicles, and hypertrophy of the water expulsion vesicle did occur, both resulting in the death of the zoospores. Conidial suspensions of Trichoderma isolates also induced zoospore lysis. It is presumed that membrane‐active peptide antibiotics (peptaibols) are involved in zoospore lysis. The peptaibol paracelsin caused lysins of zoospores at a concentration of 2.5 × 10−4 M. The effect on cysts depended on the Trichoderma isolate tested and the age of Trichoderma preculture. Old cultures (after beginning of sporulation) affected cysts more severely than young cultures (before sporulation) which usually were not lethal to the cysts but induced preferably microsporangium formation, inhibition of cyst germination, and retardation of germ tube growth.

Organization and regulation of an operon that encodes a sporulation-essential sigma factor in Bacillus subtilis

Deletion of sigE, the structural gene for the sporulation-induced RNA polymerase sigma factor, sigma E, prevented endospore formation by Bacillus subtilis. The effects of integration of plasmids into the sigE region of the chromosome and the use of complementation analyses demonstrated that sigE is part of an operon that includes a promoter-proximal gene, spoIIGA, that is essential for sporulation. Gene fusions to the promoter of this operon, spoIIG, demonstrated that transcription from this promoter is induced at the beginning of sporulation and is dependent on several spoO genes.

Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus.

Streptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.

Use of Temperature-sensitive Mutants to Study Gene Expression of Two Closely Linked Sporulation Loci in Bacillus subtilis

The spoOB and spoIVF loci are contiguous on the chromosome of Bacillus subtilis, so that genes in these loci may be parts of a single polycistronic operon. Temperature-sensitive strains having mutations in these loci were isolated, and temperature-shift experiments were carried out to investigate expression of the genes. The temperature-sensitive periods of spoOB mutants extended from the beginning of sporulation until the end of the stage II. The temperature-sensitive periods of spoIVF strains were during stage IV of sporulation. Therefore, although the spoOB and spoIVF loci are contiguous on the chromosome it is unlikely that genes in them are parts of a single polycistronic operon.

Isolation and characterization of a temperature-sensitive sporulation mutant of Bacillus subtilis.

Temperature-sensitive sporulation mutants were isolated spontaneously from Bacillus subtilis 168 TT by a sequential transfer method. A representative mutant strain, ts32, was characterized in detail. The mutant grew normally at 30°C and 42°C, but did not sporulate at 42°C. Electron microscopic observation and physiological analysis showed that the mutant was blocked at stage 0-1 of sporulation. Genetic analysis suggested that the mutation was located at the spo0B locus on the B. subtilis chromosome. Temperature-shift experiments clearly showed that the spo0B gene product functions only at the beginning of sporulation.

Alcohol-resistant sporulation mutants of Bacillus subtilis.

About 80% of Bacillus subtilis cells form spores when grown in nutrient broth. In medium containing various short-chain aliphatic alcohols, the frequency of sporulation was reduced to 0.5%. Mutants sporulated in the presence of alcohols at a frequency of 30 to 40%. Sporulation in the wild-type cells was sensitive to alcohol at the beginning of sporulation (stage zero). Sensitivity to alcohol in the mutants was also at stage zero, even though the sensitivity was considerably reduced. This sensitivity of sporulation to alcohol is the phenotypic expression of a genetic locus designated ssa. Mutations at this locus lead to a decreased sensitivity of sporulation to alcohol without modifying the sensitivity of growth. Genetic analysis by transduction was bacteriophage PBS1 revealed that ssa mutations are near the previously described spo0A locus. ssa mutants also differ from wild-type cells in the composition of membrane phospholipids. The relative amount of phosphatidylglycerol increased, whereas the relative amount of phosphatidylethanolamine and lysylphosphatidylglycerol decreased relative to the proportions in the wild type. The distribution of fatty acids in membrane lipids is the same as in the wild type. No differential sensitivity of phospholipid metabolism to alcohol could be detected in the mutant. This work therefore reveals that the extensive, pleiotropic changes in the membranes of ssa mutants are the phenotypic reflection of alterations at a specific gene locus.

Alteration of interaction with virulent bacteriophage Tal during differentiation of Thermoactinomyces vulgaris

AbstractDuring its life cycle Thermoactinomyces vulgaris 1227 and 1261 substrate mycelium showed three modes of interaction with the virulent phage Tal, each expressed at a distinct morphological stage. Primary mycelium arising from spores 50 min after start of germination was the only stage which propagated phages upon infection. However, infection of growing secondary mycelium or of late sporulation stages resulted in a loss of phage. The lack of phage production upon infection of growing secondary mycelium was not related to sporogenesis, since it appeared already 3.5 hours before the beginning of sporulation. If phages were added to the secondary mycelium at the beginning of spore formation, the phage genome became integrated in the developing spores in a heat‐stable state. Allowing outgrowth of these prophage‐carrier spores, phages were produced similarly as in germ tubes arising from normal spores infected at the time of inoculation.The growing secondary substrate mycelium was characterized by competence for the uptake of exogenous DNA. Since at the stage of competence phages were neither produced nor was phage DNA trapped, the earlier reported lack of transfection in undisturbed differentiating T. vulgaris is now understandable.

Glutamate in the initiation of spore development inBacillus polymyxa

The mechanism of initiation of spore development in bacteria is not clearly understood. It is supposed that the various metabolites that appear or disappear at the end of the growth phase and the beginning of sporulation provide the necessary signal for the sporulation process. For most of these metabolites it is not clear whether they act as sensors of nutritional deficiency, a prerequisite for microbial differentiation or they are the true signal for the activation of morphogenetic genes. Glutamate is one of the various metabolites that accumulate just before the start of the differentiation of spores in bacilli [1-3]. Although the influence of extracellular glutamate for sporulation has been emphasized [4,5] it has not been shown whether the increase in the intracellular level of glutamate might be involved directly or indirectly in the sporulation process. Sporulation in the facultative anaerobe Bacillus polymyxa cannot be induced by starvation, but requires amino acids (S. Kantengwa and M. Ojha, in preparation). Cells grown in the media containing yeast extract, arginine, glutamate or aspartate as nitrogen source sporulate rapidly with fair degree of synchrony. In this report we present the results on the influence of intracellular level of glutamate on the sporulation in Bacillus polymyxa. 2.1. Organism and cultural conditions