Sporulation of Bacillus stearothermophilus

A broth medium containing tryptone and manganese sulfate supported heavy sporulation of Bacillus stearothermophilus ATCC 7953 (NCA 1518) and four isolates identified as B. stearothermophilus. Maximal spore yields were obtained by use of inocula grown anaerobically in a medium containing glucose with aeration of sporulation medium via bubbling. After an extended stationary period, sporulation occurred concurrently with vegetative growth between 6 and 8 hr of incubation at 60 C. Omission of glucose from the inoculum or use of a “young” (2 hr) inoculum abolished the stationary period, but decreased spore yields. A requirement of oxygen for rapid vegetative growth and sporulation was demonstrated. Manganese (15 to 30 ppm) stimulated sporulation but did not enhance cell growth.     

Selection of an Asporogenous Strain of Clostridium acetobutylicum in Continuous Culture Under Phosphate Limitation

Based on the observation that cells of Clostridium acetobutylicum unable to store granulose do not initiate sporulation, a staining procedure was developed for the detection of asporogenous mutants. By application of this procedure it was shown that an asporogenous strain of C. acetobutylicum was selected in continuous culture under phosphate limitation.     

Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.     

Four linked genes participate in controlling sporulation efficiency in budding yeast

Quantitative traits are conditioned by several genetic determinants. Since such genes influence many important complex traits in various organisms, the identification of quantitative trait loci (QTLs) is of major interest, but still encounters serious difficulties. We detected four linked genes within one QTL, which participate in controlling sporulation efficiency in Saccharomyces cerevisiae. Following the identification of single nucleotide polymorphisms by comparing the sequences of 145 genes between the parental strains SK1 and S288c, we analyzed the segregating progeny of the cross between them. Through reciprocal hemizygosity analysis, four genes, RAS2, PMS1, SWS2, and FKH2, located in a region of 60 kilobases on Chromosome 14, were found to be associated with sporulation efficiency. Three of the four “high” sporulation alleles are derived from the “low” sporulating strain. Two of these sporulation-related genes were verified through allele replacements. For RAS2, the causative variation was suggested to be a single nucleotide difference in the upstream region of the gene. This quantitative trait nucleotide accounts for sporulation variability among a set of ten closely related winery yeast strains. Our results provide a detailed view of genetic complexity in one “QTL region” that controls a quantitative trait and reports a single nucleotide polymorphism-trait association in wild strains. Moreover, these findings have implications on QTL identification in higher eukaryotes.     

Novel bioreactor internals for the cultivation of spore‐forming fungi in pellet form

This study introduced an automated long‐term fermentation process for fungals grown in pellet form. The goal was to reduce the overgrowth of bioreactor internals and sensors while better rheological properties in the fermentation broth, such as oxygen transfer and mixing time, can be achieved. Because this could not be accomplished with continuous culture and fed‐batch fermentation, repeated‐batch fermentation was implemented with the help of additional bioreactor internals (“sporulation supports”). This should capture some biomass during fermentation. After harvesting the suspended biomass, intermediate cleaning was performed using a cleaning device. The biomass retained on the sporulation support went through the sporulation phase. The spores were subsequently used as inocula for the next batch. The reason for this approach was that the retained pellets could otherwise cause problems (e.g., overgrowth on sensors) in subsequent batches because the fungus would then show undesirable hyphal growth. Various sporulation supports were tested for sufficient biomass fixation to start the next batch. A reproducible spore concentration within the range of the requirements could be achieved by adjusting the sporulation support (design and construction material), and an intermediate cleaning adapted to this.     

Isolation of a Bacterial Strain Able To Degrade Branched Nonylphenol

Conventional enrichment of microorganisms on branched nonylphenol (NP) as only carbon and energy source yielded mixed cultures able to grow on the organic compound. However, plating yielded no single colonies capable, alone or in combination with other isolates, of degrading the NP in liquid culture. Therefore, a special approach was used, referred to as “serial dilution-plate resuspension,” to reduce culture complexity. In this way, one isolate, TTNP3, tentatively identified as a Sphingomonas sp., was found to be able to grow on NP in liquid culture. Remarkably, this isolate was able to be filtered through a 0.45-μm-pore-diameter filter. Moreover, isolate TTNP3 did not form visible colonies on mineral medium with NP, and it formed visible colonies on R₂A agar only after a prolonged incubation of 1 week. High-performance liquid chromatography and gas chromatography-mass spectroscopy analysis of the culture media indicated that the strain starts the degradation of NP with a fission of the phenol ring and preferably uses the para isomer of NP and not the ortho isomer. No distinct accumulation of an intermediary product could be observed.     

Asporogenous Bacillus thuringiensis Mutant Producing High Yields of δ-Endotoxin

An asporogenous Bacillus thuringiensis subsp. kurstaki strain IK mutant, strain 290-1, which produced high yields of δ-endotoxin, was obtained by ethyl methane sulfonate treatment of a spore suspension. The mutant strain produced about the same amount of δ-endotoxin as that produced by the parent strain, but 10¹⁰ to 10¹¹ cells did not form any detectable dormant spores.     

Formation of Crystalline δ-Endotoxin or Poly-β-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of δ-endotoxin on M medium, which contained 330 μg of potassium per ml, but not on ST and ST-a media, each of which contained only 11 μg of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-β-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH₂PO₄ (3.7 mM) led to the formation of crystalline δ-endotoxin. The replacement of KH₂PO₄ with equimolar amounts of KCl, KNO₃, and potassium acetate or an equivalent amount of K₂SO₄ had a similar effect, whereas the addition of an equimolar amount of NaH₂PO₄ or NH₄H₂PO₄ did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced δ-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-β-hydroxybutyric acid granules on the media containing ≤1 mM potassium. These results clearly indicate that a certain concentration of potassium is essential for the fermentative production of δ-endotoxin by these isolates of B. thuringiensis. Manganese could not be substituted for potassium. Phosphate ions stimulated poly-β-hydroxybutyric acid formation by strain 290-1. The sporulation of B. thuringiensis and several other Bacillus strains was suppressed on the potassium-deficient ST medium. This suggests that potassium plays an essential role not only in Bacillus cell growth and δ-endotoxin formation but also in sporulation.     

“Candidatus Thiobios zoothamnicoli,” an Ectosymbiotic Bacterium Covering the Giant Marine Ciliate Zoothamnium niveum

Zoothamnium niveum is a giant, colonial marine ciliate from sulfide-rich habitats obligatorily covered with chemoautotrophic, sulfide-oxidizing bacteria which appear as coccoid rods and rods with a series of intermediate shapes. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the ectosymbiont of Z. niveum belongs to only one pleomorphic phylotype. The Z. niveum ectosymbiont is only moderately related to previously identified groups of thiotrophic symbionts within the Gammaproteobacteria, and shows highest 16S rRNA sequence similarity with the free-living sulfur-oxidizing bacterial strain ODIII6 from shallow-water hydrothermal vents of the Mediterranean Sea (94.5%) and an endosymbiont from a deep-sea hydrothermal vent gastropod of the Indian Ocean Ridge (93.1%). A replacement of this specific ectosymbiont by a variety of other bacteria was observed only for senescent basal parts of the host colonies. The taxonomic status “Candidatus Thiobios zoothamnicoli” is proposed for the ectosymbiont of Z. niveum based on its ultrastructure, its 16S rRNA gene, the intergenic spacer region, and its partial 23S rRNA gene sequence.     

The early life of a leaf‐cutter ant colony constrains symbiont vertical transmission and favors horizontal transmission

Colonial organisms host a large diversity of symbionts (collectively, parasites, mutualists, and commensals) that use vertical transmission (from parent colony to offspring colony) and/or horizontal transmission to disperse between host colonies. The early life of some colonies, characterized by the dispersal and establishment of solitary individuals, may constrain vertical transmission and favor horizontal transmission between large established colonies. We explore this possibility with the miniature cockroach Attaphila fungicola, a symbiont of leaf‐cutter ants and the mutualist fungal gardens they cultivate. The early life of a leaf‐cutter colony is characterized by the dispersal of a female alate (winged “queen”) carrying a fungal pellet, and the subsequent establishment of a foundress (workerless “queen”) raising her incipient fungal garden and colony. Roaches hitchhike on female alates during leaf‐cutter nuptial flights, which strongly suggests that roaches are vertically transmitted to foundresses and their incipient colonies; however, weak compatibility between roaches and incipient gardens may constrain roach vertical transmission. Reciprocally, opportunities for horizontal transmission between large established colonies with abundant fungal gardens may weaken selection against roach‐induced harm (virulence) of incipient gardens. We use a laboratory experiment, behavioral observations, field surveys, and a transmission model to estimate the effect roaches have on the survivorship of incipient gardens and the frequency of roach vertical transmission. Contrary to traditional assumptions, our results indicate that roaches harm incipient gardens and predominantly use horizontal transmission between established leaf‐cutter colonies. Ultimately, “costs of generalism” associated with infecting disparate stages of a host''s lifecycle (e.g., incipient vs. established colonies) may constrain the vertical transmission of roaches and a broad range of symbionts.     

SYNTHESIS AND DEGRADATION OF POLY-beta-HYDROXYBUTYRIC ACID IN CONNECTION WITH SPORULATION OF BACILLUS MEGATERIUM.

Slepecky, Ralph A. (Northwestern University, Evanston, Ill.), and John H. Law. Synthesis and degradation of poly-beta-hydroxybutyric acid in connection with sporulation of Bacillus megaterium. J. Bacteriol. 82:37-42. 1961.-The production of poly-beta-hydroxybutyrate has been followed in Bacillus megaterium, a sporulating strain, and B. megaterium strain KM, a nonsporulating strain, by an improved assay procedure and by the use of C(14)-acetate.The production of polymer in the KM strain follows the growth curve very slowly and reaches a peak at the time the cells are entering the stationary phase of growth. Slow utilization of polymer follows.When the sporulating strain is grown under conditions favorable for polymer production, no spores are formed; polymer production and utilization follow kinetics similar to those observed with asporogenous strains.When the sporulating strain is grown under conditions unfavorable for polymer production but favorable for sporulation, less polymer is produced and peak production occurs during the log phase of growth. Rapid utilization of the polymer precedes sporulation.If the medium is made favorable for polymer production by the addition of glucose and acetate and vigorous aeration conditions are used, sporulation can be obtained after good polymer production and subsequent utilization.     

Adhesion of Macroconidia to the Plant Surface and Virulence of Nectria haematococca

To study spore attachment of the cucurbit pathogen Nectria haematococca (anamorph, Fusarium solani f. sp. cucurbitae), mutants with adhesion-deficient macroconidia were isolated. The adhesion-deficient mutants were selected after treatment with N-methyl-N′ -nitro-N-nitrosoguanidine followed by repeated enrichment for macroconidia which did not attach to polystyrene. Two independently derived mutants produced macroconidia with an approximately 50% reduction in attachment to polystyrene and to zucchini fruits. When macroconidia were inoculated into wounded zucchini fruits, the adhesion-deficient mutants were as virulent as the wild-type strain. However, in disease assays in which macroconidia were deposited onto the surface of unwounded zucchini, the mutants were less virulent than the wild type. Thus, adhesion of N. haematococca macroconidia to its host surface appears to be a virulence factor.     

Regulation and Self-Inhibition of Microsporum gypseum Macroconidia Germination

Germination of Microsporum gypseum macroconidia was accompanied by the release of alkaline protease, calcium ions, and inorganic phosphate into the germination fluid. The rate of germination was greatest during the first 2 hr, decreasing thereafter. This decrease in rate was accompanied by a decrease in protease activity, which was caused by an interaction of the enzyme with the inorganic phosphate released from the spores and accumulated in the germination medium after 2 hr. Germination of high spore densities was regulated by the ratio of released phosphate to protease protein, resulting in a constant percentage of germination at both high and low spore densities. A germination-defective mutant strain failed to germinate normally and released excessively high concentrations of phosphate into the germination medium during the initial 2 hr of incubation. Addition of calcium ions to germination mutant macroconidia stabilized spore morphology, prevented protease inactivation, and allowed normal germ-tube outgrowth. The germination of macroconidia appears to be regulated by the release of phosphate ions, which then inhibit the alkaline protease.     

Cellulose as an Architectural Element in Spatially Structured Escherichia coli Biofilms

Morphological form in multicellular aggregates emerges from the interplay of genetic constitution and environmental signals. Bacterial macrocolony biofilms, which form intricate three-dimensional structures, such as large and often radially oriented ridges, concentric rings, and elaborate wrinkles, provide a unique opportunity to understand this interplay of “nature and nurture” in morphogenesis at the molecular level. Macrocolony morphology depends on self-produced extracellular matrix components. In Escherichia coli, these are stationary phase-induced amyloid curli fibers and cellulose. While the widely used “domesticated” E. coli K-12 laboratory strains are unable to generate cellulose, we could restore cellulose production and macrocolony morphology of E. coli K-12 strain W3110 by “repairing” a single chromosomal SNP in the bcs operon. Using scanning electron and fluorescence microscopy, cellulose filaments, sheets and nanocomposites with curli fibers were localized in situ at cellular resolution within the physiologically two-layered macrocolony biofilms of this “de-domesticated” strain. As an architectural element, cellulose confers cohesion and elasticity, i.e., tissue-like properties that—together with the cell-encasing curli fiber network and geometrical constraints in a growing colony—explain the formation of long and high ridges and elaborate wrinkles of wild-type macrocolonies. In contrast, a biofilm matrix consisting of the curli fiber network only is brittle and breaks into a pattern of concentric dome-shaped rings separated by deep crevices. These studies now set the stage for clarifying how regulatory networks and in particular c-di-GMP signaling operate in the three-dimensional space of highly structured and “tissue-like” bacterial biofilms.     

The mold-specific MS8 gene is required for normal hypha formation in the dimorphic pathogenic fungus Histoplasma capsulatum

The dimorphic fungus Histoplasma capsulatum is the etiologic agent of one of the most common systemic mycoses of humans, histoplasmosis. In the environment, H. capsulatum grows in a differentiated mold form and shifts to an undifferentiated yeast form after mold fragments or spores are inhaled. This mold-to-yeast shift is required for disease. Little is known about the molecular biology of dimorphism in Histoplasma, and most studies have been directed toward yeast-specific genes. While it is important to examine the role of genes upregulated in the yeast morphotype, genes which are silenced in the yeast (i.e., mold-specific genes) may also play a critical role in dimorphism. To begin to examine this hypothesis, we report here the first misexpression and knockout analysis of a mold-specific gene in Histoplasma. The strongly expressed MS8 gene encodes a predicted 21-kDa protein extremely rich in glycine and glutamine. Forced expression of MS8 driven by the TEF1 promoter in yeast did not alter the yeast morphology at 37°C or mold formation at 25°C. Yeast expressing MS8 did exhibit clumping in liquid medium and formed “sticky” colonies on agar plates. Allelic replacement of MS8 was accomplished by a positive-negative selection procedure. ms8 knockout mutants formed apparently normal yeast at 37°C but gave rise to aberrant mycelia at 25°C. The mold colonies of the knockouts were less than half as large as normal, had a granular surface, produced a dark-red pigment, and formed short hyphae which were 40% wider with a distinctive twisted “zig-zag” shape.     

Role of glutamine synthetase in the initiation of sporulation in Bacillus polymyxa

The activity of glutamine synthetase (GS) was investigated during culture development of Bacillus polymyxa CN 2219 and its asporogenous mutant deficient in protease production. At 28°C, temperature permissive for sporulation, the glutamine synthetase activity was found to decline in the wild type cells which acquire the competence for sporulation. This decline was not observed in the asporogenous mutant. Incubation at 37°C (temperature non permissive) suppressed sporulation in the wild type and maintained glutamine synthetase activity. The involvement of glutamine synthetase in the repression of sporulation was further confirmied by the action of l-methionine sulfoximine a specific inhibitor of glutamine synthetase, which overcomes the catabolite repression by ammonium and induces sporulation. Intracellular proteases were measured as early markers of the initiation of sporulation and were found to be induced during sporulation.Abbreviations GS glutamine synthetase - MSO l-methionine sulfoximine - GYS glucose-yeast extract-salts - GT -glutamyltransferase - PMSF phenylmethylsulfonylfluoride     

Parasites and Pathogens of the Honeybee (Apis mellifera) and Their Influence on Inter-Colonial Transmission

Pathogens and parasites may facilitate their transmission by manipulating host behavior. Honeybee pathogens and pests need to be transferred from one colony to another if they are to maintain themselves in a host population. Inter-colony transmission occurs typically through honeybee workers not returning to their home colony but entering a foreign colony (“drifting”). Pathogens might enhance drifting to enhance transmission to new colonies. We here report on the effects infection by ten honeybee viruses and Nosema spp., and Varroa mite infestation on honeybee drifting. Genotyping of workers collected from colonies allowed us to identify genuine drifted workers as well as source colonies sending out drifters in addition to sink colonies accepting them. We then used network analysis to determine patterns of drifting. Distance between colonies in the apiary was the major factor explaining 79% of drifting. None of the tested viruses or Nosema spp. were associated with the frequency of drifting. Only colony infestation with Varroa was associated with significantly enhanced drifting. More specifically, colonies with high Varroa infestation had a significantly enhanced acceptance of drifters, although they did not send out more drifting workers. Since Varroa-infested colonies show an enhanced attraction of drifting workers, and not only those infected with Varroa and its associated pathogens, infestation by Varroa may also facilitate the uptake of other pests and parasites.     

Improvements to a Markerless Allelic Exchange System for Bacillus anthracis

A system was previously developed for conducting I-SceI-mediated allelic exchange in Bacillus anthracis. In this system, recombinational loss of a chromosomally-integrated allelic exchange vector is stimulated by creation of a double-stranded break within the vector by the homing endonuclease I-SceI. Although this system is reasonably efficient and represents an improvement in the tools available for allelic exchange in B. anthracis, researchers are nonetheless required to “pick and patch” colonies in order to identify candidate "exchangeants." In the present study, a number of improvements have been made to this system: 1) an improved I-SceI-producing plasmid includes oriT so that both plasmids can now be introduced by conjugation, thus avoiding the need for preparing electro-competent cells of each integration intermediate; 2) antibiotic markers have been changed to allow the use of the system in select agent strains; and 3) both plasmids have been marked with fluorescent proteins, allowing the visualization of plasmid segregation on a plate and obviating the need for “picking and patching.” These modifications have made the process easier, faster, and more efficient, allowing for parallel construction of larger numbers of mutant strains. Using this improved system, the genes encoding the tripartite anthrax toxin were deleted singly and in combination from plasmid pXO1 of Sterne strain 34F2. In the course of this study, we determined that DNA transfer to B. anthracis could be accomplished by conjugation directly from a methylation-competent E. coli strain.     

Isolation and characterization of two asporogenous rifampicin resistant mutants of B. thuringiensis

Two rifampicin resistant mutants, Rif^r12 and Rif^r15, of B. thuringiensis Berliner have been isolated and characterized as true asporogenous mutants. Cells of Rif^r12 were blocked between stage I and stage II in the sporulation sequence; whereas cells of Rif^r15 mutant were blocked between stage II and stage III. It has been shown that two active forms of RNA-polymerase were present at t5 in Rif^r15 cells; as for wild type strain, the subunit composition of form I and form II were respectively ββ'_mα2 and β′βα2. In Rif^r12 cells, only one enzymatic form was found at t5 ; the subunit composition was determined as β′βσ_mα2 ; such a composition was characteristic for sporulation enzyme of wild type strain at t1,5. It is concluded that the sigma modification which occurs at about t1, is anterior to the β′ modification which is closely correlated with the forespore septum completion (t2). Thus, the timing of the modifications of B. thuringiensis RNA-polymerase previously suggested was clearly confirmed through the present study.In addition, both mutants present reduced levels of intracellular proteolytic activities, as compared with wild type strain, and the role of proteases is discussed.     

The effect of sodium chloride on the growth and morphology of dermatophytes and some other keratolytic fungi.

A study of the influence of various concentrations of NaC1 on 21 species of dermatophytes and other keratolytic fungi was made. Based on the sensitivity of the species to various concentrations of NaC1, it was possible to divide them into five groups. Microsporum ferrugineum and Trichophyton concentricum and T. tonsurans were the most sensitive to NaC1 and were inhibited by 5%. The greatest number of species were inhibited by 12% NaC1. The fungi most tolerant to NaC1 were M. cookei and M. nanum, and T. mentagrophytes, T. schoenleinii, and T. terrestre. These species were inhibited by 15%. NaC1 prevented any variant change in Epidermophyton floccosum, T. mentagrophytes, and M. gypseum, but promoted a change in phenotype in M. audouinii and M. cookei, and T. gallinae. It is suggested that the word "pleomorphism" be replaced by the term sterile albinism. This term refers to that kind of cultural change when there is no evidence of sporulation and the white fluffy mycelium consists of fine sterile hyphae. Sterile albino strains of E. floccosum were induced to form a macroconidia on Sabouraud cycloheximide chloramphenicol gentamicin agar (SCCGA) containing 3-5% NaC1. Also, M. audouinii formed microconidia and macroconidia in velvety growth cultured on SCCGA containing NaC1.