Bacteriophage conversion of spore-negative mutants to spore-positive in Bacillus pumilus.

A pseudolysogenic phage, PMB1, was isolated from soil on the basis of its ability to increase the sporulation frequency of the oligosporogenic Bacillus pumilus strain NRS 576 (sporulation frequency, less than 1%). Several spore-negative mutants (sporulation frequency, less than 10-8) derived from strain NRS 576, which were converted to spore positive by infection with PMB1, were subsequently identified. PMB1 repeatedly grown on a given spore-negative mutant (e.g., GW2) converted GW2 cells to spore positive. Each plaque-forming unit initiated the conversion of a spore-positive clone in semisolid agar overlays. GW2 cells remained spore positive as long as they maintained PMB1. Return of PMB1-converted cells to the orginal spore-negative phenotype correlated with loss of PMB1. In liquid media, PMB1 infection increased the sporulation frequency of mutant GW2 over 106-fold. More than half of the spore-negative mutants we isolated from strain NRS 576 were converted to spore positive by PMB1 infection. PMB1-induced spores of the spore-negative mutant GW2 were somewhat more heat sensitive than uninfected or PMB1-infected spores of the spore positive parent of GW2. PMB1-induced spores of GW2 do not differ from wild-type spores in morphology by phase-contrast microscopy, dipicolinic acid content, or rate of sedimentation through Renografin gradients.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Nosema ceranae Can Infect Honey Bee Larvae and Reduces Subsequent Adult Longevity

Nosema ceranae causes a widespread disease that reduces honey bee health but is only thought to infect adult honey bees, not larvae, a critical life stage. We reared honey bee (Apis mellifera) larvae in vitro and provide the first demonstration that N. ceranae can infect larvae and decrease subsequent adult longevity. We exposed three-day-old larvae to a single dose of 40,000 (40K), 10,000 (10K), zero (control), or 40K autoclaved (control) N. ceranae spores in larval food. Spores developed intracellularly in midgut cells at the pre-pupal stage (8 days after egg hatching) of 41% of bees exposed as larvae. We counted the number of N. ceranae spores in dissected bee midguts of pre-pupae and, in a separate group, upon adult death. Pre-pupae exposed to the 10K or 40K spore treatments as larvae had significantly elevated spore counts as compared to controls. Adults exposed as larvae had significantly elevated spore counts as compared to controls. Larval spore exposure decreased longevity: a 40K treatment decreased the age by which 75% of adult bees died by 28%. Unexpectedly, the low dose (10K) led to significantly greater infection (1.3 fold more spores and 1.5 fold more infected bees) than the high dose (40K) upon adult death. Differential immune activation may be involved if the higher dose triggered a stronger larval immune response that resulted in fewer adult spores but imposed a cost, reducing lifespan. The impact of N. ceranae on honey bee larval development and the larvae of naturally infected colonies therefore deserve further study.     

The Ultraviolet Photochemistry and Photobiology of Vegetative Cells and Spores of Bacillus megaterium

The ultraviolet (UV) photochemistry and photobiology of spores and vegetative cells of Bacillus megaterium have been studied. The response of vegetative cells of B. megaterium appears qualitatively similar to those of Escherichia coli, Micrococcus radiodurans, and Bacillus subtilis with respect to photoproduct formation and repair mechanisms. UV irradiation, however, does not produce cyclobutane-type thymine dimers in the DNA of spores, although other thymine photo-products are produced. The photoproducts do not disappear after photoreactivation, but they are eliminated from the DNA by a dark-repair mechanism different from that found for dimers in vegetative cells. Irradiations performed at three wavelengths produce the same amounts of spore photoproduct and give the same survival curves. Variation of the sporulation medium before irradiation results in comparable alterations in the rate of spore photoproduct production and in survival.     

Disruption of a sigma factor gene, sigF, affects an intermediate stage of spore pigment production in Streptomyces aureofaciens

The Streptomyces aureofaciens sigF gene encodes a sigma factor. By integrative transformation, via double cross-over, a stable null mutant of sigF gene was obtained. This mutation appeared to have no obvious effect on vegetative growth, but affected the late stage of spore maturation. Microscopic examination showed that spores were deformed, and spore wall was thinner, compared with the wild-type spores. The spore pigment of sigF mutant was green, compared to wild-type grey-pink spore pigmentation. The plasmid-born wild-type sigF gene complemented the mutation after transformation of the mutant strain.     

THE PARASPORAL BODY OF BACILLUS LATEROSPORUS LAUBACH

On sporulation the slender vegetative rods swell and form larger spindle-shaped cells in which the spores are formed. When the spores mature they lie in a lateral position cradled in canoe-shaped parasporal bodies which are highly basophilic and can be differentiated from the surrounding vegetative cell cytoplasm with dilute basic dyes. On completion of sporulation the vegetative cell protoplasm and the cell wall lyse, leaving the spore cradled in its parasporal body. This attachment continues indefinitely on the usual culture medium and even persists after the spores have germinated. In thin sections of sporing cells the bodies are differentiated from the cell protoplasm by differences in structure. Whereas the protoplasm has a granular appearance, in both longitudinal and cross-sections the parasporal body comprises electron-dense lamellae running parallel with the membranes of the spore coat and less electron-dense material in the interstices of the lamellae. The inner surface of the body is contiguous with that of the spore coat as if it were part of the spore, rather than a separate body attached to the spore. The staining reactions of the parasporal body are not consistent with those of any substance described in bacteria. With Giemsa the bodies stain like chromatin, but the Feulgen reaction indicates that they do not contain the requisite nucleic acid. With an aqueous solution of toluidine blue they stain metachromatically, but with an acidified solution the results are variable. Neisser's stain for polyphosphate is negative. The basophilic substance is removed from the body with some organic solvents. This basophilic substance has not been specifically identified with any material seen in ultrathin sections, but it is suggested that it might be the less electron-dense material in the interstices of the lamellar structure. In contrast to the spore coat of B. laterosporus, those of its two relatives B. brevis and B. circulans take up basic stain like the parasporal body. Thin spore sections of these species have shown that the walls are thicker than those surrounding the spores of B. laterosporus, and it is suggested that the outer stainable layer of brevis and circulans spores is an accessory coat which in laterosporus may have been deformed to give a parasporal body.     

A genetic melanotic neoplasm of Drosophila melanogaster

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.     

CHEMICAL AND MORPHOLOGICAL STUDIES OF BACTERIAL SPORE FORMATION : IV. The Development of Spore Refractility

From the stage of a completed membranous forespore to that of a fully ripened free spore, synchronously sporulating cells of a variant Bacillus cereus were studied by cytological and chemical methods. Particular attention was paid to the development of the three spore layers—cortex, coat, and exosporium—in relation to the forespore membrane. First, the cortex is laid down between the recently described (5) double layers of the forespore membrane. Then when the cortex is ⅓ fully formed, the spore coat and exosporium are laid down peripheral to the outer membrane layer covering the cortex. As these latter layers appear, the spores, previously dense by dark phase contrast, gradually "whiten" or show an increase in refractive index. With this whitening, calcium uptake commences, closely followed by the synthesis of dipicolinic acid and the process is terminated, an hour later, with the formation of a fully refractile spore. In calcium-deficient media, final refractility is lessened and dipicolinic acid is formed only in amounts proportional to the available calcium. If calcium is withheld during the period of uptake beyond a critical point, sporulating cells lose the ability to assimilate calcium and to form normal amounts of dipicolinic acid. The resulting deficient spores are liberated from the sporangia but are unstable in water suspensions. Unlike ripe spores, they do not react violently to acid hydrolysis and, in thin sections, their cytoplasmic granules continue to stain with lead solutions.     

Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a

The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.     

Mitochondrial biogenesis during fungal spore germination. Development of cytochrome c oxidase activity.

Mitochondria from dormant spores of the fungus Botryodiplodia theobromae did not contain extractable cyctochrome c oxidase (EC 1.9.3.1) activity; however, this enzyme activity was elaborated rapidly after 150 min of the 240-min germination sequence. The absence of cytochrome c oxidase activity in the dormant spores apparently is not an artifact caused by spore disruption and fractionation procedures, transient enzyme instability, or insensitivity of the enzyme assay. Mitochondria from dormant spores of three other phylogenetically diverse genera of fungi were observed to contain readily detectable quantities of cytochrome c oxidase, suggesting that the absence of the enzyme in B. theobromae may be relatively novel. The elaboration of cytochrome c oxidase activity in germinating spores was abolished by cycloheximide if the drug was added at or before 95 min of germination, but development of enzyme activity was initially insensitive to inhibitors of the mitochondrial genetic system, chloramphenicol or ethidium bromide. Incubation of spores in both ethionine and S-2-aminoethyl-l-cysteine reduced the amount of extracted cytochrome c oxidase activity. Elaboration of enzyme activity was severely retarded by cerulenin, an inhibitor of fatty acid biosynthesis and of spore germination. This enzyme activity developed in water-incubated or 1% Tween 80-incubated spores in which only the cytoplasmic ribosomes are functional in translation of a stored nuclear messenger RNA. The results of this study show that cytoplasmic (but not mitochondrial) ribosome function is required for development of this enzyme activity during spore germination, and they suggest that a portion of the cytochrome c oxidase enzyme or some other protein required for its activity is synthesized de novo upon germination.     

Dimorphic spore production in the genus Acaulospora

Species of the genus Acaulospora are partly characterized by the production of ‘acaulosporoid’ spores. The simultaneous formation of glomoid and acaulosporoid spores by some species in the Glomeromycota has been used as a basis for taxonomic classification. We report the presence of both glomoid and acaulosporoid spores in four Acaulospora species. Analysis of the 18S rRNA gene confirmed that only A. spinosa was present in a pot culture that produced two morphs. The functional significance of dimorphic spore production is unknown but the production of blastic spores is not a characteristic that can have any significance as a taxonomic character.     

Description of Hyalinocysta expilatoria n. sp., a microsporidian parasite of the blackfly Odagmia ornata

Hyalinocysta expilatoria n. sp. is described from a larva of Odagmia ornata collected in Sweden. Infection was restricted to the adipose tissue which was transformed into a syncytium. The earliest stage observed was diplokaryotic merozoites, which mature directly into diplokaryotic sporonts. Each sporont produces a sporophorous vesicle (pansporoblast), which persists, also enclosing mature spores. Usually nuclear divisions result in a plasmodium with 8 nuclei, which fragments into 8 sporoblasts, each of which develops into a spore without further division. Occasionally an aberrant number of spores (2, 4, 6) is formed. The spores are pyriform with a flattened area at the posterior pole. Spores in sporophorous vesicles with 8 spores are 4.0–6.0 μm long, in vesicles with 4 spores 4.0–5.0 μm, and in vesicles with 2 spores 7.0–8.0 μm. In some vesicles the spores develop asynchronously, and 2, 4, or 6 mature spores are found together with 6, 4, or 2 immature. There was also a small number of vesicles with supernumerary spores, less than 8 normally developed. The 325–350 nm thick spore wall is composed of three layers. The polar filament is anisofilar with 7 coils in a single layer. The anterior 5–6 coils are wide, the posterior 2-1 thin. The angle of tilt of the anterior filament coil is approximately 50°. The spore has a single nucleus. The sporophorous vesicle is delimited by a thin membrane, also visible in haematoxylin stained preparations. Vesicles with mature spores are void of metabolic inclusions.     

The GerW Protein Is Not Involved in the Germination of Spores of Bacillus Species

Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores’ inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores’ levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways.     

ULTRASTRUCTURAL LOCALIZATION OF MINERAL MATTER IN BACTERIAL SPORES BY MICROINCINERATION

The fine localization of mineral matter in spores of Bacillus megaterium and Bacillus cereus was studied by the technique of microincineration adapted for use with the electron microscope. The specimens, which included intact and thin-sectioned spores as well as shed spore coats, were burned either in the conventional way at high temperature or by a new technique using electrically excited oxygen at nearly room temperature. The ash residues were examined by bright field, dark field, and diffraction in the electron microscope and also with the phase contrast microscope. In some cases, the specimen was previewed in both microscopes before incineration. The results do not support a previous report that the mineral elements of the spore are confined to a peripheral layer, but rather indicate that the spore core as well as the coat are mineral-rich. The cortex may be deficient in minerals, but the possibility of artifact prevents a clear decision on this point. Incinerated B. megaterium spores show a highly ordered fine structure displaying 100 A periodicity in the ash of the middle layer of the coat. The nature of this structure is discussed, as is the technique which demonstrated it. The fine definition of the ash patterns, particularly those obtained with the low-temperature, excited-oxygen technique, suggests that microincineration may be generally useful in the study of fine structure.     

SpoIVA and SipL Are Clostridium difficile Spore Morphogenetic Proteins

Clostridium difficile is a major nosocomial pathogen whose infections are difficult to treat because of their frequent recurrence. The spores of C. difficile are responsible for these clinical features, as they resist common disinfectants and antibiotic treatment. Although spores are the major transmissive form of C. difficile, little is known about their composition or morphogenesis. Spore morphogenesis has been well characterized for Bacillus sp., but Bacillus sp. spore coat proteins are poorly conserved in Clostridium sp. Of the known spore morphogenetic proteins in Bacillus subtilis, SpoIVA is one of the mostly highly conserved in the Bacilli and the Clostridia. Using genetic analyses, we demonstrate that SpoIVA is required for proper spore morphogenesis in C. difficile. In particular, a spoIVA mutant exhibits defects in spore coat localization but not cortex formation. Our study also identifies SipL, a previously uncharacterized protein found in proteomic studies of C. difficile spores, as another critical spore morphogenetic protein, since a sipL mutant phenocopies a spoIVA mutant. Biochemical analyses and mutational analyses indicate that SpoIVA and SipL directly interact. This interaction depends on the Walker A ATP binding motif of SpoIVA and the LysM domain of SipL. Collectively, these results provide the first insights into spore morphogenesis in C. difficile.     

Fate and Dissemination of Bacillus subtilis Spores in a Murine Model

Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action. As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice. Our results have shown that spores do not appear to disseminate across the mucosal surfaces. However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.     

Small, Acid-Soluble Spore Proteins of the α/β Type Do Not Protect the DNA in Bacillus subtilis Spores against Base Alkylation

Ethyl methanesulfonate (EMS) killed wild-type Bacillus subtilis spores as rapidly as spores lacking small, acid-soluble proteins (SASP) of the α/β type (α⁻β⁻ spores), and 20% of the survivors had obvious mutations. A recA mutation increased the EMS sensitivity of wild-type and α⁻β⁻ spores similarly but reduced their mutagenesis; EMS treatment of dormant spores also resulted in the induction of RecA synthesis during spore germination. EMS generated similar levels of alkylated bases in wild-type and α⁻β⁻ spore DNAs, in purified DNA, or in DNA saturated with α/β-type SASP. Ethylene oxide (EtO) also generated similar levels of base alkylation in wild-type and α⁻β⁻ spore DNAs. These data indicate that EMS and EtO kill spores at least in part by DNA damage but that α/β-type SASP, which protect DNA against many types of damage, do not protect spore DNA from base alkylation.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.