Refractile Spores Research Articles

Real-time FTIR monitoring and modeling Bacillus amyloliquefaciens spore germination: Effect of temperature and sub-lethal treatment by nonthermal plasma

This study uses real-time FTIR spectroscopy to probe biochemical changes occurring in spores of Bacillus amyloliquefaciens during nutrient germination at T = 23 − 50°C and after sub-lethal injury by treatment with a nonthermal plasma (NTP). The FTIR identifies several spore structural components (e.g., stretching bands of the COO− group of dipicolinic acid at 1378 and 1616 cm−1; C−N vibrations in the DPA ring at 1570 cm−1, etc.) that are correlated to steps occurring during germination. The increase in peak intensity at 1570 cm−1 is interrelated to spore germination as measured with optical density at 600 nm (OD600) of refractile spores at T = 23 − 50°C and after NTP treatment. For the OD600 germination kinetics data, the Weibull model accurately fit the observed dynamics with values for the rate parameters α = 18.1 − 52.9 s−1 and shape parameter β = 0.9 − 1.2, and the Weibull model's performance was assessed by satisfactory values of the accuracy factor (1.00 < Af ≤ 1.15) and the bias factor (Bf in the ranges of 0.90 − 1.05), with the NTP-treated spores being the lone exception (Af = 1.22, Bf = 1.06).

The effect of oxygen on the sporulation, δ-endotoxin synthesis and toxicity of Bacillus thuringiensis H14

The sporulation and toxicity of Bacillus thuringiensis H14 were studied as a function of aeration. The fed-batch cultures carried out in the similar aeration conditions were followed in four different oxygen transfer rates containing 0, 20, 100 and 250 mmol/l/h. The percentage of total cells which had formed refractile spores in these four oxygen transfer rates were 100, 93, 84 and 48%, respectively. The highest rate of sporulation was observed in the absence of oxygen and the mature spores were the only population present under this condition at the end of culture. Sporulation in a large portion of cells failed under saturated oxygenation and either mature spores or vegetative cells were present at the end of culture. In the intermediate conditions, cells in different physiological states could be observed at the end of culture. It was found that the optimal conditions for spore yield and for δ-endotoxin yield were not the same, even though sporulation and δ-endotoxin formation proceed simultaneously during the fermentation process. The 130-kDa δ-endotoxin seemed to be more sensitive to aeration conditions. The higher toxicity against Culex pipiens was obtained under the saturated condition.

The CDK-activating kinase CAK1 can dosage suppress sporulation defects of smk1 MAP kinase mutants and is required for spore wall morphogenesis in Saccharomyces cerevisiae.

Mitogen-activated protein (MAP) kinase pathways are evolutionarily conserved kinase cascades that are required for the response of eukaryotic cells to a wide variety of environmental stimuli. MAP kinase pathways are also required for the execution of developmental and differentiative programs in a variety of cell and tissue types. SMK1 encodes a developmentally regulated MAP kinase in yeast that is required for spore wall morphogenesis. Cyclin-dependent kinase-activating kinases (CAKs) phosphorylate a conserved threonine residue in the activating loop of cyclin-dependent kinases. CAK1 encodes the major CAK activity in yeast and is required for cell cycle progression. The work presented here demonstrates that CAK1 functions positively in the spore wall morphogenesis pathway. First, CAK1 has been isolated as a dosage suppressor of a conditional smk1 mutant that is defective for spore wall morphogenesis. Second, CAK1 mRNA accumulates during spore development contemporaneously with SMK1 mRNA. Third, cak1 mutant strains have been isolated that are able to complete meiosis I and II but are specifically defective in assembly of the spore wall. These results show that cell cycle progression and morphogenetic pathways can be regulated by a single gene product and suggest mechanisms for coordinating these processes during development.

Using a phase-locked mutant of Myxococcus xanthus to study the role of phase variation in development.

The bacterium Myxococcus xanthus undergoes a primitive developmental cycle in response to nutrient deprivation. The cells aggregate to form fruiting bodies in which a portion of the cells differentiate into environmentally resistant myxospores. During the growth portion of the M. xanthus life cycle, the organism also undergoes a phase variation, in which cells alternate between yellow and tan colony-forming variants. Phase variation occurs in our laboratory strain (M102, a derivative of DK1622) at a frequency high enough that a single colony of either the yellow or the tan phase already contains cells of the alternate phase. In this study we demonstrate that tan cells within a predominantly yellow population of phase variation-proficient cells are preferentially recovered as heat- and sonication-resistant spores. To further investigate the possibility of a differential role of tan and yellow cells during development, a tan-phase-locked mutant was used to compare the developmental phenotypes of a pure tan population with a predominantly yellow, phase variation-proficient population. Pure tan-phase populations did not produce fruiting bodies or mature spores under conditions in which predominantly yellow wild-type populations did so efficiently. Pure populations of tan-phase cells responded to developmental induction by changing from vegetative rod-shaped cells to round forms but were unable to complete the maturation to heat- and sonication-resistant, refractile spores. The developmental defect of a tan-phase-locked mutant was rescued by the addition of phase variation-proficient cells from a predominantly yellow culture. In such mixtures the tan-phase-locked mutant not only completed the process of forming spores but also was again preferentially represented among the viable spores. These findings suggest the intriguing possibility that the tan-phase cells within the vegetative population entering development are the progenitors of spores and implicate a requirement for yellow-phase cells in spore maturation.

Mutation of the SPS1-encoded protein kinase of Saccharomyces cerevisiae leads to defects in transcription and morphology during spore formation.

During sporulation of Saccharomyces cerevisiae, meiosis is followed by encapsulation of haploid nuclei within multilayered spore walls. Completion of the late events of the sporulation program requires the SPS1 gene. This developmentally regulated gene, which is expressed as cells are nearing the end of meiosis, encodes a protein with homology to serine/threonine protein kinases. The catalytic domain of Sps1 is 44% identical to the kinase domain of yeast Ste20, a protein involved in the pheromone-induced signal transduction pathway. Cells of a MATa/MAT alpha sps1/sps1 strain arrest after meiosis and fail to activate genes that are normally expressed at a late time of sporulation. The mutant cells do not form refractile spores as assessed by phase-contrast microscopy and do not display the natural fluorescence and ether resistance that is characteristic of mature spores. Examination by electron microscopy reveals, however, that prospore-like compartments form in some of the mutant cells. These immature spores lack the cross-linked surface layer that surrounds wild-type spores and are more variable in size and number than are the spores of wild-type cells. Despite their inability to complete spore formation, sps1-arrested cells are able to resume mitotic growth on transfer to rich medium, generating haploid progeny. Our results suggest that the developmentally regulated Sps1 kinase is required for normal progression of transcriptional, biochemical, and morphological events during the later portion of the sporulation program.

Cloning and characterization of a gene required for assembly of the Bacillus subtilis spore coat.

During endospore formation in Bacillus subtilis, approximately a dozen proteins are synthesized and assembled around the prespore to form a protective coat. Little is known about the assembly process, but several of the genes encoding these coat proteins are expressed in the mother cell compartment, where the proteins accumulate on the outer side of the developing endospore. Transcription of these genes is directed by the mother cell-specific sigma factor, sigma K, during the later stages of endospore development. sigma E may direct expression of the genes that encode proteins that function in the earliest stages of coat assembly. By screening for sigma E-dependent promoters, we cloned a gene, designated spoVID, required for assembly of a normal spore coat. Expression of spoVID was initiated at about the second hour of sporulation and continued throughout development from a sigma E-dependent promoter. The spoVID gene was located on the B. subtilis chromosome just downstream of the previously characterized hemAXCDBL operon and is predicted to encode an extremely acidic protein with 575 residues. Insertion mutants of spoVID produced refractile spores that were resistant to heat and to chloroform but were sensitive to lysozyme. Electron microscopic examination of sporulating spoVID mutant cells revealed normal morphological development up to about the third hour of sporulation. However, during the later stages of development the coat proteins assembled into aberrant structures that occurred freely in the mother cell cytoplasm and that consisted of reiterations of the single inner and outer layers that normally make up the spore coat.

Preparation of Refractile Spores of Clostridium thermosaccharolyticum Involves a Solventogenic Phase

Conversion of vegetative cells of Clostridium thermosaccharolyticum to refractile endospores was achieved by sequential transfer and dilution at each generation, with a final dilution into a sporulation medium that contained xylan supplemented with excess calcium. The subsequent growth was synchronous and resulted in elongated, solventogenic cells that were then shifted to 35 degrees C to permit further differentiation without cell division. The synchronized cells grown in xylan medium supplemented with Ca gluconate produced total solvents that reached 9.63% (vol/vol). One hundred percent of these elongated solventogenic cells (4.84 x 10 cells per ml) entered the sporangial stage and continued to differentiate into refractile spores. Only cells sequentially transferred and diluted at a critical time of the growth cycle are synchronized, induced to elongate (>/=fourfold), become highly solventogenic in the presence of excess calcium, and are converted to a homogeneous population of refractile spores.

Evidence that elevated intracellular cyclic AMP triggers spore maturation in Dictyostelium

ABSTRACT Spore maturation occurs during normal development in Dictyostelium when environmental influences induce a migrating slug to transform into a fruiting body. As the amoeboid prespore cells turn into refractile spores there is a burst of enzyme accumulation, including UDP-galactose epimerase, and at a later stage the exocytosis of preformed components of the spore coat. Evidence is presented here that this process is triggered by an elevated intracellular cAMP concentration. First, a number of rapidly developing (rde) mutants, whose cAMP metabolism had been investigated previously, are shown to be able to form spores in submerged monolayers, whereas wild-type strains are not. The phenotypes of these mutants are best explained by a derepression of the signal transduction pathway utilizing intracellular cAMP. Second and more direct, it is shown that the permeant cAMP analogues 8-Br-cAMP and 8-chlorophenylthio-cAMP, but not cAMP itself, can rapidly induce spore differentiation in wild-type amoebae incubated in submerged monolayers. These analogues also stimulate accumulation of UDP-galactose epimerase in slug cells transferred to shaken suspension. The ability to induce spore differentiation with Br-cAMP in wild-type strains provides a new technique that can be exploited in various ways. For instance, spore differentiation in strain V12M2 is induced by 8-Br-cAMP at very low cell densities, suggesting that neither cell contact nor additional soluble inducers are necessary in these conditions. In contrast NC4 cells may require an additional inducer. Spore differentiation is inhibited by the stalk-specific inducer DIF-1 suggesting that DIF-1 inhibits a target downstream of intracellular cAMP in the signal transduction pathway inducing spore differentiation.

Reexamination of the role of autolysis in the development of Myxococcus xanthus.

It has been widely reported that 80 to 90% of the cell population undergoes autolysis during sporulation in Myxococcus xanthus. A re-evaluation of the techniques used to measure autolysis in M. xanthus showed that the methods previously used to draw this conclusion are subject to artifacts, which result in a substantial underestimation of the number of cells present during development. We found that at least 80% of the cells that enter development survive throughout fruiting body formation. The cell loss that did occur appeared to be gradual over a period of at least 7 days. Our results suggest that autolysis is not an obligate stage in the development of M. xanthus. The data also showed that sporulating cells pass through a prespore stage in which they become osmotically and physically fragile and therefore difficult to harvest intact. The fragility was correlated with the change from a rod to a spherical shape. As the prespores differentiated into refractile spores, they lost fragility and became amenable to harvesting by standard protocols.

Zinc stimulates sporulation inClostridium botulinum 113B

The presence of 0.5–1.0 mM zinc (Zn) in a complex sporulation medium stimulated spore formation in certain strains ofClostridium botulinum. Zinc increased both the titer of free refractile spores (spores per liter) and the percentage conversion of vegetative cells to spores. Certain other transition metals including iron (Fe) and manganese (Mn) also improved sporulation, but not so effectively as zinc. Sporulation was drastically decreased by the addition to the medium of 0.5–1.0 mM copper (Cu). Copper was shown to compete with the acquisition of zinc by the sporulating cells. Spores were separated from their progenitor vegetative cells to ≥98% homogeneity by incorporation of a density-separation step in the extensive washing procedure. Analysis of the metal contents of the purified spores showed that zinc levels in spores were reduced considerably in culture media containing excess copper. The results imply that either the availability of zinc or the limitation of copper stimulates sporulation inC. botulinum. In addition toC. botulinum 113B, zinc also increased sporulation in several type A, B, and E strains and one proteolytic type F strain ofC. botulinum.

Netropsin stimulates the formation of an extracellular proteinase and suppresses protein turnover in sporulatingBacillus megaterium

Netropsin stimulated the rate of synthesis of an extracellular metalloproteinase in Bacillus megaterium incubated in a sporulation medium. The antibiotic delayed but did not suppress the decrease in the ability to synthesize the proteinase occurring at later sporulation stages. Netropsin also stimulated the synthesis of the proteinase when added to a growing culture; it inhibited the increase of protein turnover which was switched on between the 2nd and 3rd hour in the sporulating population. No refractile spores were developed during 6 h at 35°C in the antibiotic-treated culture. In the control 60% of sporulating cells were observed under similar conditions.

Protection of Bacillus larvae from Oxygen Toxicity with Emphasis on the Role of Catalase

Sporulation of Bacillus larvae NRRL B-3650 occurred only at aeration rates lower than those used for cultivation of most Bacillus species. One possible explanation for the requirement for a low level of aeration in B. larvae is that toxic forms of oxygen such as H(2)O(2) and superoxide are involved. The superoxide dismutase levels of strain B-3650 were similar to those of Bacillus subtilis 168 during sporulation, and no NADH peroxidase was present. Catalase activity was absent during exponential growth and first appeared near the start of the stationary phase. The catalase activity was 2,700 times less than that in B. subtilis 168 at the same stage of development. Therefore, the relative deficiency of catalase (and NADH peroxidase) might be the cause of the apparent O(2) toxicity. It was postulated that B. larvae might accumulate H(2)O(2) in the medium and exhibit more than normal sensitivity to H(2)O(2). Experimental results did not verify either postulate, but the possibilities of intracellular accumulation of H(2)O(2) and unusual sensitivity to endogenous H(2)O(2) were not excluded. The catalase present in early-stationary-phase cells was soluble, heat labile, and inhibited by cyanide, azide, and hydroxylamine. An increase in catalase activity also occurred at the time of appearance of refractile spores in both B. larvae NRRL B-3650 and B. subtilis 168. The level of catalase activity in strain B-3650 was 5,400 times less than that in B. subtilis 168 at this stage. In B. larvae, this second increase occurred primarily within the developing endospore. The activity in spore extracts was particulate, heat stable, and inhibited by hydroxylamine but not by azide or cyanide. Synthesis of catalase in B. larvae was unaffected by H(2)O(2), O(2), or glucose.

Production of intracellular serine protease during sporulation of Bacillus brevis ATCC9999

It has been reported (Slapikoff et al. 1971) that Bacillus brevis ATCC9999, the producer of gramicidin S, makes no extracellular or intracellular protease in complex medium which supports sporulation. We have found that intracellular protease is made by this strain; however, the activity requires the presence of the reducing agent dithiothreitol in the extraction buffer. B. brevis intracellular protease appears at the time that refractile spores are visible in the mother cells. Its pH optimum is 8.0. The enzyme is inhibited by ethylenediaminetetraacetic acid and phenylmethylsulfonylfluoride but not by o-phenanthroline, boronic acids, and only slightly by p-chloromercuribenzoate. This inhibitor pattern is similar to that of intracellular proteases of B. megaterium and B. subtilis, classifying the B. brevis protease as an intracellular seryl protease.

Synthesis of acid-soluble spore proteins by Bacillus subtilis.

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

Stimulation of Clostridium perfringens enterotoxin formation by caffeine and theobromine.

In the presence of 100 micrograms of caffeine per ml or 200 micrograms of theobromine per ml, sporulation of Clostridium perfringens NCTC 8679 rose from less than 1 to 80 or 85%. Enterotoxin concentration increased from undetectable levels to 450 micrograms/mg of cell extract protein. Heat-resistant spore levels increased from less than 1,000 to between 1 X 10(7) and 2 X 10(7)/ml. These effects were partially reversible by the addition of adenosine or thymidine. In the case of NCTC 8238, caffeine and theobromine caused a three- to fourfold increase in the percentages of cells possessing refractile spores and a similar increase in enterotoxin concentration. Heat-resistant spore levels, however, were unaffected. Inosine was ineffective in promoting sporulation in NCTC 8679.

Stimulation of endogenous proteolysis in macrophages exposed to sporulating bacteria

The relationship between degradation of endogenous macrophage protein and exposure to bacteria at various stages of sporulation was studied. Rabbit pulmonary macrophages were obtained by lavage, attached to plastic tissue culture dishes, and prelabeled with [ 14C]- or [ 3H]-phenylalanine for 1 or 20 h, respectively. Bacillus licheniformis (ATCC 14580) was grown in Bacto Marine Broth until at least 50% of the bacilli contained refractile spores (19 h), washed and lyophilized. The lyophilized bacilli were washed, counted and applied for 1.5 h to the washed prelabeled macrophages at a ratio of 300 bacteria/macrophage. Rates of degradation of both rapidly and slowly turning over proteins were increased. The effect was not mediated by soluble components of the incubation medium. Free spores, vegetative cells and autoclaved sporulating bacilli did not affect rates of proteolysis. We propose that proteases from the lyophilized sporulating bacilli participate in endogenous macrophage protein degradation following engulfment.

Induction of accelerated sporangial lysis by basic peptide antibiotics. A novel method of preparation of free spores of bacilli.

An accelerated release of free spores from sporangia of Bacillus cereus NCIB‐8122 and Bacillus subtilis SMYW was induced by the addition of the basic peptide antibiotics, polymyxin B or colistin (100 μg/ml), to sporangia formed in liquid Bactopeptone medium. Destruction of sporangial cell walls of B. cereus prelabelled with 3H‐4‐diaminopimelic acid commenced shortly after the addition of either antibiotic, the label being gradually released into the medium. Normal free spores were released following the addition of antibiotics to sporangia containing refractile spores (stages IV‐V of sporogenesis). Earlier additions induced the lysis of both compartments of the sporangium, accompanied by the release of already‐synthesized dipicolinic acid and alreadyaccumulated 45calcium. The heat resistance and germination ability of spores released in the presence of the antibiotics were the same as those of control spores released by long‐term spontaneous lysis of sporangia. Similar effects of the antibiotics were observed with B. subtilis SMYW. Results obtained were used firstly for fast preparation of relatively clean free spores and secondly for the characterization of the developmental stage of sporogenesis at which the spore becomes independent of the maternal cell. It reaches this property at the end of stage IV and during stage V.

Increased Numbers of Heat-resistant Spores Produced by Two Strains of Clostridium perfringens Bearing Temperate Phage s9

Sporulation kinetics and spore heat resistance data were compared for a lysogenic strain of Clostridium perfringens, s9, before and after curing with ultraviolet irradiation. The cured strain showed the same growth rate in broth media as the lysogenic strain but took 6 h longer to form refractile spores. For lysogenized and cured strains the percentages of refractile spores produced that were heat-resistant (80 degrees C for 15 min) were 50 and 0.2, respectively. When reinfected with the temperature phage, the cured strain produced spores in 2 to 3 h, like the original lysogenic culture, and 10% of the spores produced were heat-resistnat.

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain of Bacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respect to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3-5 h of incubation. After 7 h 20-50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteins in vitro at a rate up to 150 mug mg-1 h-1 and native proteins at a rate up to 70 mug mg-1 h-1. Degradation of proteins in vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 mug mg-1 h-1 and the inactivation of beta-galactosidase at a rate of 70 mug mg-1 h-1. The intracellular proteolytic activity was inhibited to 65-88% by EDTA, to 23-76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50-65% of the activity were localized in protoplasts. Another strain of Bacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain of Bacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3-4 h. The intracellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.

Conditional spore cortex-less mutants of Bacillus sphaericus 9602.

Lysine-requiring mutants of Bacillus sphaericus 9602 were isolated and classified into three groups by their mutation site in the pathway of lysine biosynthesis. The Group I mutant lacks meso-alpha, epsilon-diaminopimelic acid (meso-Dap) decarboxylase activity, but Group II and III mutants have a normal level of Dap decarboxylase activity. A Group II mutant makes dipicolinic acid from an intermediate in the lysine pathway, but Group III mutants do not. In the absence of meso-Dap in the culture, muramic lactam content in the spore cortex of Group II and III mutants is very low, compared to the wild type content. Addition of meso-Dap to the culture causes an increase of muramic lactam content. Since meso-Dap is detectable only in the spore cortex of B. sphaericus 9602, almost all of the muramic lactam in the spore is probably also located in the cortex. Group I mutants grown in the presence of L-lysine sporulate normally. Group II and III mutants produce oval and nonrefractile spores under the same conditions but the addition of meso-Dap to the culture results in the production of round and refractile spores. Thus, the presence of cortex in the spore is essential to give the round and refractile spores in B. sphaericus. The presence of cortex is also required for the accumulation of dipicolinic acid in the sporulating cells. Furthermore, 1-octanol resistance of the spore depends only on the presence of cortex but both cortex and dipicolinic acid are required for heat resistance of the spore.