The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.Abbreviation GS Gramicidin S     

Germination initiation and outgrowth of spores ofBacillus brevis strain Nagano and its gramicidin S-negative mutant

Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to germination of their spores produced in several media. Germination initiation occurred in the presence of nutrient broth orL-alanine but not with inosine, glucose, glycerol or fructose; the process was activated by heat. Parental and mutant spores behaved similarly in these experiments. During outgrowth, parental spores remained in this phase of germination much longer than did mutant spores, but only when the parental spores had been harvested from a sporulation medium where significant gramicidin S synthesis had occurred. When parental spores were extracted or treated with an enzyme that hydrolyzes gramicidin S, rapid outgrowth occurred. Adding exogenous gramicidin S or the extract from parental spores to mutant spores lengthened the outgrowth in a dose-dependent manner. The uptake of labeledL-alanine by parental spores was delayed compared to mutant spores in the presence or absence of chloramphenicol. These data suggest a mechanism of action for gramicidin S whereby it interferes in membrane function, such as transport or energy metabolism, in outgrowing spores.Abbreviations GS Gramicidin S - CFU colony-forming units     

Differentiation of species in the Bacillus brevis group and the Bacillus aneurinolyticus group based on the electrophoretic whole-cell protein pattern

Ninety strains of eleven Bacillus species in the Bacillus brevis group and the Bacillus aneurinolyticus group were compared with the electrophoretic whole-cell protein pattern. The strains were separated into two clusters at the similarity of 55%. One cluster (cluster 1) was consisted of strains from the B. brevis group, and another cluster (cluster 2) was composed of strains from the B. aneurinolyticus group. The cluster 1 was separated into eight subclusters. Out of eight subclusters, seven subclusters contain strains from B. brevis, B. laterosporus, B. agri, B. reuszeri, B. choshinensis, B. formosus, and B. borstelensis. Another subcluster was further separated into two related clusters, which corresponded to B. centrosporus and B. parabrevis, and they were fused at the similarity of 76%. Cluster 2 was separated into two subclusters, which corresponded to B. aneurinolyticus and B. migulanus. The above eleven species showed characteristic patterns distinct from one another, and this correlated well with the published DNA relatedness data. The comparison of the electrophoretic whole-cell protein pattern proved to be useful for evaluation of taxonomic relationships among these taxa.     

Induction of microcycle sporulation in Bacillus brevis spp. AG4 by amethopterin

Addition of amethopterin to medium before inoculation inhibited DNA synthesis and induced microcycle sporogenesis in Bacillus brevis spp. AG4. Synthesis of RNA and proteins occurred at a considerably reduced rate.Abbreviations TVC total viable counts - HSC heat stable counts - CDGS chemically defined medium for growth and sporulation - TCA trichloro acetic acid     

Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis

AIMS: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations. METHODS AND RESULTS: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells. This spore osmoresistance is higher on richer media. Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al. 1994). Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, sigmaB. CONCLUSIONS: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose. SIGNFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation.     

Effect of mechanical abrasion on the viability, disruption and germination of spores of Bacillus subtilis

AIMS: To elucidate the factors influencing the sensitivity of Bacillus subtilis spores in killing and disrupting by mechanical abrasion, and the mechanism of stimulation of spore germination by abrasion. METHODS AND RESULTS: Spores of B. subtilis strains were abraded by shaking with glass beads in liquid or the dry state, and spore killing, disruption and germination were determined. Dormant spores were more resistant to killing and disruption by abrasion than were growing cells or germinated spores. However, dormant spores of the wild-type strain with or without most coat proteins removed, spores of strains with mutations causing spore coat defects, spores lacking their large depot of dipicolinic acid (DPA) and spores with defects in the germination process exhibited essentially identical rates of killing and disruption by abrasion. When spores lacking all nutrient germinant receptors were enumerated by plating directly on nutrient medium, abrasion increased the plating efficiency of these spores before killing them. Spores lacking all nutrient receptors and either of the two redundant cortex-lytic enzymes behaved similarly in this regard, but the plating efficiency of spores lacking both cortex-lytic enzymes was not stimulated by abrasion. CONCLUSIONS: Dormant spores are more resistant to killing and disruption by abrasion than are growing cells or germinated spores, and neither the complete coats nor DPA are important in spore resistance to such treatments. Germination is not essential for spore killing by abrasion, although abrasion can trigger spore germination by activation of either of the spore's cortex-lytic enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanisms of the killing, disruption and germination of spores by abrasion and makes the surprising finding that at least much of the spore coat is not important in spore resistance to abrasion.     

The preparation,germination properties and stability of superdormant spores of Bacillus cereus

Aims: To determine yields, germination and stability of superdormant Bacillus cereus spores. Methods and Results: Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or l ‐alanine in 2–5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca‐DPA, dodecylamine, l ‐alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat‐activated spores were germinated. Superdormant spores stored at 4°C slowly recovered some germination capacity, but recovery was slowed significantly at ?20°C and ?80°C. Conclusions: Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Significance: Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.     

Comparision between Bacillus subtilis RP24 and its antibiotic-defective mutants

Bacillus subtilis RP24, a promising plant growth-promoting rhizobacterium and a potent biocontrol agent isolated from pigeonpea rhizosphere was mutagenized with ethyl methanesulphonate to study the possible mechanism/s involved in the potential antagonistic properties of the strain. Over 10,000 mutants were screened against the phytopathogenic fungus Macrophomina phaseolina on potato dextrose agar plates to select ten mutants showing partial antagonism as compared to the parent strain and one negative mutant showing no antagonism. The parent strain RP24 was compared with its mutants for the presence of different possible mechanisms behind antagonism. Production of hydrogen cyanide, ammonia, siderophores, and hydrolytic enzymes like lipase, amylase, and protease were detected in all the mutants as well as the parent strain, whereas fungal cell-wall-degrading enzymes, β-1, 3-glucanase and chitosanase were not detected in any of the mutants and the parent strain, indicating that none of these mechanisms was involved in the antagonistic trait of the strain. Two possible mechanisms detected behind the antifungal trait of the strain RP24 were production of antifungal volatiles and extra-cellular diffusible antibiotics. An attempt was made for extraction, partial characterization of the extra-cellular diffusible antifungal metabolite/s by thin layer chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The extracellular, methanol soluble, hydrophobic, ninhydrin-negative, thermostable and pH-stable antifungal metabolites were characterized as cyclic lipopeptides belonging to the iturin group of peptide antibiotics.     

Fermentation and toxin studies of the molluscicidal strains ofBacillus brevis

Summary Strain SS86-4 was one of 40Bacillus brevis strains shown to be molluscicidal to the schistosomiasis snail vectorBiomphalaria glabrata. When grown in mB4 medium in 2-L fermentors, SS86-4 was molluscicidal only if fructose or phenylalanine was present in the medium. This is reminiscent of secondary fermentation factor effects, in this case an antioxidant effect. In vivo proteases also were capable of reducing molluscicidal activity. The molluscicidal toxin has an LC₅₀ of 1 g toxin protein ml^–1 (approx. 1 p.p.m.) and may be described as a small proteinaceous, heat-stable, oxygen-sensitive entity associated with the particulate portion of the cell wall fraction ofB. brevis that is formed prior to sporulation. Initial information indicates that its HPLC signature shows major peaks at 148.37 and 163.96 s and consists of two bands of approximately 5.3 kDa and 8.7 kDa on PAGE gel.     

Geosmin production in the cyanobacterium Oscillatoria brevis

The cyanobacterium Oscillatoria brevis (Kütz.) Gom., strain NIVA CYA 7, was used to investigate how geosmin production is related to the synthesis of chlorophyll a, phycobiliproteins and β-carotene under nitrogen (NH ₄ ⁺ ) and light limiting conditions. Chemostat samples were used to inoculate batch cultures that were treated with inhibitors of isoprenoid synthesis, norflurazon and dimethazone, and gabaculine that inhibits tetrapyrrole synthesis. Dimethazone decreased and norflurazon increased geosmin production under light limited conditions, as was expected due to their sites of action in the isoprenoid pathway. This effect was not so pronounced in nitrogen limited cultures due to the additional effect of increasing nitrogen deficiency during the experimental period. Norflurazon was the only inhibitor that uncoupled geosmin production completely from β-carotene formation which indicates a strikt coupling between geosmin and β-carotene biosynthesis. From the observed increase of geosmin production relative to pigment synthesis after norflurazon treatment it was suggested that isoprenoid precursors are directed to geosmin synthesis when the demand for pigment precursors is very low. Within the framework of this study the data strongly support the hypothesis of geosmin formation via the isoprenoid pathway in Oscillatoria brevis as was found for actinomycetes. This research was performed at the Department of Microbiology, University of Amsterdam, with financial support provided by the royal Norwegian Ministry of Foreign Affairs and the Royal Norwegian Council for Scientific and Industrial Research     

Germination and outgrowth of Bacillus subtilis and Bacillus licheniformis spores in the gastrointestinal tract of pigs

Aims: To determine if orally ingested Bacillus spores used as probiotics or direct‐fed microbial feed additives germinate and the vegetative cells grow in the gastrointestinal (GI) tract. Methods and Results: Three independent experiments were done to determine if spores of Bacillus licheniformis and Bacillus subtilis germinate and grow in the GI tract of pigs. After a 2 weeks spore‐feeding period, spores were detected in all segments of the GI tract. The lowest number of spores was found in the stomach, increasing in the small intestine to approx. 55% of the dietary inclusion. When spores were withdrawn from the feed, faecal excretion of spores reflected the dietary inclusion, but decreased gradually to the background level after 1 week. By containing spores in short, sealed pieces of dialysis membrane that were orally administered to the pigs, both the number of spores and vegetative cells could be determined by flow cytometry. Spores accounted for 72% of the total counts after 4–6 h in the stomach and proximal part of the small intestine. After 24 h, spores constituted only 12% of the total counts in the stomach, caecum, and mid‐colon. Less spores and more vegetative cells were detected after 24 h, but total counts increased only 2·14‐fold compared to time zero. Conclusions: The experiments showed that 70–90% of dietary‐supplemented Bacillus spores germinate in the proximal part of the pig GI tract, and that only limited outgrowth of the vegetative cell population occurs. The two Bacillus strains can temporarily remain in the GI system, but will be unable to permanently colonize the GI tract. Significance and Impact of the Study: A substantial population of growing vegetative cells in the GI tract is not a prerequisite for the mode of action of Bacillus feed additives and probiotics.     

Inter-simple sequence repeat (ISSR) diversity within Monarda fistulosa var. brevis (Lamiaceae) and divergence between var. brevis and var. fistulosa in West Virginia

Inter-simple sequence repeat (ISSR) banding patterns were used to examine genetic diversity within and among populations ofMonarda fistulosa var.brevis, a rare taxon restricted to several populations in limestone glades and barrens in eastern West Virginia and Virginia. More than 34% of the total ISSR diversity in var.brevis occurred among populations, which is high when compared to the few other rare species that have been examined for ISSR variation. Prior studies demonstrated that var.brevis is morphologically distinct from the more widespread var.fistulosa, and that the differences are maintained when the two varieties are grown together in a uniform environment. The present study utilizing ISSR markers indicated that the two varieties are distinct, though quite similar genetically, and this is concordant with prior investigations documenting their morphological and habitat differences. However, the ISSR results suggest that the two varieties have diverged relatively recently and/or there is a low level of gene flow between them.     

Interactions between Bacillus subtilis early spore coat morphogenetic proteins

When challenged by stresses such as starvation, the soil bacterium Bacillus subtilis produces an endospore surrounded by a proteinaceous coat composed of >70 proteins that are organized into three main layers: an amorphous undercoat, lightly staining lamellar inner coat and electron-dense outer coat. This coat protects the spore against a variety of chemicals or lysozyme. Mutual interactions of the coat's building blocks are responsible for the formation of this structurally complex and extraordinarily resistant shell. However, the assembly process of spore coat proteins is still poorly understood. In the present work, the main focus is on the three spore coat morphogenetic proteins: SpoIVA, SpoVID and SafA. Direct interaction between SpoIVA and SpoVID proteins was observed using a yeast two-hybrid assay and verified by coexpression experiment followed by Western blot analysis. Coexpression experiments also confirmed previous findings that SpoVID and SafA directly interact, and revealed a novel interaction between SpoIVA and SafA. Moreover, gel filtration analysis revealed that both SpoIVA and SpoVID proteins form large oligomers.     

Interactions between Psilocybe fasciata and its companion fungus Acremonium strictum

Interactions between Psilocybe fasciata and its companion fungus Acremonium strictum were analysed. The conidia of A. strictum were observed on stipe, flesh and gill of P. fasciata, which suggested that A. strictum is the fungicolous fungi or mycophilic fungi of P. fasciata. The microscopic observations of the interacting hyphae of P. fasciata and A. strictum in dual culture and the negligible activities of chitinase and β-1,3-glucanase in inducing or non-inducing media of the pure and mixed cultures of the two fungi indicated that A. strictum is not the mycoparasite of P. fasciata. In addition, the co-existence, no pigmentation and dew formation in dual culture of both fungi were observed, which implied that the interference competition between the two fungi is weak. The activities of cellobiase, filter paper enzyme, endoglucanase and xylanase of pure and mixed cultures of P. fasciata and A. strictum were the same or similar, which may allow the co-existence of the two fungi. As a consequence of coevolution, the relationship between P. fasciata and A. strictum is commensalism: A. strictum showed no clear benefit to P. fasciata; however, P. fasciata may shelter A. strictum by its psychoactive tryptamines and may be helpful to conidium dispersal of A. strictum. The relationship between P. fasciata and A. strictum is different from that of A. strictum and other fungi.     

Isolation and biochemical characterization of a new NADH oxidase from Lactobacillus brevis

A new NADH oxidase, useful for the regeneration of NAD⁺, was isolated and characterized from Lactobacillus brevis. In crude extracts the activity was from 10–15 U mg^–1. After purification by four chromatographic steps, an activity of 116 U mg^–1 was obtained with 14% yield. Highest activity was from pH 5.5–7 and at 40°C. The enzyme requires dithiothreitol to prevent oxidative deactivation. The K _m value for NADH was 24 M.     

Thermostable extracellular protease of Bacillus stearothermophilus: factors affecting its production

A strain of protease-producing Bacillus stearothermophilus has been isolated. Glycerol was the best carbon source for production whereas yeast extract was the best nitrogen source. The bacterium could grow up to 70°C but optimum protease production was at 60°C. Best initial pH for protease production was 5. Alkaline pH inhibited production. The enzyme was stable at 60°C for 18 h and was inhibited by EDTA, PMSF and HgCl₂.The authors are with the Enzyme and Microbial Technology Group, Faculty of Science and Environmental Studies, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor, Malaysia     

General Stress Protein CTC from Bacillus subtilis Specifically Binds to Ribosomal 5S RNA

Two recombinant proteins of the CTC family were prepared: the general stress protein CTC from Bacillus subtilis and its homolog from Aquifex aeolicus. The general stress protein CTC from B. subtilis forms a specific complex with 5S rRNA and its stable fragment of 60 nucleotides, which contains internal loop E. The ribosomal protein TL5 from Thermus thermophilus, which binds with high affinity to 5S rRNA in the loop E region, was also shown to replace the CTC protein from B. subtilis in its complexes with 5S rRNA and its fragment. The findings suggest that the protein CTC from B. subtilis binds to the same site on 5S rRNA as the protein TL5. The protein CTC from A. aeolicus, which is 50 amino acid residues shorter from the N-terminus than the proteins TL5 from T. thermophilus and CTC from B. subtilis, does not interact with 5S rRNA.     

Reaction of substituted phenols with thermostable laccase bound to Bacillus subtilis spores

A strain of B. subtilis produced 1.8 times more laccase on sporulation medium than on non-sporulation medium. Spores oxidized mono- and di-methoxyphenols (0.1 mM) at 50 °C. The half-life of laccase bound to spores was about 2 d and the substrate was repeatedly removed by spores recovered from the reaction mixture.     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference