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     

Transition of outgrowing spores of Bacillus brevis into vegetative cells is not dependent on destruction of gramicidin S

Summary Certain Bacillus brevis strains produce gramicidin S (GS) during sporulation, and germination of such spores is delayed at the stage of outgrowth by endogenous or exogenous GS. Claims have been made that the transition from germinating spores into vegetative cells is dependent on GS destruction. We observed no such destruction of either exogenous or endogenous GS. Thus, in our hands, the recovery of the inhibited germinating spores must be dependent on something other than GS elimination.Offprint requests to: G. Bentzen     

Coexistence of parentalBacillus brevis and its gramicidin S-negative mutant during spore germination stages

Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared in separate as well as in mixed cultures with respect to germination of their spores in several media. Mixed-culture experiments were facilitated by the observation that colonies of wild and mutant cultures are distinctly different in appearance on nutrient agar. We found that there was complete coexistence in both strains throughout the outgrowth phase of germination, during which gramicidin S-induced suicide normally occurs in the wild-type prior to vegetative growth. Coexistence was also observed in media supporting germination but not growth, i.e., alanine-salts and alanine-water. The same was found when spores of the two strains were incubated in a soil suspension. We found that both strains become sensitive to starvation in a salts mixture only after development into vegetative cells, the mutant strain being more sensitive than the parent in this regard, but again coexistence was observed in mixed culture.     

Biological role of gramicidin S in spore functions. Studies on gramicidin-S-negative mutants of Bacillus brevis ATCC9999.

Gramicidin-S-negative mutants of Bacillus brevis ATCC9999 have been isolated with a remarkly higher yield after ethidium bromide or acridine orange treatment, than after N-methyl-N'-nitro-N-nitrosoguanidine treatment. Four (MIV, Smr170, R5 and EB 16) of 38 isolated mutants were characterized with respect to the lesion in gramicidin-S-synthesizing activity. The mutants sporulate to the same extent as the parental strain except mutant Smr 170 which sporulates less. However, mutant spores were more heat-sensitive and possessed a reduced level of dipicolinic acid content. No significant difference was observed in the germination time of wild-type and mutant spores. All spores germinated after 80--110 min, but the outgrowth time was different: all gramicidin-S-negative mutants grew out immediately after germination whereas wild-type spores required a lag period of 9--10 h. When the mutants were allowed to sporulate in the presence of gramicidin S, the spores were found to be heat-resistant and their outgrowth postponed to the same period as the parent spores. The addition of gramicidin also eliminated the deficiency of dipicolinic acid. A new class of gramicidin-S-negative mutant, R5, which only activates L-valine and L-leucine, is described. A possible biological function of gramicidin S in the heat-resistance and in the timing of spore outgrowth is discussed.     

Permeability of dormant spores of Bacillus subtilis to gramicidin S

Abstract Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis , had a partial inhibitory effect on l-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.     

Induction of petite mutations during germination and outgrowth of Saccharomyces cerevisiae ascospores.

The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.     

Mutual Relationship between Antibiotics and Resting Spores of Bacillus subtilis: Binding of Cyclic Polypeptide and Aminoglycoside Antibiotics to Spores and Their Inhibitory Effect on Outgrowth and Vegetative Growth

Not only cyclic polypeptide antibiotics such as polymyxin B, colistin and gramicidin S but also aminoglycoside antibiotics such as streptomycin, kanamycin, gentamicin and kanamycin derivatives combined with the resting spores of Bacillus subtilis and inhibited outgrowth or vegetative growth after germination. All the antibiotics other than gramicidin S were released from the resting spores and their inhibitory action was reversed by the addition of Ca²⁺ and Fe³⁺. As the above antibiotics have free amino (or guanidine) groups in common, it was assumed that such groups play an important role in binding of the antibiotics to the resting spores. Moreover, it was shown that protamine and poly-l-lysine were also bound to the resting spores and were released from them by Ca²⁺. On the other hand, free carboxyl groups had been demonstrated in the outermost surface of the resting spores in a previous study. Thus, we assume that the mode of binding of the antibiotics to the resting spores may be due to the formation of reinforced ionic bonds between amino (or guanidine) groups in the antibiotics and carboxyl groups on the spore surface.     

Resting Spore Dormancy and Infectivity Characteristics of the Potato Powdery Scab Pathogen Spongospora subterranea

The soil‐borne potato pathogen Spongospora subterranea persists in soil as sporosori, which are aggregates of resting spores. Resting spores may germinate in the presence of plant or environmental stimuli, but direct evidence for resting spore dormancy is limited. A soilless tomato bait plant bioassay and microscopic examination were used to examine features of S. subterranea resting spore dormancy and infectivity. Dried sporosori inocula prepared from tuber lesions and root galls were infective after both short‐ and long‐term storage (1 week to 5 years for tuber lesions and 1 week to 1 year for root galls) with both young and mature root galls inocula showing infectivity. This demonstrated that a proportion of all S. subterranea resting spores regardless of maturity exhibit characteristics of stimuli‐responsive dormancy, germinating under the stimulatory conditions of the bait host plant bioassay. However, evidence for constitutive dormancy within the resting spore population was also provided as incubation of sporosorus inoculum in a germination‐stimulating environment did not fully exhaust germination potential even after 2.4 years. We conclude that S. subterranea sporosori contain both exogenous (stimuli‐responsive) and constitutively dormant resting spores, which enables successful host infection by germination in response to plant stimuli and long‐term persistence in the soil.     

Germination-initiated spores of Bacillus brevis Nagano retain their resistance properties.

Initiated spores and vegetative cells of the gramicidin S-producing Bacillus brevis Nagano were compared with respect to their resistance to various forms of stress (osmotic shock-starvation, exposure to ethanol, sonic oscillation, and heat). The resistance of initiated spores to all of these stress situations was considerably greater than that of vegetative cells and approached that of dormant spores. The period during which the initiated spores remained resistant to heat was extended by addition of gramicidin S. The antibiotic may therefore be of survival value to the species in nature by slowing down the development of initiated spores in the outgrowth phase of germination, thereby extending the period during which the cells are resistant to environmental stress.     

Chloride dependence of endospore germination in Halobacillus halophilus

The ion requirement for germination and outgrowth of endospores from the moderately halophilic salt marsh bacterium Halobacillus halophilus was studied. Germination and outgrowth of endospores plated onto nutrient broth was dependent on the salt concentration in the artificial seawater used as the source of ions. Maximal germination and outgrowth were observed when double-concentrated artificial seawater was used. Replacement of chloride salts in the artificial seawater by other salts resulted in a complete loss of germination and outgrowth that was restored upon addition of chloride. To analyze the role of chloride more directly and quantitatively, a defined growth medium was used in which the artificial seawater was substituted by a solution of magnesium sulfate and sodium chloride. Spore germination and outgrowth were strictly dependent on the chloride concentration; maximal germination and outgrowth were observed at ≈ 1.3 M Cl^–. Chloride could be substituted by bromide, but not by sulfate or nitrate. Microscopic examinations of single spores clearly showed that germination is the chloride-dependent step. This first report on chloride dependence of spore germination in any endospore-forming bacterium adds another function to chloride in H. halophilus apart from its being essential for the physiology of the vegetative cell. Received: 21 May 1999 / Accepted: 26 July 1999     

Die Beeinflussung der Farnsporen-Keimung [Osmunda cinnamomea (L.) undO. claytoniana (L.)] über das Phytochromsystem und die Photosynthese

Summary The control by light of the spore germination ofOsmunda cinnamomea L. andO. claytoniana L. has been investigated.This light control is complex, and in order to investigate this control quantitatively the process of germination had to be divided into at least two stages. The first stage of germination leads to the rupture of the exospore, the second step is characterized by the outgrowth of a rhizoid. In a very few percent of our spore population the first stage can occur in total darkness, the second step however does not ever occur in darkness.In the present paper it has been demonstrated, usingO. cinnamomea spores, that the first step of germination is phytochrome controlled, whereas the second step is under photosynthetic control. This was observed withO. claytoniana by a rough action spectrum and in experiments with a CO₂-less atmosphere.The germinating spores of these two species ofOsmunda show a very similar behaviour as the spores of the mossFunaria hygrometrica (L.) Sibth. (Bauer andMohr 1959). They behave very differently compared with spores of other ferns, e.g.Dryopteris filix-mas (L.) Schott, where the whole process of germination is exclusively phytochrome controlled (Mohr 1956).

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潮间带盐沼植物种子及实生苗漂浮起动临界剪切应力研究

盐沼是分布在海陆过渡区域,以大型草本植物为优势种的潮间带生态系统。在潮间带生态系统中,盐沼植物的种子和实生苗在潮流作用下的漂浮起动是盐沼植物自然扩散的重要前提,决定了盐沼植物能否实现有效扩散和长距离传播。然而,目前缺乏有关盐沼植物繁殖体在复杂潮滩环境下漂浮起动扩散过程的定量研究。以长江口典型盐沼先锋物种海三棱藨草(Scirpus mariqueter)为研究对象,应用U-GEMS微观侵蚀系统,测定了在不同沉积物底质条件下,海三棱藨草种子及不同萌发阶段实生苗漂浮起动时的临界剪切应力,定量分析不同萌发阶段和沉积物底质对海三棱藨草种子和实生苗漂浮起动的影响。研究结果表明：(1)不同种子萌发阶段和沉积物底质对海三棱藨草种子和实生苗漂浮起动的临界剪切应力均有极显著影响(P<0.01);(2)随着萌发阶段的进展,海三棱藨草实生苗漂浮起动所需的临界剪切应力逐渐减小,因而随潮流漂浮起动进而扩散的机会也相应增大;(3)相比于淤泥质潮滩底质,海三棱藨草种子和实生苗在粉砂质潮滩更易于漂浮起动。研究结果丰富了盐沼植物在潮间带自然扩散过程和机理的研究,也为今后长江口及其他区域开展高效、低成本、以种子为修复...     

Stabilization of the polarity axis in the zygotes of some Fucaceae

Summary The existence of a period of latent but stable polarity, i.e. a period in which the polarity axis has been irreversibly established but no morphological asymmetry can be detected, was studied in germinating populations of zygotes of Pelvetia fastigiata. We found that the time course of the loss of sensitivity to a single polarity-axis-determining light stimulus coincided with the time course of germination (rhizoid outgrowth), up to the germination of about one-third the population, showing that the cells remained responsive to the light stimulus until or almost until the appearance of visible asymmetry. In the later part of the germination period, some of the zygotes may have lost their light sensitivity somewhat before rhizoid outgrowth, suggesting that at least some of the ungerminated cells may at this time possess, for a brief period, a latent but stable polarity axis.The loss of responsiveness with time to an antagonistic, second light stimulus followed the same time course as the loss of sensitivity to a single light stimulus. There was no suggestion of the existence of a latent but stable polarity axis in any members of the population in this experiment.An analysis of data of Jaffe (1968) on polarity-axis determination and germination in P. fastigiata following a single light stimulus yields essentially the same conclusions as our own single-light-stimulus experiments.In contrast, analysis of data of Whitaker and Lowrance (1936) on Fucus furcatus indicated that in these zygotes latent but stable determination of the polarity axes had taken place 3–4 hours before germination. A similar situation emerges from the analysis of another experiment of Jaffe (personal communication) with P. fastigiata zygotes in which the loss of sensitivity to an orienting light stimulus appeared accelerated and germination may have been delayed in comparison with his 1968 data.We conclude, therefore, that populations of Fucaceae zygotes may vary with regard to the existence of a latent but stable polarity axis. However, when existence of such a latent, stable polarity axis can be inferred, its duration usually is brief, and it seems to be in most cases limited to a small fraction of the individuals of the total population at any particular time. In order to infer rigorously the existence of latent but stable polarity axes in populations of germinating zygotes and similar cells, it is essential to obtain the time courses for axis stabilization and for the development of visible asymmetry simultaneously.     

Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water

Biofilms were used to produce gramicidin S (a cyclic decapeptide) to inhibit corrosion-causing, sulfate-reducing bacteria (SRB). In laboratory studies these biofilms protected mild steel 1010 continuously from corrosion in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant, which was augmented with reference SRB. The growth of both reference SRB (Gram-positive Desulfosporosinus orientis and Gram-negative Desulfovibrio vulgaris) was shown to be inhibited by supernatants of the gramicidin-S-producing bacteria as well as by purified gramicidin S. Electrochemical impedance spectroscopy and mass loss measurements showed that the protective biofilms decreased the corrosion rate of mild steel by 2- to 10-fold when challenged with the natural SRB of the TMI process water supplemented with D. orientis or D. vulgaris. The relative corrosion inhibition efficiency was 50–90% in continuous reactors, compared to a biofilm control which did not produce the antimicrobial gramicidin S. Scanning electron microscope and reactor images also revealed that SRB attack was thwarted by protective biofilms that secrete gramicidin S. A consortium of beneficial bacteria (GGPST consortium, producing gramicidin S and other antimicrobials) also protected the mild steel.     

Complexation and phase transfer of nucleic acids by gramicidin S

A novel interaction between gramicidin S (GrS) and nucleic acids is characterized which, like that between GrS and nucleotides, exploits both the dicationic and amphiphilic properties of the peptide. Complex formation between calf thymus DNA and GrS is demonstrated by (i) phase transfer to CHCl3 of ultrasonically irradiated DNA and (ii) inhibition of phase transfer to CHCl3 of adenosine 5'-triphosphate by either native or ultrasonically irradiated DNA. The stoichiometry of the interaction is 2:1 (DNA-P:GrS), which is consistent with a predominantly electrostatic mode of binding. The apparent affinity of GrS for DNA is considerably higher than it is for free nucleotides. The interaction of the monocationic derivative [2-N delta-acetylornithyl]gramicidin S with calf thymus DNA is considerably weaker. DNA binding by GrS provides a rationale for the lag between germination and RNA synthesis exhibited by wild-type spores of producer strains of Bacillus brevis but not by GrS-negative mutants. On the basis of these results in vitro, a protective role is proposed for GrS in the dormant spore.     

Localization of gramicidin S on the cytoplasmic membrane of Bacillus brevis and its effect on the activity of membrane enzymes

It was shown that malate dehydrogenase of isolated membranes of the gramicidin S producer Bacillus brevis var. G.-B. (R.-form) is completely inhibited by the antibiotic (approximately 200 mkg/mg of protein). Succinate and NADH dehydrogenases at concentration up to 1 mg per mg of protein are insensitive to it, while corresponding oxidases are inhibited by the antibiotic not more than by 65 -- 75% apparently due to partial damage of the terminal parts of the respiratory chain. The respiration of the producer intact cells is inhibited by exogenous gramicidin S by not more than 55 -- 60%, while the respiration of antibiotic-sensitive cells of M.lysodeikticus is inhibited completely. It was shown that phosphatidyl ethanolamine (50%), phosphatidyl glycerol (15% and diphosphatidyl glycerol (25%) are the major phospholipid components of the membranes of the given strain of Bac. brevis. It was assumed that the resistance of Bac. brevis cells to gramicidin S is partly due to the constant ratio of the charged and amphoteric phospholipids. Using 31P-NMR spectroscopy, the kinetics of free phosphoric compounds in the cells and cell extracts of Bac. brevis during culture growth and gramicidin S synthesis were studied. The content of carbohydrate monophosphate, remained unaffected, while that of nucleoside di- and triphosphates and dinucleotides was low and at definite density and gramicidin S content (above 100 mkg/ml) fell down below the resolution capacity of the method employed. Evidence for gramicidin S localization of the Bac. brevis membrane and possible causes for the manifestation of the NADH dehydrogenase activity at a certain stage of culture growth are discussed.     

Sulfinylated azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils

Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultra‐high resolution electrospray ionization (ESI), Fourier‐transform ion cyclotron resonance (FT‐ICR) and MS/MS techniques to accurately determine the mass (202.126 Da) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C₁₀H₁₉NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N‐methanesulfinyl 1‐ and 2‐azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 μm stimulated approximately 50% germination) and elicit accession‐specific response. Although N‐methanesulfinyl 2‐azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin‐like molecules to ensure rapid fertilization by the appropriate pollen.     

Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.