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.     

Spore formation by Bacillus polymyxa during the inhibition of polymyxin biosynthesis

When amino acids are added to the culture of Bacillus polymyxa growing under the conditions of active spore formation, the rate of polymyxin biosynthesis and the quantity of spores being formed decrease. All cells in the population start to produce spores under these conditions, but only some of them pass through all of the differentiation stages. Most of the cells remain in the state of prospores. Some cells which have started to differentiate are characterized by ultrastructural anomalies, do not form prospores and undergo early lysis.     

Enzyme activity of Bacillus polymyxa 153, the producer of polymyxin B, during sporulation and biosynthesis

Metabolic properties of Bacillus polymyxa 153 were studied during vegetative growth, polymyxin B biosynthesis and active sporulation. In the cell extracts there was detected activity of exoproteases, endoproteases, tricarboxylic acid cycle dehydrogenases and pyruvate dehydrogenase. The enzymes activity in the cells growing into spores was higher than that in the cells of the vegetative developmental type. The activity of the enzymes depended on the culture age.     

Polymyxin biosynthesis by Bacillus polymyxa during growth limitation by the nutritional sources

The synthesis of the antibiotic polymyxin M was studied under the conditions of batch and continuous cultivation of Bacillus polymyxa var. Ross whose growth was limited with glucose, phosphate and ammonium nitrogen. Polymyxin M was synthesized when the culture growth decelerated as a result of its limitation with the above compounds. Different amounts of the antibiotic were synthesized depending on the type of a limiting factor. The highest productiveness was found in the case of glucose limitation. The optimal conditions for polymyxin M synthesis were established under the conditions of one-step continuous cultivation.     

Increased susceptibility of injured spores of Bacillus subtilis to cationic and other stressing agents

Sublethally heated spores of Bacillus subtilis NCTC 8236 were susceptible to posttreatment concentrations in agar of polymyxin B sulphate, sodium hydroxide, cetylpyridinium chloride and sodium lauryl sulphate that did not prevent colony formation to untreated spores. The non-ionic surfactants polysorbates 20 and 80 were not inhibitory when used at high concentrations against both heated and unheated spores. The method has been developed for detecting sublethal injury in biocide-exposed spores, since iodine-treated spores became highly susceptible to polymyxin contained in recovery agar.     

Ethanol sensitivity of sporulation in Bacillus subtilis: a new tool for the analysis of the sporulation process.

The growth rate of Bacillus subtilis is lowered but the final cell yield is unchanged when certain concentrations of ethanol are present in the culture medium. At the concentration allowing growth at half-maximal rate, practically no spores are formed. Blockage of spore formation generally occurs at stage 0-I. Sensitivity to ethanol of the capacity to form spores is limited, in a nonsynchronized culture, to a period of at most 45 min around t1. Postexponential events such as excretion of certain enzymes and modification of ribonucleic acid polymerase are altered or suppressed in the presence of ethanol, possibly as the results of a physical change upon the cell membrane. In effect, ethanol is turning wild-type cells into phenocopies of spoO mutants.     

Quantitative Analysis of Polymyxin B Released from Polymyxin B-Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 10⁸ cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl₂ solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.     

Spores of microorganisms

Preexisting¹⁴C-DAP in vegetative cells ofBacillus cereus is not incorporated into the spores, but is released into the medium after sporogenesis is complete. Exogenous¹⁴C-DAP added to the medium before sporulation is incorporated intensively into the sporangia and practically all of it is taken up by the spores. During sporogenesis, two periods of increased incorporation of¹⁴C into hot TCA-precipitate of cells are found after adding¹⁴C-DAP— one before formation of the spores, when¹⁴C-lysine formed by decarboxylation is incorporated together with¹⁴C-DAP, and one during the “whitening” phase, when any¹⁴C-lysine is no longer incorporated. The incorporation of exogenous¹⁴C-lysine into the sporangial proteins is also markedly elevated during the presporulation phase and at the outset of sporogenesis.     

Synthesis and characterization of the colistin peptide polymyxin E1 and related antimicrobial peptides.

Two strategies were developed to synthesize the acylated cyclic peptides know as polymyxins. Synthesis of polymyxin E1 and several analogs enabled us to evaluate the minimum inhibitory concentration of individual compounds against Gram-negative bacteria. In this study we also report the first identification of two component peptides in the complex polymyxin fermentation product colistin, a Thr2Ser isoform and an acyl group isomer. Both of these peptides, as well as a known component peptide, Leu7Ile, were similar to polymyxin E1 in potency, suggesting that conservative mutations in the colistin family are functionally inconsequential. In contrast, the acyclic analogs of all of these peptides were inactive, indicating that the characteristic lariat structure of the polymyxins is necessary for antimicrobial activity.     

Development of dilipid polymyxins: Investigation on the effect of hydrophobicity through its fatty acyl component

Continuous development of new antibacterial agents is necessary to counter the problem of antimicrobial resistance. Polymyxins are considered as drugs of last resort to combat multidrug-resistant Gram-negative pathogens. Structural optimization of polymyxins requires an in-depth understanding of its structure and how it relates to its antibacterial activity. Herein, the effect of hydrophobicity was explored by adding a secondary fatty acyl component of varying length onto the polymyxin structure at the amine side-chain of l-diaminobutyric acid at position 1, resulting to the development of dilipid polymyxins. The incorporation of an additional lipid was found to confer polymyxin activity against Gram-positive bacteria, to which polymyxins are inherently inactive against. The dilipid polymyxins showed selective antibacterial activity against Pseudomonas aeruginosa. Moreover, dilipid polymyxin 1 that consists of four carbon-long aliphatic lipids displayed the ability to enhance the antibacterial potency of other antibiotics in combination against P. aeruginosa, resembling the adjuvant activity of the well-known outer membrane permeabilizer polymyxin B nonapeptide (PMBN). Interestingly, our data revealed that dilipid polymyxin 1 and PMBN are substrates for the MexAB-OprM efflux system, and therefore are affected by efflux. In contrast, dilipid polymyxin analogs that consist of longer lipids and colistin were not affected by efflux, suggesting that the lipid component of polymyxin plays an important role in resisting active efflux. Our work described herein provides an understanding to the polymyxin structure that may be used to usher the development of enhanced polymyxin analogs.     

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.     

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     

分子模拟在多黏菌素药理机制研究中的应用

多黏菌素是一种膜靶向的脂肽类抗生素，是临床上治疗革兰氏阴性多重耐药菌感染的最后一道防线。通过与脂多糖相互作用，多黏菌素破坏细菌外膜结构并导致细菌死亡。然而，受限于生物化学和结构生物学手段对细胞膜-药物相互作用的表征能力，目前对多黏菌素药理机制的认识还不充分，从而限制了新一代多黏菌素药物的设计和开发。为此，本文总结了近年来利用分子动力学方法对细胞膜系统与多黏菌素相互作用的研究进展，为深入理解多黏菌素药理机制与细胞膜系统的内在联系，加快新型抗生素药物研发提供新思路。     

Spores of microorganisms

The addition of penicillin (300–1,000 units/ml.) to a culture ofBacillus cereus during formation of the refractive prespores leads to lysis of the sporangia and to the release of spore components (calcium and dipicolinic acid) from the cells. Penicillin mildly raises the incorporation of amino acids, including diaminopimelic acid, into hot-TCA precipitate of cells, while chloramphenicol lowers it. In the later phases of penicillin inhibition, DAP-containing structures are also destroyed, including the fraction firmly bound to the envelope structures of the spore (in the control culture this fraction is not released until later, during digestion by enzymes localized in the envelope structures themselves). Penicillin inhibition of sporogenesis can be reversed by adapting the culture to penicillin or by simultaneously adding chloramphenicol. After the presporulation phase, sporogenesis is relatively resistant to chloramphenicol, but the whole process is considerably slowed down. Chloramphenicol also affects the morphology of the spores during their formation and inhibits their release from the sporangia until the late phase of sporulation.     

Permeability of gentamicin and polymyxin B into the inside of Bacillus subtilis spores

The penetration of gentamicin and polymyxin B into the inside of Bacillus subtilis spores was examined by an immunoelectron microscopy method with colloidal gold--immunoglobulin G (IgG) complex. The colloidal gold particles were located predominantly in the coat region of both gentamicin-treated and polymyxin B-treated spores and were hardly observed in the other regions, i.e., the cortex and core regions. When these antibiotic-treated spores were subsequently treated with CaCl2, the number of gold particles bound to the coat region was greatly decreased. These results suggest that these two antibiotics are able to penetrate into the spore coat but not into the cortex or core, that is, the primary permeability barrier to them exists between the coat and the cortex regions.     

The effect of carbohydrates on the sporogenesis of Clostridium perfringens and Bacillus anthracis

The authors studied the effect of a number of carbohydrates on the sporogenesis of Clostridium perfringens and Bacillus anthracis (vaccine strain STI) as probable soil factors capable of influencing the duration of survival of these causative agents in the external environment. Differences in the effect of the same sugars on the formation of spores by these microorganisms and clearly expressed sporogenesis-inhibiting effect of glucose (and also of lactose in clostridia) have been demonstrated. The analysis of the peculiarities of sporogenesis under unadjusted and stabilized pH values provides a basis for regarding the "glucose effect" as repression of sporogenesis in the given causative agent, but not as inhibition resulting from considerable acidification of the culture medium. This is essential for the soil conditions characterized by high buffer capacity. The ecological value of substances of carbohydrate nature consists in their important role in the energetics and trophicity of microbial coenoses of the soil which cannot fail reflecting on the fate of pathogenic microorganisms in the soil.     

Polymyxin B biosynthesis and tricarboxylic acid cycle enzymatic activity]

Activity of pyruvate dehydrogenase (PDG), isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase was found in the extracts of the cells of Bac. polymyxa 153, an organism producing polymyxin B. Dependence of the activity of the above enzymes on the carbon source in the medium, aeration conditions, strain features and culture age was shown. A low level of polymyxin B biosynthesis was observed at high activity of PDG and dehydrogenases of the tricarbonic acid cycle. Increased antibiotic production was recorded against the background of decreases values of the above enzyme activities.     

MEIOSIS IN VITRO: SPOROGENESIS IN CULTURED FERN PLANTS

Sporangia containing mature, functional spores can be induced to form on pinnae of Todea barbara sporophytes maintained in nutrient culture. Meiosis is initiated in the sporogenous cells and proceeds to completion in vitro with the formation of viable spores. Sporangial development, meiosis and the production of spores occur in plants which exhibit signs of senescence. Fertile leaves are characterized by a reduction in chlorophyll concentration and total cellular nitrogen when compared to sterile leaves. The data suggest that the process of sporogenesis may be correlated with a reduction in metabolic activity of the leaf.     

Isolation of bioactive antibiotic peptides from Bacillus brevis and Bacillus polymyxa against Botrytis grey mould in strawberry

Abstract

Grey mould caused by Botrytis cinerea is a devastating disease that results in extensive yield losses to strawberry. Bacillus brevis (Brevibacillus brevis) and Bacillus polymyxa (Paenibacillus polymyxa), which showed strong antifungal activity against B. cinerea, were isolated from the phyllosphere of strawberry plants. The advantage of using these bacteria is that the biochemistry and physiology of production of antibiotic peptides antimicrobial substances is well documented. A study was conducted to assess the activity of both Bacilli and their antibiotic peptides produced against B. cinerea in strawberry plants in vitro and in vivo. In vitro bioassay, both Bacilli have strongly inhibited pathogen germination, growth and extra-cellular enzyme production. Bacillus brevis was generally the most effective in reducing Botrytis growth. Gramicidin S and polymyxin B peptide antibiotics were extracted from culture filtrate of B. brevis and B. polymyxa, respectively, purified by silica thin chromatography and identified by high performance liquid chromatography. Germination, growth rate and production of extra-cellular enzymes were more sensitive to both antibiotics. Gramicidin S was the most active against B. cinerea with a minimal inhibitory concentration of 15 μmol/l. Polymyxin B also showed activity against B. cinerea at 25 μmol/l. Under controlled conditions (18 – 22°C, 90% relative humidity and 12 h photoperiod), strawberry plants were sprayed with pathogens (10⁵ spores/ml), antagonists (from 10⁵ to 10⁸ cells/ml) and antibiotic peptides (0 – 30 μmol/l) for reducing grey mould. Disease incidence was decreased in the presence of B. brevis. Both antibiotic peptides inhibited Botrytis growth that was observed by scanning electron microscopy. The plant leaves adsorbed significant amounts of antibiotics which reached from 46.1 to 67.5% of the original solution. Under natural field conditions, these biocontrol and antibiotic peptides at different concentrations were evaluated in 2003/2004 and 2004/2005 seasons against Botrytis grey mould. Treating plants with B. brevis exhibited a significant high activity against the development of Botrytis disease on strawberry. Gramicidin S showed a strong potential in reducing disease incidence, followed by polymyxin B, and acted as a fungicide to the pathogen growth. Inhibition of B. cinerea by both Bacilli was similar to equivalent levels of their antibiotics produced. In addition, these treatments significantly reduced the development of Botrytis and increased fruit yield. It can be suggested that B. brevis and B. polymyxa may be considered as potential biocontrol agents against Botrytis grey mould on strawberry based on the production of antifungal peptides. Therefore, gramicidin S and polymyxin B products are considered as biocontrol agents and may play a significant role in the future for practical applications in strawberry management systems.