Oxidation of Manganese and Formation of Mn3O4 (Hausmannite) by Spore Coats of a Marine Bacillus sp

Isolated spore coats of a marine Bacillus species were incubated in 25 mM MnCl₂ at pH 7.5. Manganese precipitates, formed on the coat surfaces, were analyzed by transmission electron microscopy, electron diffraction, and energy-dispersive X-ray spectroscopy. Initially, an amorphous manganese oxide was observed on the coats which recrystallized to hausmannite after prolonged incubation in the MnCl₂ solution. The spore coats catalyze the oxidation of Mn(II) and have no structural influence on the final mineral phase precipitated.     

红盖鳞毛蕨孢子发育的超微结构和细胞化学研究

采用透射电镜和细胞化学技术对红盖鳞毛蕨(Dryopteris erythrosora(Eaton)O.Ktze.)的孢子发育过程进行了研究,根据超微结构和细胞化学特征可将其孢子发育过程分为3个阶段:(1)孢子母细胞及其减数分裂阶段:孢子母细胞壳在孢原细胞末期开始形成,位于孢子母细胞及其减数分裂形成的四分体外侧,PAS反应显示其为多糖性质,与胼胝质壁为同功结构;在减数分裂形成的四分孢子之间产生孢子外壳,从功能、形成位置和时间上看与胼胝质壁相似,但苏丹黑B反应显示其可能含有脂类物质,与孢子母细胞壳和胼胝质壁不同。(2)孢子外壁形成阶段:外壁为乌毛蕨型(Blechnoidal-type),由薄的多糖性质的外壁内层和表面平滑的孢粉素外壁外层构成;小球参与外壁外层的形成,组织化学分析显示小球的中央区域和外壁外层内侧部分由红色(多糖)变为黄色,小球的表面区域和外壁外层部分始终被染成黑色(脂类),可知小球与外壁同步发育。(3)孢子周壁形成阶段:周壁为凹陷型(Cavate-type),包括2层,内层薄,紧贴外壁,外层隆起形成孢子脊状褶皱纹饰的轮廓,以少见的向心方向发育;苏丹黑B和PAS反应观察周壁被染成橙色,推测其可能由多糖等成分构成;孢子囊壁细胞参与周壁的形成。本研究为揭示蕨类植物孢子发生的细胞学机制提供了新资料。     

家蚕微孢子虫孢壁蛋白与其发芽的相关性

为了研究孢壁蛋白与家蚕微孢子虫发芽（孢原质弹出）的相关性,我们采用碳酸钾诱导微孢子虫体外发芽结合密度梯度离心的方法（简称GDGC法）,收集纯化发芽后的孢子空壳（简称孢壳）,对发芽液、纯化的孢壳及成熟孢子的孢壁蛋白组分进行了分析。结果表明：GDGC法可以获得高纯度孢壳,计算出其密度为1．130g/cm^3;与发芽前成熟孢子提取的孢壁蛋白相比,空孢壳可以提取到主要孢壁蛋白SWP32、SWP30、SWP25,同时发现SWP32、SWP25丰度有所降低;结合碳酸钾发芽液的蛋白电泳分析,发现孢壳上丰度降低的SWP32在发芽液蛋白样品中存在,LC—MS／MS数据分析也发现SWP32、SWP30、SWP25在碳酸钾处理液中都有存在;而用碳酸钾溶液处理冷冻孢子时,未观察到发芽现象,电泳结果显示此时K2CO,溶液中只有SWP30条带,说明在碳酸钾溶液诱导的发芽过程中SWP32和SWP25从孢壳上脱落可能与发芽相关而不是被碱性的碳酸钾溶解下来的[动物学报54（6）：1068—1074,2008]。     

Identification of a Microsporidian Polar Tube Protein Reactive Monoclonal Antibody

ABSTRACT. The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analyis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.     

Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat

The hydrophobic characteristics of Clostridium perfringens NCTC 8679 spores were demonstrated by adherence to toluene in a toluene-aqueous partition system. Spores and spore coat preparations were hydrophobic. Vegetative cells and spores extracted with a dithiothreitol-sodium dodecyl sulfate treatment known to remove spore coats were not hydrophobic. A heat activation treatment (75 degrees C for 20 min) which promotes more rapid spore germination increased the hydrophobicity of intact spores and decreased that of isolated spore coats. The hydrophobic changes were reversed by washing and stabilized by 0.5% glutaraldehyde. Heat-induced hydrophobic changes were observed in spore coats prepared from spores that were preheated and washed before rupturing in a buffer containing glutaraldehyde. These results suggest the occurrence of a heat-induced change in the spore coat (possibly in the conformation of a macromolecule) which was stable only within the architectural confines of the intact spore.     

The Presence of Keratin-like Substance in Spore Coat of Bacillus subtilis

Unique crystalline structures were found by X-ray diffractometry to be present in spore coats of Bacillus subtilis. By crystallographical and chemical studies of the purified spore coats it was found that these crystalline structures of the spore coats were essentially similar to those of α- and β-keratin, and that the spore coats were composed of keratin-like substance (or keratin). This keratin-like substance was found to be synthesized during sporogenesis from sulfur-containing water-soluble substances in the cells.     

苏芸金杆菌伴孢晶体和芽孢孢衣的蛋白质组分及其与毒力的关系

研究了苏芸金杆菌(Bacillus thuringiensis)3个变种6个菌株的提纯伴孢晶体和芽孢对大蜡螟(Galleria mellonella)和大菜粉蝶(Pieris brassicae)的毒力、晶体的蛋白质和多肽成分及芽孢衣中的类晶体蛋白质成分.生物测定表明,晶体毒力高于芽孢,在总数量相同的情况下,晶体和芽孢近1:1的混合物的毒力高于单独的晶体或芽孢.芽孢衣中存在一种类似晶体蛋白质的成分,无晶体突变株及无效野生株的芽孢则无此种蛋白质且对两种昆虫无毒.变种wuhanensis和变种galleriae的晶体含MW138000的主要蛋白质和63000的次要蛋白质,经碱性缓冲液溶解后,上清液含MW138000的蛋白质,沉淀中含MW63000的蛋白质;变种aizawai的晶体中仅含138000的蛋白质.对大蜡螟无毒的HD-11(var.aizawai)晶体的蛋白质成分有别于上述晶体,其胰蛋白酶消化物的SDS凝胶电泳图型显示少2条多肽带,但对大菜粉蝶仍有效.结果表明,苏芸金杆菌的芽孢在昆虫病理中有重要作用,伴孢晶体的蛋白质和多肽成分与它们对昆虫的毒力特性之间有密切关系.     

Properties of Hg- and Cd-spores of Bacillus megaterium QM B1551

Hg- and Cd-spores of Bacillus megaterium QM B1551 were produced in Schaeffer's medium containing mercuric chloride and cadmium chloride respectively. Metals were added to the medium at 9 hr of incubation (Stage V) to give a final concentration of 50 microM. It was found by electron microscopic and biochemical studies that the coats of both Hg- and Cd-spores were thinner than those of control spores. Of the total Hg and Cd in the spores, 77% of the Hg and 63% of the Cd were detected in the spore coats. Hg- and Cd-spores were less resistant to heat and more sensitive to germinants than control spores. Other properties of Hg- and Cd-spores were similar to those of control spores. These results suggest that the spore coat has some relationship to the heat resistance and germinability of spores.     

Unicapsula marquesi n. sp. (Mysxosporea, Multivalvulida) a gill parasite of Polydactylus quadrifilis (Cuvier, 1829) (Pisces, Polynemidae) from the senegalese coast (West Africa)]

Unicapsula marquesi n. sp. (Myxosporea) is described from gill filaments of Polydactylus quadrifilis (Pisces, Polynemidae) obtained from coats of Senegal. The cysts were elongated and their length was 1 to 3 mm. The spores were pyramidal and composed of three valves. Only one of theses valves contained a developed polar capsule measuring 3.01 +/- 0.09 microns in diameter. Length of spore was 6.13 +/- 0.21 microns and the width was 7.18 +/- 0.17. No filament like appendage at the extremity of shell valves. Data on ultrastructure of spores are presented.     

FORMATION AND STRUCTURE OF THE SPORE OF BACILLUS COAGULANS

Spore formation in Bacillus coagulans has been studied by electron microscopy using an epoxy resin (Araldite) embedding technique. The developmental stages from the origin of the initial spore septum to the mature spore were investigated. The two forespore membranes developed from the double layer of cytoplasmic membrane. The cortex was progressively deposited between these two membranes. The inner membrane finally became the spore protoplasmic membrane, and the outer membrane part of the inner spore coat or the outer spore coat itself. In the mature spore the completed integuments around the spore protoplasm consisted of the cortex, a laminated inner coat, and a dense outer coat. No exosporium was observed. The method of formation of the cortex and the spore coats is discussed.     

Optimization of medium composition for the production of mosquitocidal toxins from Bacillus thuringiensis subsp. israelensis

Optimization of chicken feather (CF) based culture medium for the production of Bacillus thuringiensis subsp. israelensis (Bti) biomass in combination with the agro industrial by-product (coconut cake, CC) and manganese chloride (MnCl2) has been evaluated. The biomass yield of Bti spore/crystal toxin was highest (12.06 g/L) from the test medium (CF+CC+MnCl2) compared to the reference medium (Luria Bertani, LB). Toxicity assay with Bti produced from the test medium against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) was also satisfactory and results were comparable with bacteria produced from LB. The results suggest that Bti can be produced to the maximum extent possible as a potential mosquitocidal activity as suggested by the test medium (CF+CC+MnCl2).     

独行菜属植物玛咖(Lepidium meyenii)的微形态研究

利用光学显微镜和环境扫描电子显微镜,对不同颜色、不同产地玛咖(Lepidium meyenii)的叶表皮、花粉粒以及种皮进行了微形态学研究。结果表明:(1)不同颜色玛珈的叶表皮、花粉粒微形态具有高度的一致性,但与我国部分独行菜属植物有着明显的差异;(2)不同产地、不同颜色玛珈的种皮微形态有明显的差异。该研究为新植物资源玛咖的种质鉴定、分类提供了一些新的依据。     

Cytology of Spore Germination in Clostridium pectinovorum

The process of spore germination in Clostridium pectinovorum has been followed by phase-contrast and electron microscopy. Unlike most other Bacillaceae, germination of this species takes place within the sporangium. Under phase-contrast, the spore darkens and swells slightly, and then the vegetative rod slips out through the end opposite the collar-like extension of the sporangium. In thin sections, a spore from an early stage in germination consists of a central protoplast, core membrane, germ cell wall, cortex, and two coats. Within a short period, the cortex disintegrates and the young cell develops. It possesses a large fibrillar nucleoplasm and several mesosomes. Subsequently, the young cell elongates, becomes somewhat deformed, and then emerges through a narrow aperture in the inflexible coats of the spore, finally rupturing the sporangium. Free vegetative cells of C. pectinovorum resemble in their structure other gram-positive rods.     

Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.     

Interaction of the Bacillus subtilis spore protoplast, cortex, ion-exchange and coatless forms with glutaraldehyde

G orman , S.P. S cott , E.M. H utchinson , E.P. 1984. Interaction of the Bacillus subtilis spore protoplast, cortex, ion-exchange and coatless forms with glutaraldehyde. Journal of Applied Bacteriology 56 , 95–102.
Bacillus subtilis spores with altered ionic content were tested for their susceptibility to lysis with lysozyme or sodium nitrite following treatment with glutaraldehyde. The Ca-form was more sensitive to glutaraldehyde (pH 4.0.and pH 7.9) than the untreated or H-form. Removal of spore coat dramatically increased sensitivity of the spore to glutaraldehyde. Pretreatment of spores, the coats of which had been extensively removed, with glutaraldehyde (pH 7.9) reduced the rate of lysis by lysozyme and by sodium nitrite, whereas glutaraldehyde at pH 4.0.had little effect. Glutaraldehyde pretreatment (pH 4.0 and pH 7.9) reduced the amount of hexosamine released by lysozyme but not by nitrite from isolated cortical fragments. Spore protoplasts were more susceptible to 0.01% (w/v) glutaraldehyde at pH 4.0 and isolated spore coats adsorbed alkaline glutaraldehyde more rapidly. These results are discussed in terms of a possible mode of action of glutaraldehyde on the bacterial spore.     

Reevaluation of Bacitracin as a Spore Coat Component

Analysis of hydrolysates of highly purified spore coats revealed only small quantities of ornithine, a component of bacitracin. We conclude that the peptide, bacitracin, is not a significant component of spore coats.     

Localization of Mn(II)-oxidizing activity and the putative multicopper oxidase,MnxG, to the exosporium of the marine Bacillus sp. strain SG-1

Dormant spores of the marine Bacillus sp. strain SG-1 catalyze the oxidation of manganese(II), thereby becoming encrusted with insoluble Mn(III,IV) oxides. In this study, it was found that the Mn(II)-oxidizing activity could be removed from SG-1 spores using a French press and recovered in the supernatant following centrifugation of the spores. Transmission electron microscopy of thin sections of SG-1 spores revealed that the ridged outermost layer was removed by passage through the French press, leaving the remainder of the spore intact. Comparative chemical analysis of this layer with the underlying spore coats suggested that this outer layer is chemically distinct from the spore coat. Taken together, these results indicate that this outer layer is an exosporium. Previous genetic analysis of strain SG-1 identified a cluster of genes involved in Mn(II) oxidation, the mnx genes. The product of the most downstream gene in this cluster, MnxG, appears to be a multicopper oxidase and is essential for Mn(II) oxidation. In this study, MnxG was overexpressed in Escherichia coli and used to generate polyclonal antibodies. Western blot analysis demonstrated that MnxG is localized to the exosporium of wild-type spores but is absent in the non-oxidizing spores of transposon mutants within the mnx gene cluster. To our knowledge, Mn(II) oxidation is the first oxidase activity, and MnxG one of the first gene products, ever shown to be associated with an exosporium.     

The surface coats on spores

There are surface coats on the sporocytes and spores of some pteridophytes and bryophytes which will bind one or more of the stains generally used to demonstrate the presence of an acid mucopolysaccharide surface coat in animal cells, viz. Alcian blue, colloidal iron, lanthanum, thorium, silver, SO,-coriphosphine and phosphotungstic acid. This suggests that the composition of the coat substances in the ferns and bryophytes agrees with that in animal cells. It has been found that thorium-staining in the sporocyte and the coat of the young spore of one fern, Botrychium lunaria , can be abolished with neuraminidase, indicating that sialic acid is a principal component of these coats. The spore wall in the pteridophytes (and probably also in the bryophytes) is constructed within these mucopolysaccharide surface coats, and it is suggested that differences in wall form are attributable to qualitative, quantitative and functional differences of the coats.     

Killing of Bacillus subtilis spores by a modified Fenton reagent containing CuCl2 and ascorbic acid

Bacillus subtilis spores were killed by CuCl(2)-ascorbic acid, chloride ions were essential for killing of spores, and spores with defective coats were killed more rapidly. CuCl(2)-ascorbic acid did not damage spore DNA, and spores killed by this reagent initiated germination. However, spores killed by CuCl(2)-ascorbic acid may have damage to their inner membrane.