枯草芽孢杆菌芽孢中的芽孢衣

芽孢衣是赋予芽孢对有机溶剂和溶菌酶的抗性,以及对外界出芽诱导物的感应能力的保护性结构。该主要介绍由枯草芽孢杆菌(bacillus subtilis)所形成的芽孢的芽孢衣中已鉴定的主要蛋白质．及其基因表达调控和应用等方面的研究进展。     

Spore probiotics

Data on probiotic preparations prepared from spore-forming bacteria is reviewed. Information on indications for use, effectiveness and the mechanism of action is given. Preparations being produced in Russia (Biosporin, Sporobacterin and Bactosporin) are evaluated in detail as well as the prospects of the application of probiotics from spore-forming bacteria.     

Spore germination

The germination of dormant spores of Bacillus species is the first crucial step in the return of spores to vegetative growth, and is induced by nutrients and a variety of non-nutrient agents. Nutrient germinants bind to receptors in the spore's inner membrane and this interaction triggers the release of the spore core's huge depot of dipicolinic acid and cations, and replacement of these components by water. These latter events trigger the hydrolysis of the spore's peptidoglycan cortex by either of two redundant enzymes in B. subtilis, and completion of cortex hydrolysis and subsequent germ cell wall expansion allows full spore core hydration and resumption of spore metabolism and macromolecular synthesis.     

Microsporidian Spore Envelope Keratins Phosphorylate and Disassemble During Spore Activation

The microsporidian spore stage of the nerve parasite, Spraguea lophii, consists of outer envelope stabilized in part by keratins, including K4 and K13. The nonepidermal K4 and K13 keratins were found only in the spore envelope and were absent in the internal microsporidian sporoplasm. At the time of spore activation, the keratin-based outer spore envelope assemblage dissociated and became phosphorylated when the spores were placed in the presence of labeled ATP. Verapamil or lanthanum, agents which block S. lophii spore activation, also blocked spore envelope keratin disassembly and phosphorylation when the spores were incubated in activation medium with labeled ATP. However, after the removal of the verapamil or lanthanum, the spores regained the capacity to activate in discharge medium and the keratin analogues appeared to dissociate and phosphorylate.     

Spore productivity in Cladosporium

The high incidence ofCladosporia in the airspora indicates a prolific production of spores. Six species ofCladosporium were sampled over a period of 9 weeks, using dry and wet (mist-laden) air, and over a period of 4 weeks using humid air. Many more spores were released in wet air than in dry air: numbers released in humid air were generally intermediate between those of wet and dry samples. None of the cultures was exhausted of spores at the end of the sampling periods although samples generally decreased in size from the fifth or sixth week onwards. Removal of spores would seem to be conducive to further sporulation provided the substrate is not exhausted. Maximum productivities recorded for the six species (all in mist-laden air) ranged from 730 to 26 100 spores per mg dry weight of mycelium. Differences in the levels of spore production in culture by the six species do not correlate with their individual frequencies in the airspora, indicating that the latter are more dependent on the distribution and substrate relationships of each species.     

Bacillus subtilis Spore Coat

In response to starvation, bacilli and clostridia undergo a specialized program of development that results in the production of a highly resistant dormant cell type known as the spore. A proteinacious shell, called the coat, encases the spore and plays a major role in spore survival. The coat is composed of over 25 polypeptide species, organized into several morphologically distinct layers. The mechanisms that guide coat assembly have been largely unknown until recently. We now know that proper formation of the coat relies on the genetic program that guides the synthesis of spore components during development as well as on morphogenetic proteins dedicated to coat assembly. Over 20 structural and morphogenetic genes have been cloned. In this review, we consider the contributions of the known coat and morphogenetic proteins to coat function and assembly. We present a model that describes how morphogenetic proteins direct coat assembly to the specific subcellular site of the nascent spore surface and how they establish the coat layers. We also discuss the importance of posttranslational processing of coat proteins in coat morphogenesis. Finally, we review some of the major outstanding questions in the field.     

Spore Discharge in Basidiomycetes: III. Spore Liberation from Narrow Hymenial Tubes

The hymenial tubes of Polyporus betulinus were shown to be approximatelycylindrical,and to have a sporulating hymenium over the greater part oftheir surface. Sporophores were tilted through measured anglesfrom the vertical in the field and in the laboratory, and theeffect on spore liberation was measured by determining the numberof spores liberated from particular tubes in standard time.All tilting caused a reduction in spore liberation. The amountof this reduction was found to compare closely with that predictedfrom calculation of the amount of hymenium lying directly abovethe orifice at various angles of tilt. It is therefore concludedthat liberation from the tube can be accounted for by the initialviolent discharge of the spore and gravitational attractionalone. An addendum corrects an earlier paper (1959) in thisseries. It shows that the variation in density of spore depositscollected from Trametes gibbosa was due to air currents in theapparatus, not to variation in sporulation rate.     

Roles of Low-Molecular-Weight Penicillin-Binding Proteins in Bacillus subtilis Spore Peptidoglycan Synthesis and Spore Properties

The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by d,d-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparent d,d-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.Peptidoglycan (PG) is the structural element of the bacterial cell wall which determines cell shape and which resists the turgor pressure within the cell. The bacterial endospores produced by species of Bacillus, Clostridium, and several other bacterial genera are modified cells that are able to survive long periods and extreme conditions in a dormant, relatively dehydrated state. The PG wall within the endospore is required for maintenance of the dehydrated state (10, 11), which is the major determinant of spore heat resistance (2, 17, 22). Spore PG appears to be comprised of two distinct though contiguous layers. The thin inner layer, the germ cell wall, appears to have a structure similar to that of the vegetative wall and serves as the initial cell wall of the germinated spore (1, 20, 21, 31). The thicker outer layer, the spore cortex, has a modified structure which may determine its ability to carry out roles specific to the spore, and is rapidly degraded during spore germination (1, 20, 35, 37). The most dramatic of the cortex structural modifications results in partial cleavage or complete removal of ∼75% of the peptide side chains from the glycan strands. Loss of these peptides limits the cross-linking potential of the PG and results in the formation of only one peptide cross-link per 35 disaccharide units in the spore PG, compared to one peptide cross-link per 2.3 to 2.9 disaccharide units in the vegetative PG (1, 20, 36). This low degree of cross-linking has been predicted to give spore PG a flexibility that allows it to have a role in attainment of spore core dehydration (14, 34) in addition to its clear role in maintenance of dehydration. We are studying the structure and mechanism of synthesis of spore PG in an attempt to discern the roles of this structure and its individual components in determining spore properties.A family of proteins called the penicillin-binding proteins (PBPs) polymerizes PG on the external surface of the cell membrane (reviewed in reference 7). The high-molecular-weight (high-MW) members of this family (generally ≥60 kDa) carry the transglycosylase and transpeptidase activities involved in polymerization and cross-linking of the glycan strands. The low-MW PBPs have commonly been found to possess d,d-carboxypeptidase activity. This activity can remove the terminal d-alanine of the peptide side chains and thereby prevent the side chain from serving as a donor in the formation of a peptide cross-link. Analysis of the B. subtilis genome reveals six low-MW PBP-encoding genes: dacA (33), dacB (4), dacC (19), dacF (38), pbpE (23), and pbpX (accession no. {"type":"entrez-nucleotide","attrs":{"text":"Z99112","term_id":"32468745","term_text":"Z99112"}}Z99112). The four dac gene products exhibit very high sequence similarity to proven d,d-carboxypeptidases, and this activity has been demonstrated in vitro for the dacA and dacB products, PBP5 (12) and PBP5* (32), respectively. The sequences of the pbpE and pbpX products are more distantly related, and no activity has yet been established or ruled out for them.PBP5 is the major penicillin-binding and d,d-carboxypeptidase activity found in vegetative cells (12). Although dacA expression declines significantly during sporulation, a significant amount of PBP5 remains during the time of spore PG synthesis (29). A dacA-null mutation results in no obvious effects on vegetative growth, sporulation, spore characteristics, or spore germination (3, 33). However, loss of PBP5 does result in a reduction of cleavage of peptide side chains from the tetrapeptide to the tripeptide form in the spore PG (20). PBP5* is expressed only during sporulation and only in the mother cell compartment of the sporangium, under the control of the RNA polymerase ς^E subunit (4, 5, 28, 29). A dacB-null mutation leading to loss of this d,d-carboxypeptidase results in a fourfold increase in the effective cross-linking of the spore PG (1, 20, 22). This structural change is accompanied by only slight decreases in spore core dehydration and heat resistance (3, 22). The suspected d,d-carboxypeptidase activities of the products of the dacC and dacF genes have not been demonstrated. The latter two genes are expressed only during the postexponential growth phase: dacC is expressed during early stationary phase under the control of ς^H (19) and dacF is expressed only within the forespore under the control of ς^F (27, 38). Null mutations effecting either gene result in no obvious phenotype and no change in spore PG structure (19, 38).The multiplicity of these proteins in sporulating cells and the lack of effect of loss of some of them suggested redundancy of function among these proteins, a situation observed previously with PBPs of a high-MW class (25, 30, 39). In order to examine this possibility we have constructed mutants lacking multiple low-MW PBPs and have examined their sporulation efficiency, spore PG structure, spore heat resistance and wet density, and spore germination and outgrowth. The present study demonstrates a role for the dacF gene product in synthesis of spore PG, and we also present a model for the roles of the dacB and dacF gene products in spore PG formation.     

Simulated Self-assembly of Spore Exines

The spores and pollen of higher plants have enormous value intaxonomic studies by contributing to our understanding of pastand present biodiversity, and of environmental change. The criticalfeatures used in species identification are wall structure andsurface pattern. However, ultrastructural and histo-developmentalstudies of spore and pollen walls have, so far, provided limitedexplanation of wall construction and surface pattern formation.The consistency of pattern form within any species suggestsa high degree of genetic regulation, and yet few templates orother mechanisms of control have been demonstrated. Our experimentsshow that all layers and organizations within the spore wallof our test plant group (lycopodiopsid megaspores) can be simulatedby the flocculation of mixed colloidal systems. This leads usto a possible explanation of the mode of genetic control overpattern formation. It also provides a feasible, largely self-assembling,mechanism of construction which has the potential to reflectthe diversity of structure known to exist in all spore and pollenwalls.Copyright 1998 Annals of Botany Company Simulated self-assembly, spore exine development, sporopollenin, Lycopodiopsida, polyballs.     

细菌芽胞染色方法改进研究

改进微生物学实验教材上介绍的Schaeffer-Fulton氏芽胞染色法。将涂片的初染用水浴蒸汽加热和烘箱加热,通过显微镜观察不同温度下不同染色时间的染色效果。以5%孔雀绿为染色剂,100℃蒸汽加热4m in或80℃蒸汽加热6 m in能达到好的染色效果。改进方法操作简单易行,加热过程不需添加染料,更符合环保要求;将改进的染色方法运用于微生物学实验教学,收到了良好的教学效果。     

Evolutionary Dynamics of Spore Killers

Spore killing in ascomycetes is a special form of segregation distortion. When a strain with the Killer genotype is crossed to a Sensitive type, spore killing is expressed by asci with only half the number of ascospores as usual, all surviving ascospores being of the Killer type. Using population genetic modeling, this paper explores conditions for invasion of Spore killers and for polymorphism of Killers, Sensitives and Resistants (which neither kill, nor get killed), as found in natural populations. The models show that a population with only Killers and Sensitives can never be stable. The invasion of Killers and stable polymorphism only occur if Killers have some additional advantage during the process of spore killing. This may be due to the effects of local sib competition or some kind of ``heterozygous' advantage in the stage of ascospore formation or in the short diploid stage of the life cycle. This form of segregation distortion appears to be essentially different from other, well-investigated forms, and more field data are needed for a better understanding of spore killing.     

Spore ultrastructure in Sporothrix schenckii

Pathogenic strains of Sporothrix schenkii may show triangular spores, whose angular shape is maintained by a tiebeam effect in the inner cell wall structure. This difference in wall structure lies adjacent to a folded and possibly more active part of the spore cytoplasm. The supposed generation of asci in old cultures was simulated by the death of hyphae which are reinvaded by intrahyphal growth with intrahyphal spore production, while true asci were not seen.     

A Simplified Bacterial Spore Stain

A simplification of the Schaeffer-Fulton spore stain for bacteria is presented. It is shown that omission of the heating step during staining with malachite green resulted in spore stains as good as when the heat was applied. The simplified procedure involves (1) heat fixation of the smear by 20 passages through the flame, (2) staining with saturated aqueous malachite green for 10 minutes, (3) rinsing, and (4) counterstaining with 0.25% aqueous safranin for 15 seconds. The omission of the heating step in staining has obvious advantages, particularly in the classroom.     

Spore Dormancy Mutants of Phycomyces

Rivero, F., and Cerdá-Olmedo, E. 1994. Spore dormancy mutants of Phycomyces. Experimental Mycology 18, 221-229. The spores of the Zygomycete Phycomyces blakesleeanus are called dormant because few of them germinate when placed in a medium that sustains mycelial growth and development. Nearly all the spores germinate after activation, that is, exposure to heat or certain chemicals. We have looked for mutants whose spores would not need activation. Nine mutants formed authentic, but transient spores, which germinated spontaneously in the sporangium. Mutant mycelia had lower alcohol and aldehyde dehydrogenase activities and less glycogen than wild-type mycelia. The spontaneous germination and the metabolic alterations are attributed to the same recessive mutations. No differences were found between mutants and wild type in the cyclic AMP and fructose 2,6-bisphosphate concentrations in immature sporangia and the trehalase activity in the mycelia. In another mutant the spore primordia did not form spores, but remained viable for some time in the sporangium. The mutants were difficult to keep in the laboratory (except as lyophils); this stresses the importance of preventing spore germination in the sporangium.     

Spore discharge in Entomophthora grylli

Entomophthora grylli Fres. has been found on larvae of Bradysia (Sciaridae, Diptera) on wood in Kansas State University greenhouses since November 1967. Infected larvae crawled to an exposed site in the night and by morning spores of E. grylli were being discharged. In the greenhouse and under controlled environments spore discharge showed a marked periodicity; spore discharge occurred in the light with peaks in the first 1–4 h; discharge then gradually declined but extended into the dark. On the second and third days peaks occurred in the light but were progressively smaller. In a greenhouse under fluctuating conditions twin peaks occurred at 10·00 and 22·00; in a growth chamber, at constant 21° C. 90% r.h., alternating 12 h light and dark periods, spore discharge was similar, with maximum numbers about 21/2 h after the onset of the photo-period. In continuous dark and continuous light no endogenous pattern was evident. Temperature range for spore discharge was 2–28·, with optimum at 15·. Decreasing humidities resulted in decreased spore production and cessation of discharge below 40% r.h.     

假冷蕨属孢子形态研究

利用光学显微镜和扫描电子显微镜对假冷蕨属4种植物的孢子进行了详细观察,并与亲缘关系较近的冷蕨属、蹄盖蕨属植物孢子形态进行比较。结果表明,假冷蕨属孢子均为单裂缝,两侧对称,极面观为椭圆形,赤道面观为肾形,周壁表面具不规则脊状褶皱,连接成网状。假冷蕨属与蹄盖蕨属孢子形态特征相似,亲缘关系较近。