Changes in the Fine Structure of Microbial Cells Induced by Chaotropic Salts

The electron microscopic examination of thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37°C for 3–5 h or at 100°C for 5–6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, the ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

Comparative Study of the Elemental Composition of Vegetative and Resting Microbial Cells

X-ray microanalysis showed that vegetative cells, viable resting forms, and nonviable forms (micromummies) of the bacteria Bacillus cereus and Micrococcus luteus and the yeast Saccharomyces cerevisiae differ in the content of elements S, P, Ca, and K and Ca/K and P/S ratios. Viable resting forms (cystlike refractive cells and bacillar endospores) had more calcium and less phosphorus and potassium than vegetative cells, the difference being higher for bacilli than for micrococci and yeasts. The distinctive feature of all viable resting microbial forms was their low P/S ratios and high Ca/K ratios. The differences revealed in the cellular content and ratios of elements probably reflect changes in ionic homeostasis accompanying the transition of vegetative microbial cells to the dormant state. Relevant potassium parameters indicate that the membranes of viable resting forms retain their barrier function. At the same time, the nonviable micromummies, even those morphologically intact, of B. cereus and S. cerevisiae exhibited an anomalously low content of potassium, while those of M. luteus had an anomalously high content of this element. This suggests that the cellular membranes of micromummies lose their barrier function, which results in a free diffusion of potassium ions across the membranes. The possibility of using the elemental composition parameters for the quick analysis of the physiological state of microorganisms in natural environments is discussed.     

Synthesis of Anabiosis Autoinducers by Non-Spore-Forming Bacteria as a Mechanism Regulating Their Activity in Soil and Subsoil Sedimentary Rocks

Non-spore-forming bacteria of the genera Arthrobacterand Micrococcus, isolated from permafrost subsoil, were found to produce greater amounts of the d ₁extracellular factor than closely related collection strains isolated from soil. The effect of this factor, responsible for cell transition to anabiosis, was not species-specific. Thus, the d ₁preparation isolated from the culture liquid of the permafrost isolate Arthrobacter globiformis245 produced an effect on the collection strain Arthrobacter globiformisB-1112 and also on Micrococcus luteusand Bacillus cereus.The d ₁preparation from the permafrost isolate of Arthrobacterdiffered from the chemical analogue of this factor, 4-n-hexylresorcinol, in the level of the induced cell response, which may have resulted from different cell sensitivity to various homologs of alkylhydroxybenzenes contained in the d ₁preparation. Thus, additional evidence was obtained indicating that autoregulation of bacterial growth and development is implemented at the level of intercellular interactions in microbial communities. Abundant production of the d ₁anabiosis-inducing factors by bacteria isolated from permafrost subsoil is probably a result of special antistress mechanisms responsible for the survival of these bacteria under extreme conditions of natural long-term cooling.     

Production of Resting Forms by the Gram-Negative Chemolithoautotrophic Bacteria Thioalkalivibrio versutusand Thioalkalimicrobium aerophilum

The haloalkaliphilic chemolithoautotrophic gram-negative bacteria Thioalkalivibrio versutus, strain AL2, and Thioalkalimicrobium aerophilum, strain AL3, were shown to possess the capacity to produce resting forms, namely cystlike refractile cells (CRC), whose production was controlled by the level of the d₁ extracellular factors exhibiting the function of anabiosis autoinducers. The conditions were elucidated that promote the formation of CRC in the developmental cycles of the cultures studied, in condensed cell suspensions undergoing autolysis, and under the action of exogenously introduced chemical analogues of anabiosis autoinducers (alkylhydroxybenzenes). The peculiarities of the fine structure of the resting cells obtained were studied. Distinctions were revealed (with respect to viability and thermotolerance) between the CRC formed under different conditions. The relationship between the growth strategy and survival strategy of extremophilic bacteria is discussed taking into account the effect of the d₁ autoregulatory factors. A new model of CRC formation is proposed: CRC production in the life cycle of bacteria developing under conditions of increased concentration of anabiosis autoinducers.     

桃儿七种子休眠解除过程中细胞壁代谢及种皮超微结构的变化北大核心CSCD

为探究低温层积过程中桃儿七种子细胞壁代谢及种皮超微结构与休眠解除的内在联系,该研究通过低温层积解除桃儿七种子休眠,分析休眠解除过程中种子不同部位细胞壁组分及相关代谢酶的变化,同时利用扫描电镜对种皮的超微结构进行观察。结果表明,(1)桃儿七种皮主要由角质层、栅状石细胞层及海绵组织层3层构成,在层积过程中,种皮内部的海绵组织逐步疏松膨胀,种皮表面破损加剧;(2)种子不同部位的细胞壁组分具有明显差异,整个层积过程中,种胚、种皮和胚乳中的纤维素含量均在层积中期(45 d和60 d)降至最低,3个部位的纤维素酶活性在层积中期对应升高;种胚和种皮内的半纤维素含量均在层积中期显著下降,种皮中甘露聚糖酶活性和木糖苷酶活性在层积中期时相应达到最大;3个部位的果胶含量均在层积后期(75 d和90 d)时显著下降,而种皮和胚乳中多聚半乳糖醛缩酶活性也在层积后期相应升高;(3)种胚和胚乳内过氧化物酶活性在层积75 d和90 d时明显下降,而SOD活性在此时显著上升。(4)种子不同部位3种木质素单体的组成比例具有明显区别,同时3种木质素单体含量均随层积时间的延长而显著降低,且胚乳和种皮中的S-木质素含量对种子萌发存在显著的负向影响关系。研究认为,在低温层积过程中,桃儿七种子内细胞壁组分纤维素、半纤维素及木质素的逐步酶解,活性氧作用下的细胞壁松弛以及海绵组织层的疏松膨胀和种皮的破裂,破坏了细胞壁的刚性结构,促使种子机械束缚力降低,吸水性能提高、胚根生长能力增强,最终导致其休眠解除。     

抗菌肽CM4抗K562癌细胞的超微结构研究

报道了家蚕抗菌肽CM4抗K562癌细胞的体外实验研究.结果表明：纯化后的家蚕抗菌肽CM4对培养的K562(人髓样白血病细胞)有很强的杀伤作用.用扫描和透射电镜观察超微结构以及用激光共聚集显微断层图像分析,表明微量纯化的抗菌肽CM4能使K562癌细胞产生一系列的病理变化,可造成细胞高度肿胀,膜与胞质分离,细胞器和膜结构排列紊乱,细胞表面微绒毛消失,出现不规则的孔洞,细胞骨架严重破坏,膜局部结构破裂,缺损,胞浆内容物大量外泄,最终细胞解体,崩解成碎片.     

苗期低温胁迫对不同棉花品种叶肉细胞超微结构的影响

以早熟、中熟、晚熟三种不同熟性的6个棉花品种幼苗为试材,分析4℃低温胁迫处理对叶肉细胞超微结构的影响。结果表明：4℃低温处理3 d后,中熟品种N177和N181及晚熟品种K-1和N203的细胞器均受到严重破坏,表现为叶绿体受损严重,部分类囊体解体,线粒体膜模糊,失去完整性,细胞核膜基本消失,染色质凝聚,淀粉粒的数量和体积增大,嗜锇颗粒增多;而早熟品种中50和N52受损较轻,叶绿体变化不明显,线粒体结构完整,细胞壁完整。说明早熟品种中50和N52具有与抗冷性相适应的结构特点,其抗冷性较强。     

盐胁迫对玉米叶片叶肉细胞生物膜超微结构的影响

研究了NaCl胁迫对玉米叶肉细胞生物膜超微结构的影响. 结果表明：NaCl胁迫破坏了玉米叶片叶肉细胞生物膜的正常结构,50 mmol·L^-1 NaCl处理胁迫下,玉米叶肉细胞核膜,线粒体膜,细胞膜,叶绿体膜,液泡膜都受到不同程度的破坏,叶绿体基粒类囊体膨胀,间质片层空间增大,片层紊乱。100 mmol·L^-1 NaCl处理胁迫下,质膜,液泡膜,线粒体,叶绿体都受到严重的破坏。细胞质膜破坏,破损的叶绿体充斥在细胞间隙中;叶绿体外膜破坏,甚至解体消失,叶肉细胞中充满膜结构,基粒排列方向改变,垛叠层数减少,基粒和基质片层界限模糊不清,有的基粒解体消失,甚至叶绿体完全解体;核膜破坏、解体,核中的染色质高度凝缩;线粒体的数量增多,线粒体膜破坏,脊的数量减少,甚至整个线粒体破损解体;液泡膜破坏;由于各种生物膜的破坏,使细胞内充满许多囊状小泡、多泡体或斑层小体;叶肉细胞发生严重的质壁分离,严重时发生细胞壁断裂;甚至整个细胞溶解。     

不同光温条件下紫背浮萍叶肉细胞和叶绿体超微结构的研究

以浮萍科紫萍属紫背浮萍为试验材料,利用透射电子显微镜研究了紫背浮萍叶肉细胞和叶绿体的超微结构。结果发现:强光下,紫背浮萍叶肉细胞内的叶绿体数明显增加;弱光时虽然叶绿体数减少,但叶绿体基粒片层结构变厚,增加了光合作用反应面积。与25℃适温条件相比,10℃低温和35℃高温下,紫背浮萍细胞均出现一定程度的逆境胁迫作用。表现在低温时部分叶绿体皱缩成带状,叶肉细胞之间出现较大空隙;高温时叶绿体外膜溶解,基质外渗,脂质小球数量增多。紫背浮萍通过改变细胞内部形态结构的方式来适应不同光强和温度环境条件,对逆境光温条件具有一定的耐受性。     

Ultrastructure and surface topography of aggregates of human limb mesenchymal cells (HLM15) in vitro

Summary Tissue-like aggregates of human embryo fibroblasts can be created in vitro by limited aspiration of cells released from substrate during subcultivation. Aggregates increase in size, exhibit intercellular junctions, display a surface topography characteristic of cellular movement, elaborate an extracellular matrix and possess features of cellular death and phagocytosis. These cells, when introduced to a new culture environment, do not migrate away from one another as is common when a primary culture is started from tissue fragments. Instead, cells exhibit continued contact with each other, and develop complex junctional structures during that association. Cellular aggregates generated in this manner may provide a useful system for providing further information on cellular adhesion, intercellular communication, morphogenetic cell movements and the mechanisms of cell death. Dr. Kelley is the recipient of a Research Career Development Award (HD70407).     

低温下的绿豆下胚轴细胞超微结构变化

采用透射电子显微镜观察经10℃低温处理2d的绿豆下胚轴细胞超微结构变化的结果表明,细胞受到不同程度的伤害,其中大部分细胞的损伤是可逆的而非致死性的,如内质网膜模糊和呈颗粒状;线粒体呈现不同程度的膨胀,其数量增加,且聚集分布在内质网和高尔基体附近,核糖体聚集成为多聚核糖体等。也有小部分细胞受到的伤害是致死性的,如细胞质电子密度显著升高,质体中淀粉粒过度积累,内质网和高尔基体膨胀和解体,小液泡频繁吞噬细胞质和细胞器,细胞自溶死亡等。     

利用异化金属还原菌构建含糖微生物燃料电池

环境中的一些微生物通过还原金属氧化物进行无氧呼吸,而石墨电极与金属氧化物相似,也可以作为这类微生物呼吸作用的最终电子受体,利用这类微生物构建微生物燃料电池,以糖类物质为燃料,对电池产电情况、产电原理进行研究。实验结果表明,以Rhodoferaxferrireducens为产电微生物,在外接电阻510Ω条件下,以葡萄糖为燃料,常温下产生的电流密度达158mAm2(平台电压为0.46V,电极有效接触表面积为57cm2),且循环性能良好。更换燃料为其它糖,发现微生物可以利用多种糖进行产电;通过SEM观察发现大量微生物吸附在石墨电极上,用Bradford法对运行20d后电池的细胞量进行定量,测得悬浮细胞蛋白浓度为140mgL,吸附在电极上的生物量为1180mgm2。通过数据采集分析和细菌还原实验,发现吸附在电极上的微生物对电压的产生贡献最大,具有电化学和生物学活性;悬浮细胞对产电贡献很小,不具有电化学和生物学活性。     

Electron microscopic detection and in situ characterization of bacterial nanoforms in extreme biotopes

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1–3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29–43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of ≤ 0.3 μm and volume of ≤ 0.014 μm³) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18–0.20 × 0.20–0.30 μm in size, forming in situ spherical aggregates (microcolonies) of dividing cells. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.     

Ultrastructure of Chloroplasts in Phloem Companion Cells and Mesophyll Cells as Related to the Stimulation of Sink Activity by Cytokinins

Changes in the chloroplast ultrastructure and starch and lipid content in the mesophyll and phloem companion cells of the phloem were studied after induction of source and sink functions in leaf tissues. A detached sugar-beet leaf, one half of which was treated with water (source part) and the other half of which was treated with 10^–4 M benzyladenine (BA) (acceptor part), was used as a model. After 65-h exposure to diffuse light, starch disappeared and lipid content increased in the source part of the leaf, with simultaneous disorganization of the chloroplast structure, which was most pronounced in the companion cells. Changeover from the source to sink function, induced by BA treatment, did not lead to marked destructive changes in the chloroplast structure of companion cells and resulted in the appearance of starch and in further increase in the level of lipids. Smaller amounts of starch also appeared in the mesophyll chloroplasts in the sink part of the leaf. We suppose that: (1) BA promotes the storage of assimilates, which are imported from the source part of the leaf to the companion cells, in the form of starch and lipids within chloroplasts; and this storage contributes to the maintenance of the sucrose concentration gradient in the conducting system between donor and sink parts of the leaf and, thus, activates metabolite inflow and (2) a barrier exists in the sink part of the leaf for assimilates destined to mesophyll cells, which restricts their export from the phloem.     

Assessment of Keratinase and Other Hydrolytic Enzymes in Thermophilic Bacteria Isolated from Geothermal Areas in Patagonia Argentina

Thermophilic aerobic bacteria were isolated from two geothermal areas in Neuquén province using two different enrichment methods and a total of 30 isolates were obtained. From chicken feather enrichment cultures, strains affiliated to Bacillus cytotoxicus and Bacillus licheniformis were isolated and all of them demonstrated the capability to degrade completely chicken feather. A preliminary research on biotechnological enzymes' potential demonstrated that all the isolates displayed at least one of the extracellular hydrolytic enzymes tested. Most of the isolates showed protease, inulinase and/or pectinase activities, while cellulase and xylanase activities were less common. In light of these findings, geothermal areas of Argentina may be considered as a potential source of thermophilic bacteria able to produce many industrially relevant enzymes.     

Ultrastructure of follicular atresia in the rat

Observations were made on the sequence of morphologic changes in atresia of medium-sized preantral follicles in the rat. Ultrastructural studies indicated that in both control and hormonally treated animals granulosa cell changes, including nuclear condensation and alterations in cytoplasmic organelles, occurred prior to effects on the oocyte. In more advanced stages of atresia, extensive disruption of granulosa cell cytoplasm was associated with loss of microvilli and cytoplasmic vacuolization in oocytes. The findings are consistent with the view that follicular atresia begins with alterations in granulosa cells, effects on the oocyte occurring later in the atretic process.     

Ultrastructure and chemistry of the cell wall of the moss Rhacocarpus purpurascens (Rhacocarpaceae): a puzzling architecture among plants

The architectural, compositional and functional characteristics of the cell walls of the leaves of the moss Rhacocarpus purpurascens (Brid.) Par. have been analysed by scanning and transmission electron microscopy, wall-extraction methods, nuclear magnetic resonance (NMR) spectroscopy, and water-retention experiments. Four-layered cell walls with a peculiar architecture which, so far, appears to be unique among plants were apparent. The architecture of the walls was not affected by sequential wall-extraction procedures. Subsequent analysis of the residual pre-extracted walls by classical spectro-photometrical methods revealed that the walls are composed of mainly lignin, hemicellulose and cellulose in a ratio of about 9:8:5, determining their integrity. This was supported by NMR spectroscopy. The resonance spectrum showed various characteristics typical of lignin; however, some specific peaks associated with lignin were missing. The walls exhibited no particular properties for external water conduction but seem to be adapted to rapid absorption of fog, dew, or rain. Received: 3 June 1997 / Accepted: 25 February 1998