Formation of giant and ultramicroscopic forms of Nostoc muscorum CALU 304 during cocultivation with Rauwolfia tissues

The study of heteromorphic Nostoc muscorum CALU 304 cells, whose formation was induced by 6- to 7-week cocultivation with the Rauwolfia callus tissues under unfavorable conditions, revealed the occurrence of giant cell forms (GCFs) with a volume which was 35-210 times greater than that of standard cyanobacterial cells. Some GCFs had an impaired structure of the murein layer of the cell wall, which resulted in the degree of impairment of the cell wall ranging from the mere loss of its rigidity to its profound degeneration with the retention of only small peptidoglycan fragments. An analysis of thin sections showed that all GCFs had enlarged nucleoids. The photosynthetic membranes of spheroplast-like GCFs formed vesicles with the contents analogous to that of nucleoids (DNA strands and ribosomes). About 60% of the vesicles had a size exceeding 300 nm. With the degradation of GCFs, the vesicles appeared in the intercellular slimy matrix. It is suggested that the vesicles are analogous to elementary bodies, which are the minimal and likely primary reproductive elements of L-forms. The data obtained in this study indicate that such L-forms may be produced in the populations of the cyanobionts of natural and model syncyanoses. Along with the other known cyanobacterial forms induced by macrosymbionts, L-forms may represent specific adaptive cell forms generated in response to the action of plant symbionts.     

Ultrastructure of cyanobacterium <Emphasis Type="Italic">Nostoc</Emphasis> sp. f. <Emphasis Type="Italic">Blasia</Emphasis> cell forms in persisting populations

Cell clusters formed in persistent populations of Nostoc sp. f. Blasia, a cyanobacterium capable of cell differentiation, under prolonged storage in the dark at low temperatures were studied for the first time. Cell reorganization was observed, including changes in the ultrastructure of thylakoids, the cell wall peptidoglycan layer, and carboxysomes. Subcellular structures involved in intercellular communication within the clusters were revealed (structures similar to microplasmodesms and contact pores, secretory vesicles, etc.) Persistence of cyanobacterial populations was concluded to result from formation not only of specialized dormant cells (akinetes), but also L-forms, as well as from the modification changes of the clustered vegetative cells. A cluster containing the vegetative cells and L-like forms within a common intercellular matrix is considered a structural unit at the supracellular level, which is responsible for survival of cyanobacterial populations when mass akinete formation does not occur.     

Surface Ultrastructure of the Heteromorphic Cells of Nostoc muscorumCALU 304 in a Mixed Culture with the RauwolfiaCallus Tissue

The ultrastructure of the heteromorphic cells (HMCs) of the cyanobacterium Nostoc muscorumCALU 304 grown in pure culture, monoculture, and a mixed culture with the Rauwolfiacallus tissue was studied. The comparative analysis of the cell surface of HMCs, the frequency of the generation of cell forms with defective cell walls (DCWFs), including protoplasts and spheroplasts, and the peculiarities of their ultrastructure under different growth conditions showed that, in the early terms of mixed incubation, the callus tissue acts to preserve the existing cyanobacterial DCWFs, but begins to promote their formation in the later incubation terms. DCWFs exhibited an integrity of their protoplasm and were metabolically active. It is suggested that structural alterations in the rigid layer of the cell wall may be due to the activation of the murolytic enzymes of cyanobacteria and the profound rearrangement of their peptidoglycan metabolism caused by the Rauwolfiametabolites diffused through the medium. These metabolites may also interfere with the functioning of the universal cell division protein of bacteria, FtsZ. In general, the Rauwolfiacallus tissue promoted the unbalanced growth of the cyanobacterium N. muscorumCALU 304 and favored its viability in the mixed culture. The long-term mixed cultivation substantially augmented the probability of the formation of L-forms of N. muscorumCALU 304.     

Primary Lysosomes of the Ciliate Pseudomicrothorax dubius: Cytochemical Identification and Role in Phagocytosis*

SYNOPSIS. Filamentous cyanobacteria are ingested through the cytopharynx of the ciliate Pseudomicrothorax dubius. The cytopharynx is a complex of microtubules and microfilaments located in a highly vesiculated cytoplasm, the phagoplasm. Two types of membrane-bounded phagoplasmic vesicles can be distinguished by their differences in size, fine structure, and acid phosphatase (AcPase) content. One type has a homogeneous, electron-dense interior which is AcPase-positive. These vesicles are present in fed cells and in unfed cells devoid of food vacuoles, and thus appear to be primary lysosomes. During phagocytosis, exocytosis within the cytopharynx of the primary lysosomes results in the elaboration of a food vacuole. The vacuole grows by incorporation of lysosomal membrane; lysosomal hydrolases are liberated into the vacuole. Within less than 1 second of AcPase's entry into the food vacuole, it is detectable within the cyanobacterial cytoplasm, and within 5 seconds, destruction of the cyanobacterial filament is observed. It is hypothesized that the rapidity of hydrolase penetration of the cyanobacterial cell wall is the result of the action of molecules analogous to the “killing agents” of neutrophil leukocytes, which rapidly render bacterial envelopes permeable. AcPase, and presumably other hydrolases, are present in the cyanobacterial filament when filament destruction occurs; they thus appear implicated in this process. Hydrolases may activate an autodestruction mechanism in the cyanobacterium. Firm adherence of the food vacuole membrane to the cyanobacterial filament is demonstrated, and its role in phagocytosis is discussed.     

Ultrastructure of Brucella abortus L-forms induced by penicillin in a liquid and in a semisolid medium

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.     

Distribution and dynamics of mitochondrial nucleoids in animal cells in culture

Mitochondrial (mt) nucleoids were visualized in living cells in culture by staining with the fluorochrome picoGreen. The cell types included a line derived from Xenopus heart endothelial cells (XTH-2), 3T3 cells, SV40-transformed 3T3 cells and primary cultures of Xenopus tadpole epidermis cells. In the permanent cell lines 6-60% of the mitochondria were found to be devoid of DNA. The peaks of the frequency distribution of mtDNA content, as revealed by microfluorometry, were not very distinct, indicating the presence of a high amount of aneuploid mt nucleoids. The maximum size of nucleoids (as derived from fluorescence intensity) was 10-12 times that of the minimum peak value in proliferating cell cultures. A linear ratio was found between the volume of the nucleoids and their DNA content, which is interpreted as a uniform package density. In terminally differentiating tadpole epidermis cells mitochondria form large bodies containing giant nucleoids, while in mitotic cells the mt nucleoids are small and of uniform size. Fusion and fission of the nucleoids were observed to occur either for no visible reason or in connection with fusion and fission events of the mitochondria.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Atypical Cell Forms Overproducing Extracellular Substances in Populations of Cycad Cyanobionts

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. The cyanobiont microcolonies grown in the intercellular space of the cyanobacterial zone of cortical parenchyma in the cycad coralloid roots contained two specific forms of vegetative cells with a reduced cell wall, namely, protoplasts and spheroplasts. The protoplasts and spheroplasts exhibited ultrastructural properties indicating the overproduction of two extracellular substances, one of which resembled the mucilage polysaccharides and the other was protein-like. The substances were likely to be synthesized intracellularly and then be excreted with the aid of surface vesicles or by ruptures in the cytoplasmic membrane to form, respectively, a mucilagious extracellular matrix and an additional electron-opaque envelope around the cell. At the late developmental stages, the excretion of these substances was accompanied by degradative changes in the cells, leading eventually to cell death. The physiological role of these specific cell forms and the factors that induce their development and death in the cell populations of cyanobionts are discussed.     

Ultrastructure of L forms. IV. L forms of nonagglutinating vibrios]

A study was made of the ultrastructure of stable L-forms of Nag vibrios aged 24 hours. Cells of all types of the L-forms had cytoplasmic membranes, and a three-layered structure, which was found not everywhere. Externally of the cytoplasmic membrane, in some areas of the individual cells there were revealed a plastic layer of cell wall and a basal membrane. However, in difference to bacterial forms of the vibryos, rigidity of the cell wall was disturbed, and the links between the cell wall and the cytoplasmic membrane were indetectable. There were regularly revealed lamellar of myelin-like membranous structures in the cytoplasm, which did not occur in bacterial forms, and also lamellar mesosomes. The latter were found in the sites of cell division. Viability of small bodies as the minimal reproductive forms of the L-cultures is confirmed by the presence in them of a nucleoid and of the binary division.     

免疫酶技术鉴定 El Tor 型霍乱弧菌稳定 L 型

细菌稳定 L 型的形态、培养特性以及生化反应常与原菌不同,其菌落在盐水中不能乳化,故不能通过玻片凝集测定其抗原。对于这种一时不能回复为原菌的 L 型很难进行鉴定。本文采用免疫酶技术对由鳝鱼和鲫鱼胆汁诱导的 El Tor 型霍乱弧菌稳定 L 型进行了鉴定。实验证明 L 型的细胞壁可有不同程度的缺失,稳定 L 型仍可能有少量“O”抗原存在。PAP 法比较敏感,即使少量抗原亦可以检出。     

Endophyte differentiation inCasuarina actinorhizae

Summary This is an ultrastructural study of development of infected cells in nitrogen fixing root nodules ofCasuarina spp. While several aspects of development are similar to those found in many other actinorhizae, unusual aspects of development of the host cell and differentiation of the endophyte inCasuarina are correlated with unusual changes in the wall of the infected cell. Instead of vesicles the endophyte forms atypical hyphae in mature infected cells. These unusual hyphal forms are termed intracellular hyphae. Intracellular hyphae are nonseptate hyphae which originate and terminate within the same host cell, and have a varying diameter and a multidirectional growth and branching pattern. A laminate surface layer previously undescribed on hyphae ofFrankia is a feature common to mostCasuarina endophytic hyphae and is probably similar chemically to the laminae comprising the multilamellate envelope of endophytic vesicles in other actinorhizae.This paper is Florida Agricultural Experiment Station Journal Series No. 7350.     

Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria

Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.     

Nuclear and cell division in Bacillus subtilis: cell development from spore germination.

The changes in the morphology of the nucleoids and the mesosomes in Bacillus subtilis cells during synchronous outgrowth after spore germination were followed in large-scale three-dimensional cell reconstructions. Shortly after outgrowth of the cell begins in Spizizen medium with glucose, the mesosome becomes an elongated structure in close contact with a rounded nucleoid. When nuclear replication reaches full activity, the mesosome develops into a single, complicated versatile system, with tubules that traverse the cytoplasm and have elaborations in and near the nucleoplasm. Later the system may retract to form large rounded mesosomes; the tubules and strings of vesicles within these mesosomes probably have been collected from the cytoplasm. Shortly after the first cell division, both sister cells have two nucleoids, but with longer generation times induced by growth in media containing acetate instead of glucose; these sister cells have only one nucleoid each. In acetate-grown cells rounded nucleoids that have no contact with a mesosome may represent nucleoids in a temporary stage of rest. On the other hand, the nucleoids of cells growing in glucose-containing medium are always penetrated by mesosomal material, superficially or deeply. Since the mesosome appears capable of traversing the nuclear fibrils, and even reaching the last strands connecting the dividing nucleoids, it is suggested that this organelle may play a vital role in the Bacillus division cycle.     

Submicroscopic structure of the streptococcal L forms isolated from a living body]

The authors studied in the L-forms of streptococcus induced in the living organism. Submicroscopic structure of the L-forms under study was analogous to the L-forms of the museum streptococcus strain and to the L-forms of some other bacteria. As revealed on the ultra-thin sections of the protoplast-like cells the intracytoplasmic membrane structures were located close to the cytoplasmic membrane and also passed through the whole cell in the form of a band. The latter was in contact with the nucleotide. The elemental bodies were found in the vesicular and the protoplastic cells, and also in the space between the cells; sometimes they formed groups surrounded by a membrane.     

Ultrastructure of LLR-mutants of E. coli K12]

The author studied the ultrastructure of two spherical E. coli K12 mutants (llr) obtained under the effect of N-nitroso-N-methylurea. Seven morphological types of cells differing from one another by shape, size and cytoarchitectonics were distinguished. Superficial structures of the majority of the cells were represented by the membranes of the cell wall and the cytoplasmic membrane of common structure. Some of the cells had only one membrane coat and a high electron optic density of the cytoplasm. Transitional forms of cells were also encountered. The ultrastructure of each morphological type in the population of the llr-mutants was described in detail. The capacity of the mutants to vacuolization, to the intra- and extracellular budding, and also the ability to form multiple membrane structures resembled analogous structures of stable L-forms of the Gram-negative microbes. The problems of morphological differentiation of the L-forms and of the llr-mutants, and also problems connected with the formation of the multiple membrane structures and small elemental bodies in the cells of the llr-mutants are discussed.     

Induction, growth and antibiotic production of Streptomyces viridifaciens L-form bacteria

AIMS: To induce, cultivate and investigate the characteristics of L-form bacteria derived from the filamentous actinomycete Streptomyces viridifaciens. METHODS AND RESULTS: L-forms were induced in a liquid medium supplemented with lysozyme and penicillin. A stable culture which no longer required inducing agents but could still revert, was obtained by the twelfth subculture. The specific growth rate of stable L-forms was faster (0.751) than unstable L-forms (0.361). After the exponential growth phase, the cell diameter continued to increase, as did the percentage of vacuoles. Morphologically, the L-forms appeared as spherical bodies with no signs of differentiation and were sensitive to osmotic stress, indicating removal of the cell wall. The L-forms produced secondary metabolites although much lower levels of antibiotic were assayed in the L-forms compared with the cell walled forms. CONCLUSION: Stable L-form bacteria were induced from S. viridifaciens and their growth characterized. The L-forms produced secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Stable Streptomyces L-forms were induced and have potential as biocontrol agents.     

Brefeldin A effects on the trans-Golgi network and Golgi bodies inBotryococcus braunii are not uniform during the cell cycle

Summary Using cryo-fixation and freeze-substitution electron microscopy, the effects of brefeldin A (BFA) on the structure of the trans-Golgi network (TGN), the endoplasmic reticulum (ER), and Golgi bodies in the unicellular green algaBotryococcus braunii were examined at various stages of the cell cycle. In the presence of BFA, all the TGNs of interphase and dividing cells aggregated to form a single tubular mass. In contrast, the TGNs decomposed just after cell division and disappeared during cell wall formation. Throughout the cell cycle, the TGN produced at least six kinds of vesicles, of which two were not formed in the presence of BFA: vesicles with a diameter of 200 nm and fibrillar substances, which formed in interphase cells; and vesicles with a diameter of 180–240 nm, which may participate in septum formation. In addition, the number of clathrin-coated vesicles attaching to the TGN decreased. In interphase cells, BFA induced the disassembly of Golgi bodies and an increase in the smooth-ER cisternae at the cis-side of Golgi bodies. This result may suggest the existence of retrograde transport from the Golgi bodies to the ER in the presence of BFA. These drastic structural changes in the Golgi bodies and the ER of interphase cells were not observed in BFA-treated dividing cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TGN trans-Golgi network     

Mutations disturbing the condensation of plastid nucleoids inChlamydomonas reinhardtii

Summary To study the mechanism of condensation of dispersed plastid (pt) nucleoids into a single pt nucleoid with aging of the cells ofChlamydomonas reinhardtii, two mutants, designated cond-1 and cond-2, were isolated. A plastid of a wild type cell, 6.5 m in diameter, contained ten dispersed spherical pt nucleoids within one week of culture on an agar plate. At about one week of culture, the cell number was saturated and pt nucleoids began to associate with each other, condensing into a single pt nucleoid at three weeks of culture. In contrast, cond-1 and cond-2 cells, which had about 20 and 45 pt nucleoids and whose cell diameters were 7.8 and 9.5 m at one week of culture respectively, still had about 10 and 20 pt nucleoids at even 7 weeks of culture. Doubling times of the three cell types were similar. From genetic analysis, each of the two mutants had one gene mutation. The two mutations are probably linked. The measurement of O₂ evolution showed that the two mutations did not affect the photosynthetic system. Lipid contents of the two mutant cells were clearly higher than that of wild type cells. The role of a higher number of pt nucleoids is probably to increase the activity of lipid and/or membrane synthesis for lipid storage.     

Ultrastructure of the cell surface of heteromorphic Nostoc muscorum CALU 304 cocultured with Rauwolfia tissue]

The ultrastructure of the heteromorphic cells (HMCs) of the cyanobacterium Nostoc muscorum CALU 304 grown in pure culture, monoculture, and a mixed culture with the Rauwolfia callus tissue was studied. The comparative analysis of the cell surface of HMCs, the frequency of the generation of cell forms with defective cell walls (DCWFs), including protoplasts and spheroplasts, and the peculiarities of the cell surface ultrastructure under different growth conditions showed that, in the early terms of mixed incubation, the callus tissue acts to preserve the existing cyanobacterial DCWFs, but begins to promote their formation in the later incubation terms. DCWFs exhibited an integrity of their protoplasm and were metabolically active. It is suggested that structural alterations in the rigid layer of the cell wall may be due to the activation of the murolytic enzymes of cyanobacteria and the profound rearrangement of their peptidoglycan metabolism caused by the Rauwolfia metabolites diffused through the medium. These metabolites may also interfere with the functioning of the universal cell division protein of bacteria, FtsZ. In general, the Rauwolfia callus tissue promoted the unbalanced growth of the cyanobacterium N. muscorum CALU 304 and favored its viability in the mixed culture. The long-term incubation of the Rauwolfia tissue with the N. muscorum CALU 304 cells led to their transformation to L-forms.     

聚合酶链反应-单链构型多态性检测细胞壁缺陷型淋病奈瑟菌cppB基因变异

目的：检测与分析淋病奈瑟菌L型的cppB基因,探讨细胞壁缺陷对淋病奈瑟菌cppB基因的影响。方法：用青霉素诱导淋病奈瑟菌成为L型并获得稳定L型纯培养物,用cppB基因特异性引物以聚合酶链反应(PCR)检测稳定L型纯培养物的cppB基因和进行单链构型多态性(SSCP)分析。结果：淋病奈瑟菌的细菌型及其L型都具有cppB基因扩增产物,但PCR—SSCP分析可见异常泳动DNA带型(细菌型有2条带、L型有3条带)。结论：细胞壁缺陷淋病奈瑟菌仍然具有cppB基因,但其碱基序列可以发生改变。     

Male gametophyte development inPlumbago zeylanica: cytoplasm localization and cell determination in the early generative cell

Summary The behavior of the generative cell during male gametophyte development inPlumbago zeylanica was examined by epifluorescence microscopy and electron microscopy with organelle nucleoid as a cytoplasm marker. When the thin sections stained with 4,6-diamidino-2-phenylindoIe (DAPI) were observed under an epifluorescence microscope, two types of fluorescence spots were detected in the cytoplasm of the pollen cells before the second mitosis. The spots emitting stronger fluorescence were confirmed as plastid nucleoids and those emitting dimmer fluorescence were mitochondrial nucleoids. Before the first mitosis, both plastid and mitochondrial nucleoids distributed randomly in the cytoplasm of the microspore. A small lenticular generative cell formed with attachment to the interior of the intine after the mitosis. Small vacuoles were found in the lenticular cell. In the cytoplasm of the lenticular cell, both plastid nucleoids and the small vacuoles were distributed randomly at the very beginning but began to migrate in opposite directions immediately. Plastid nucleoids aggregated to the side of the cell that faces the pollen center and the small vacuoles aggregated to the side of the cell that attaches to the inline. As the result, the lenticular generative cell appeared highly polarized in cytoplasm location soon after the first mitosis. In accordance with the definition of the cytoplasm polarization, the primary wall between the generative and the vegetative cells began to flex and the lenticular generative cell started to protrude towards the pollen center. When the generative cell peeled away from the inline, it was spherical in shape with the pole that aggregated plastids towards the vegetative nucleus. But the cell direction appeared to be transformed immediately. The pole that aggregated small vacuoles turned to the position towards the vegetative nucleus and the pole that aggregated plastid nucleoids turned to the position countering to the vegetative nucleus. A cellular protuberance formed at the edge of the pole that aggregated small vacuoles and elongated into a tapered end that got into contact with the vegetative nucleus. The polarization of the cytoplasm kept constant throughout the second mitosis. The small vacuoles that apportioned to the sperm cell which attached the vegetative nucleus (the leading sperm cell) disappeared during sperm cell maturation. Plastid nucleoids were apportioned to the other sperm cell (the trailing sperm cell) completely. Mitochondrial nucleoids became undetectable after the second mitosis.