Tomoko Ehara

Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena

Euglena complex chloroplasts evolved through secondary endosymbiosis between a phagotrophic trypanosome host and eukaryotic algal endosymbiont. Cytoplasmically synthesized chloroplast proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the Euglena chloroplast. Euglena chloroplast preprotein pre-sequences contain a functional N-terminal ER-targeting signal peptide and a domain having characteristics of a higher plant chloroplast targeting transit peptide, which contains a hydrophobic stop-transfer membrane anchor sequence that anchors the precursor in the vesicle membrane. Pulse-chase subcellular fractionation studies showed that 35S-labeled precursor to the light harvesting chlorophyll a/b binding protein accumulated in the Golgi apparatus of Euglena incubated at 15°C and transport to the chloroplast resumed after transfer to 26°C. Transport of the 35S-labeled precursor to the chlorophyll a/b binding protein from Euglena Golgi membranes to Euglena chloroplasts and import into chloroplasts was reconstituted using Golgi membranes isolated from 15°C cells returned to 26°C. Transport was dependent upon extra- and intrachloroplast ATP and GTP hydrolysis. Golgi to chloroplast transport was not inhibited by N-ethylmaleimide indicating that fusion of Golgi vesicles to the chloroplast envelope does not require N-ethylmaleimide-sensitive factor (NSF). This suggests that N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are not utilized in the targeting fusion reaction. The Euglena precursor to the chloroplast-localized small subunit of ribulose-1,5-bisphosphate carboxylase was not imported into isolated pea chloroplasts. A precursor with the N-terminal signal peptide deleted was imported, indicating that the Euglena pre-sequence has a transit peptide that functions in pea chloroplasts. A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with the hydrophobic membrane anchor and the pre-sequence region C-terminal to the hydrophobic membrane anchor deleted was imported localizing the functional transit peptide to the Euglena pre-sequence region between the signal peptidase cleavage site and the hydrophobic membrane anchor. The Euglena precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase and the deletion constructs were not post-translationally imported into isolated Euglena chloroplasts indicating that vesicular transport is the obligate import mechanism. Taken together, these studies suggest that protein import into complex Euglena chloroplasts evolved by developing a novel vesicle fusion targeting system to link the host secretory system to the transit peptide-dependent chloroplast protein import system of the endosymbiont.

BEHAVIOR OF CHLOROPLAST NUCLEOIDS DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) IN SYNCHRONIZED CULTURE1

Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12:12 h light: dark regimen. They increased in size during the light period, while nuclear division, chloroplast division and cytokinesis occurred during the dark period. Zoospores were liberated toward the end of the dark period. Changes in profile and distribution of chloroplast nucleoids were followed with a fluorescence Microscope after fixation with 0.1%(w/v) glutaraldehyde followed by staining with 4′.6‐diamidino‐2‐phenylidole (DAPI), a DNA fluorochrome. About ten granular nucleoids were dispersed in the chloroplast at the beginning of the light period (0 h). Within 4 h the nucleoids aggregated around the pyrenoid giving a compact profile. The formation of the compact aggregate of cp‐nucleoids around the pyrenoid occurred with maximal frequency twice during the light period. Toward the end of the light period the nucleoids were transformed into the form of threads interconnected with fine fibrils spreading throughout the chloroplast. Initially the thread‐like nucleoids fluoresced only faintly. The fluorescence of some parts of the threadlike form became brighter over a period of 6 h; these nucleoids were divided into daughter chloroplasts during chloroplast division. Soon after chloroplast division, these thread‐like nucleoids were transformed into about 20 granular forms, which were gradually combined to form about ten larger granular bodies in zoospores immediately prior to liberation from mother cells. Fixation of cells with glutaraldehyde at high concentrations or treatment of cells with protease significantly modified the profiles of DAPI‐stained nucleoids. The different morphologies of chloroplast nucleoids are discussed in relation to changes in configuration of their protein components.

Electron microscopic studies on bactericidal effects of electrolyzed acidic water on bacteria derived from kendo protective equipment.

ObjectivesKendo protective equipment is used without washing for a long time.Staphylococcus saprophyticus, Micrococcus luteus, andBacillus sphaericus are frequently isolated from the mask (‘men’ in Japanese) of kendo protective equipment during one year. To investigate the bactericidal effects of electrolyzed acidic water on these three bacteria, we observed their cellular structures by electron microscopy after treatment with such water.MethodsEach bacterium isolated from ‘men’ was treated with electrolyzed acidic water and then observed under scanning and transmission electron microscopes.ResultsWhenS. saprophyticus was treated with electrolyzed acidic water and its cellular structures were observed under a transmission electron microscope, ghost cytoplasm was observed, in which no ribosomal granules or fibrous DNA structures were present, and the cell wall inner layer was detached from the outer layer. Under a scanning electron microscope, the structure of the cell wall surface layer was wrinkled, and round pores were partially formed, indicating that the cytoplasmic structures were flushed out of the cells treated with electrolyzed acidic water through the pores formed in the cell wall. InM. luteus, the destruction of ribosomal granules and that of DNA fibers were observed to be similar to those ofS. saprophyticus. ForB. sphaericus, the effect of electrolyzed acidic water was investigated using vegetative cells. A dissociation between the cytoplasm and cell wall wrinkled the cell surface layer.ConclusionOn the basis of above findings, electrolyzed acidic water was found to destroy the cellular structures of the three bacterial species frequently isolated from kendo men within a short time. Electrolyzed acidic water may be useful for disinfecting of kendo equipment.

The occurrence of non-specific lipid transfer proteins in developing castor bean fruits

Abstract The occurrence of non-specific lipid transfer proteins (nsLTPs) in developing castor bean fruits was examined by immunoblot quantitation and immunogold electron microscopy. The levels of nsLTPs increased with fruit development, being higher in the pericarp than the seed. The distribution of nsLTPs was localized throughout the development in the peripheral tissues, the outer layer of the endosperm in the seed and the epicarp in the pericarp. nsLTPs in the endosperm were localized only in the dense vesicle in the cytoplasm at 14-days after flowering (14-DAF). At 28-DAF the dense vesicles containing nsLTPs were assembled to the central vacuole to form an aggregate, which in turn disappeared at 35-DAF. On the other hand, nsLTPs were scattered in the cell walls of sclereid surrounding the seed and in the cell walls of trichome and sclerenchyma in the pericarp. These results suggest that gene-expressed nsLTPs are transported by two routes; one is to the vacuole by the dense vesicles in the endosperm and the other is to the cell wall and around there in the pericarp, probably by Golgi route.

Modulation of Fatty Acid Synthesis in Plants by Thiolactomycin

The synthesis of fatty acids and lipids in plants are complicated because of involvement in both the chloroplast and the endoplastic reticulum for de novo synthesis, elongation and desaturation of fatty acids, accompanied by their esterification to glycerides. Nevertheless, a controlled flow of the metabolic path proceeds in a plant cell, as shown by a given ratio of lipid and fatty acid composition in a organelle membrane. Administration of the inhibitor for the synthesis of fatty acids and lipids is used as a tool to elucidate the mechanism of the controlled flow. However, even when an inhibitor is specific for a definite reaction of fatty acid synthesis, the effect of the inhibition on overall synthesis of fatty acids are different in different lipids and different plants. For example, cerulenin inhibits 3-ketoacyl-ACP synthase in de novo synthesis of fatty acids and not that in elongation of fatty acids2, but the effect of cerulenin on lipid synthesis in greening barley leaves indicates an increase of 16: 0 and a marked decrease of 18: 3 in MGDG, and conversely a decrease of 16: 0 and an increase of 18: 3 in PC, although the synthesis of both glycolipids and phospholipids are considerably inhibited3.

Behaviour of proplastids and their nucleoids in dark-organotrophically grown cells of Euglena gracilis transferred to an inorganic medium

Euglena gracilis Z cells in the stationary phase of dark-organotrophic growth in a culture without agitation were rich in lipid, and contained six to eight proplastids dispersed in the cytoplasm (A-type proplastids). When these cells were transferred to an inorganic medium and aerated in darkness, they showed, with the disappearance of lipid, diphasic increase in number after an induction phase, concurrently with the development of prolamellar bodies and some primary thylakoids in their proplastids. The proplastids and their nucleoids, examined under a fluorescence microscope with and without staining with the DNA fluorochrome, 4′,6-diamidino-2-phenylindole, showed dynamic and sequential morphological changes. Before the first cell doubling, the A-type proplastids gathered to form three to four aggregates (B-type), that were themselves connected into a single elongated form with intricate windings and branchings (C-type). The nucleoid in the C-type proplastid was present as a branched string winding throu...

Investigation of the Role of Bacteria in the Development of Acanthamoeba Keratitis.

Purpose: Recently, much interest has been shown in bacteria extracted from Acanthamoeba strains isolated from patients with Acanthamoeba keratitis (AK). We hypothesized that the bacteria in Acanthamoeba strains may be a contributing factor in the development of AK. To prove this hypothesis, we investigated the involvement of bacteria harbored by Acanthamoeba in causing progressive ocular infection in rabbit corneas. Methods: One Acanthamoeba strain (T4 genotype) that harbored bacteria was isolated from a patient with AK. The Acanthamoeba strain pretreated or not pretreated with levofloxacin (LVFX) was inoculated into rabbit corneas. We also tested the effect of LVFX eye drops on keratitis induced by the Acanthamoeba strain. The infected rabbit eyes were evaluated for clinical scores, Acanthamoeba 18S rDNA and bacterial 16S rDNA numbers were analyzed by the real-time polymerase chain reaction, and the presence of Acanthamoeba was analyzed by histological examination. Results: Inoculation of nonpretreated Acanthamoeba resulted in severe keratitis. In contrast, inoculation of LVFX-pretreated Acanthamoeba did not induce keratitis (mean clinical score, 17.3 vs. 2.3; P < 0.05). Rabbit corneas inoculated with nonpretreated Acanthamoeba followed by topical LVFX therapy developed severe keratitis. In corneas inoculated with nonpretreated Acanthamoeba followed by LVFX therapy, the number of Acanthamoeba 18S rDNA copies was significantly higher than in other groups (P < 0.05), whereas the bacterial 16S rDNA gene was undetectable. Acanthamoeba cysts were detected by Fungiflora Y staining only in corneas inoculated with nonpretreated Acanthamoeba followed by LVFX therapy. Conclusions: These results suggest that the presence of bacteria in Acanthamoeba may be required for the development of AK.

Measuring antimicrobial susceptibility of Pseudomonas aeruginosa using Poloxamer 407 gel

Pseudomonas aeruginosa is a Gram-negative bacterium that causes various opportunistic infections. Chronic and intractable infections with P. aeruginosa are closely related to the high levels of resistance displayed by this organism to antimicrobial agents and its ability to form biofilms. Although the standard method for examining antimicrobial resistance involves susceptibility testing using Mueller–Hinton agar or broth, this method does not take into account the influence of biofilm formation on antimicrobial susceptibility. Poloxamer 407 is a hydrophilic, nonionic surfactant of the more general class of copolymers that can be used to culture bacteria with similar properties as cells in a biofilm environment. Therefore, the aim of this study was to compare the antimicrobial susceptibility of bacteria cultured in Poloxamer 407 gel to those grown on Mueller–Hinton agar using the Kirby–Bauer disk diffusion method with 24 strains of P. aeruginosa. Antimicrobial sensibility differed between the two mediums, with >60% of the strains displaying increased resistance to β-lactams when cultured on Poloxamer 407 gel. In addition, scanning electron microscopy revealed that typical biofilm formation and extracellular polymeric substance production was only observed with bacteria grown on Poloxamer 407 gel. Therefore, antimicrobial susceptibility test using Poloxamer 407 gel may provide more accurate information and allow the selection of suitable antimicrobial agents for treating patients infected with biofilm-forming pathogens.

Stage-dependent localization of mitochondrial DNA during the cell cycle of Euglena gracilis Z by immunogold electron microscopy

Examination of Chlamydomonas and Euglena mitochondrial morphology in synchronized cells by high resolution fluorescence microscopy, serial ultra-thin sectionelectron microscopy, and computer graphic analysis has found dynamic morphological changes such as fusion, fission, and branching during the cell cycle. Giant Euglena and Chlamydomonas mitochondria are formed by fusion of smaller reticular mitochondria at specific stages of the cell cycle. Giant mitochondria formation is associated with a temporary reduction of respiratory function suggesting a role in the cell cycle for giant mitochondria other than their primary respiratory function[1,2,3]. We examined the localization of mitochondrial DNA in synchronized cells of Euglena gracilis Z [2]. Mitochondrial DNA was localized by fixation of cells, staining with an anti-DNA monoclonal antibody, immuno-electron microscopy, and computer graphic analysis. Cross sections of normal size reticulate mitochondria in cells sampled 4 hours after the onset of light showed only a few colloidal gold particles confined to specific regions of the matrix while in other mitochondria, DNA molecules were not observed. Serial ultra-thin sectioning and immuno-electron microscopy found colloidal gold particles homogeneously distributed throughout the matrix of giant mitochondria. This demonstrates for the first time the distribution of mitochondrial DNA throughout the matrix of giant mitochondria suggesting that individual DNA molecules are not spatially separated as found in reticulate mitochondria. If mitochondria differ in their genetic composition, the temporary formation of giant mitochondrion through mitochondrial fusion would provide an opportunity during the cell cycle for gene exchange between individual mitochondrial DNA molecules and for mitochondrial DNA replication prior to mitochondrial division.

Analysis of bacterial flora in dohyo soil

ObjectivesSumo wrestling is one of the most popular sports in Japan. Injuries are not uncommon as this is a vigorous contact sport. Sumo wrestlers have little in the way of protective clothing; their main garb is the mawashi, making them prone to exposure to any microorganisms in the dohyo. The bacterial flora of the dohyo has received little attention. If the constituent flora is identified, then appropriate treatment or prevention of any bacterial lesions or infections incurred by the wrestlers is possible.MethodsThe Vitek AMS system used in this study was developed by McDonnell Douglas Corporation. In this system, the physiological and biochemical properties of Gram-positive and negative bacilli, Gram-positive and-negative cocci, and fungi isolated from clinical materials and environments are examined using test cards specifically for each microorganism group, and the results are automatically read by a computer and encoded. Obtained codes are compared with a built-in database, and bacterial species of test strains are identified.ResultsIn this study, using the automatic identification kit VITEK or ATB, we describe the aerobic bacterial flora found in the dohyo over the four seasons of the year. We also investigated the effect of salt on the bacterial flora as sumo wrestlers toss salt on the dohyo before each match. We show the relationship between salinity changes and variations in the flora observed upon the addition of salt. Without salt, at the beginning of a match, Gram-negative bacteria predominate. When salt is added, there is a transient decrease in the incidence of flora followed by an increase in the incidence Grampositive cocci.ConclusionsSixteen bacterial genera were identified using the bacterial identification systems in dohyo soil samples during the year. The number of identified bacterial species was 32. Even in the presence of salt, there is a measurable amount of bacterial flora in dohyo soil; salt does not act as an antibacterial agent.