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PubMed2005

Isolation and characterization of light-dependent hemolytic cytotoxin from harmful red tide phytoplankton Chattonella marina.

Chattonella marina (C. marina), a raphidophycean flagellate, is a causative organism of red tide, and highly toxic to fish. In this study, we found that the cell-free methanol extract prepared from this flagellate exhibited potent hemolytic activity against rabbit erythrocytes. Interestingly, the hemolytic activity of the extract was absolutely light-dependent, and no hemolytic activity was detected in the dark even at very high concentration. Gel filtration chromatography of the methanol extract on a column of Sephadex LH-20 revealed that the extract contained hemagglutinin as well as hemolytic agents, and the substances responsible for these activities were separately eluted. These results suggest that the hemagglutinating and hemolytic activities were derived from distinct compounds. The hemolytic fraction obtained after gel filtration (F4) caused marked inhibition of the growth of C. marina itself and other species of phytoplanktons. Furthermore, F4 showed a potent cytotoxicity toward various mammalian cultured cell lines including human tumor cells (HeLa cells) in a dose-dependent manner. The cytotoxicity was also light-dependent, and no cytotoxic effect was exhibited in any cell lines tested in the dark. After further purification procedures via preparative thin-layer chromatography and subsequent HPLC, a major hemolytic agent was obtained as highly purified form. Since the methanol extracts prepared from other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica showed light-dependent hemolytic activity toward rabbit erythrocytes, it was suggested that the light-dependent hemolytic agents commonly exist at least in these raphidophycean flagellates.

Animals↗
PubMed2003

Colonic spirochetosis of colony-raised rhesus macaques associated with Brachyspira and Helicobacter.

Colonic spirochetosis is an inflammatory bowel disease that affects a broad range of hosts, including human and non-human primates. The disease in humans and non-human primates is characterized by intimate attachment of the anaerobic spirochetes Brachyspira aalborgi and B. pilosicoli, and some unclassified flagellated microbes along the apical membrane of colonic enterocytes. Although the presence of spiral-shaped bacteria with single polar flagella and blunted ends in colonic spirochetosis is well established, the identities of many of these organisms is still unknown. Recently, Helicobacter species with a morphology similar to the flagellated bacteria present in colonic spirochetosis have been cultured from intestinal specimens obtained from rhesus macaques, some with idiopathic colitis. The purpose of the present study was to determine whether or not the flagellated bacteria seen in the colons of rhesus macaques with colonic spirochetosis are Helicobacter. The presence of flagellated bacteria alone (n=2) or together with spirochetes (n=1) in formalin-fixed and paraffin-embedded colons of three rhesus macaques with the naturally occurring disease was demonstrated by immunohistochemical staining and ultrastructural examination. Total DNA extracted from affected and control intestinal specimens was amplified by polymerase chain reaction (PCR) using Helicobacter 16S rRNA gene-specific primers. Comparative nucleotide sequence analysis of PCR products cloned from positive reactions indicated that two distinct Helicobacter genomospecies were present either alone or in combination with Brachyspira in the colons of rhesus macaques with microscopic lesions indicative of colonic spirochetosis.

Journal Article↗
PubMed1985

Investigations on the role of flagella in the colonization of infant mice with Campylobacter jejuni and attachment of Campylobacter jejuni to human epithelial cell lines.

The biochemical and biological properties of the flagella of Campylobacter jejuni have been investigated using two variants selected from a flagellate, motile clinical isolate (strain 81116): a flagellate, non-motile variant (SF-1) and an aflagellate variant (SF-2). Phenotypic and biochemical analysis of the strains and amino acid analysis of the isolated flagella suggest that the variants differed from the wild-type strain only in the absence of flagella and/or motility. The aflagellate variant poorly colonized the gastrointestinal tract of infant mice but the flagellate, non-motile variant colonized the mice as successfully as the wild-type strain. 35S-labelled organisms were used to investigate the attachment of the variants to human epithelial cell monolayers in vitro. The flagellate, non-motile strain attached more efficiently to the cells than the wild-type strain or the aflagellate strain. Differences in attachment suggest that an adhesin is intimately associated with flagella of C. jejuni and that active flagella mediate only a tenuous association with host cells. This adhesin attached most efficiently to cells of intestinal epithelial origin and was not specifically inhibited by various sugars.

Adhesiveness↗
PubMed2003

The flagellum of Legionella pneumophila and its link to the expression of the virulent phenotype.

Legionalla pneumophila is a human pathogen causing atypical pneumonia. It is a monopolar flagellated gram-negative bacterium. Flagellation of L. pneumophila is life cycle dependent and the expression of flagella is genetically linked to the virulence phenotype. Non-flagellated mutants of L. pneumophila are less infectious for macrophages and amoebae compared to the wild type. The flagellar operon is expressed in a hierarchical manner, and different sigma factors and transcriptional regulators are involved in this cascade of gene regulation. The genome sequence of L. pneumophila was used to identify putative regulatory elements of various flagellar operons. Preliminary reports about regulators which are involved in the link between virulence gene regulation and flagellation are discussed.

Animals↗
PubMed1995

Reversible expression of motility and flagella in Clostridium chauvoei and their relationship to virulence.

Clostridium chauvoei strain Okinawa produced spontaneous non-motile variants at an unusually high rate (approx. 10(-4) per generation) under normal conditions without mutagen. Revertants of non-motile variants were detected at a rate of approximately 10(-3). Biochemically, every variant corresponded well with the parental strain. By transmission electron microscopy, three of nine non-motile variants of strain Okinawa were found to be flagellate, while the other six were found to be aflagellate. These phenotypes were confirmed by Western blot analysis using monoclonal antibodies directed against the flagella of C. chauvoei. Moreover, the parental flagellate strain and non-motile flagellate variants were significantly more virulent in mice than non-motile, aflagellate variants. Our results demonstrated that phase variation in motility and flagellation occurs in C. chauvoei, and that the flagella are associated with the full expression of virulence.

Animals↗
PubMed2004

Major alteration of the expression site of endogenous cellulases in members of an apical termite lineage.

Termites are among the most important cellulose-digesting animals on earth, and are well-known for the symbiotic relationship they have with cellulolytic trichomonad and oxymonad flagellates (unicellular eukaryotes). Perhaps less well-known is the fact that approximately 75% of the approximately 2600 described termite species -- those belonging to the family Termitidae -- do not harbour such flagellates. Unlike most termites from other families, the majority of termitids do not consume wood, feeding instead on soil, leaf litter, fungi, grass, or lichen. Recent years have seen the characterization of the endogenous cellulase enzymes that help termites digest cellulose, from one flagellate-harbouring species (Reticulitermes speratus), as well as one termitid (Nasutitermes takasagoensis). The genes encoding the enzymes in these two termites are similar. However, their site of expression differs markedly -- the salivary glands in R. speratus and the midgut in N. takasagoensis. To investigate this difference further, we performed a comparative study of cellulase expression in various termitid and flagellate-harbouring species, using enzyme assays and reverse transcription polymerase chain reactions. Taxa from phylogenetically basal lineages were consistently found to express endogenous genes specifically in the salivary glands, whilst those from a relatively apical lineage containing termitids expressed cellulases solely in the midgut. Relatively low levels of cellulase activity were found in nonwood-feeding species, while the wood-feeding Coptotermes formosanus -- arguably the most destructive pest species world-wide -- was found to have high levels of activity in all parts of the gut when compared to all other termites. In the light of these results, as well as recently accumulated phylogenetic data, we discuss scenarios for the evolution of cellulose digestion in termites.

Animals↗
PubMed2004

Microcolonies, quorum sensing and cytotoxicity determine the survival of Pseudomonas aeruginosa biofilms exposed to protozoan grazing.

This study was based on the hypothesis that biofilms of the opportunistic pathogen Pseudomonas aeruginosa are successfully adapted to situations of protozoan grazing. We tested P. aeruginosa wild type and strains that were genetically altered, in structural and regulatory features of biofilm development, in response to the common surface-feeding flagellate Rhynchomonas nasuta. Early biofilms of the wild type showed the formation of grazing resistant microcolonies in the presence of the flagellate, whereas biofilms without the predator were undifferentiated. Grazing on biofilms of quorum sensing mutants (lasR and rhlR/lasR) also resulted in the formation of microcolonies, however, in lower numbers and size compared to the wild type. Considerably fewer microcolonies than the wild type were formed by mutant cells lacking type IV pili, whereas no microcolonies were formed by flagella-deficient cells. The alginate-overproducing strain PDO300 developed larger microcolonies in response to grazing. These observations suggest a role of quorum sensing in early biofilms and involvement of flagella, type IV pili, and alginate in microcolony formation in the presence of grazing. More mature biofilms of the wild type exhibited acute toxicity to the flagellate R. nasuta. Rapid growth of the flagellate on rhlR/lasR mutant biofilms indicated a key role of quorum sensing in the upregulation of lethal factors and in grazing protection of late biofilms. Both the formation of microcolonies and the production of toxins are effective mechanisms that may allow P. aeruginosa biofilms to resist protozoan grazing and to persist in the environment.

Adaptation, Biological↗
PubMed2005

Grazing resistance of Pseudomonas aeruginosa biofilms depends on type of protective mechanism, developmental stage and protozoan feeding mode.

In a previous study we identified microcolony formation and inhibitor production as the major protective mechanisms of Pseudomonas aeruginosa biofilms against flagellate grazing. Here we compared the efficacy of these two key protective mechanisms by exposing biofilms of the non-toxic alginate overproducing strain PDO300 and the wild-type toxic strain PAO1 to a range of feeding types commonly found in the succession of protozoans associated with natural biofilms. Alginate-mediated microcolony formation conferred effective protection for strain PDO300 against the suspension feeding flagellate Bodo saltans and, as reported earlier, the surface feeding flagellate Rhynchomonas nasuta, both of which are considered as early biofilm colonizers. However, microcolonies of mature PDO300 biofilms were highly susceptible to late biofilm colonizers, the surface-feeding amoeba Acanthamoeba polyphaga and the planktonic ciliate Tetrahymena sp., resulting in a significant reduction of biofilm biomass. Mature biofilms of strain PAO1 inhibited growth of flagellates and A. polyphaga while the grazing activity of Tetrahymena sp. remained unaffected. Our findings suggest that inhibitor production of mature P. aeruginosa biofilms is effective against a wider range of biofilm-feeding predators while microcolony-mediated protection is only beneficial in the early stages of biofilm formation.

Acanthamoeba↗
PubMed1975

Reduced growth of Blastocrithidia culicis and Crithidia oncopelti freed of intracellular symbiotes by chloramphenicol.

The intracellular symbiotes of Blastocrithidia culicis and Crithidia oncopelti can be eliminated from cultures of the flagellates by a single chloramphenicol (CAP) treatment. Effective dosages were determined to be 0.01-0.08 per cent (w/v) CAP after a treatment for 2 weeks or more for B. culicis and 0.08 per cent (w/v) after 1 month for C. oncopelti in most cases. Ineffective dosages only lowered the numbers of symbiote-bearing flagellates. Growth of both species of flagellates in the presence of CAP was reduced in proportion to the drug concentration. Repeated subcultures at effectie dosages yielded symbiote-free flagellates, which maintained a low level of growth rate. After repeated subcultures at ineffective dosages, the growth rate rose and the symbiote-bearing cells, initially very few, increases in number. The lowest effective dosages proved to be marginal, often producing symbiote-free cultures, but occasionally cultures with a few symbiote-bearing cells. After repeated subcultures at these drug concentrations, symbiote-containing cultures grew faster than the symbiote-free cultures. Hence, the symbiotic bacteria benefit the growth of their hosts, perhaps by supplying essential factors that are adequate even in a rich blood medium.

Chloramphenicol↗
PubMed1983

Ultrastructure of the amoeboflagellate Tetramitus rostratus.

The life-cycle of the amoeboflagellate Tetramitus rostratus includes amoeboid, cyst, and flagellate stages. The ultrastructure of these three stages is illustrated, with particular emphasis on flagellate morphology. Amoeba morphology is typical of that of limax amoebas. Cysts, forming from trophic amoebas, are enclosed by a wall made up of two layers: ectocyst (ca. 70 nm), and endocyst (200 nm). The wall apparently forms from precursor material present in vesicles in the pre-cyst stage cytoplasm. Flagellate morphology is characterized by a well-defined top-shaped profile, maintained by microtubules under the plasma membrane. The flagellar apparatus or mastigont consists of four flagella, their basal bodies, sheaves of microtubules associated with two of the basal bodies, and several rhizoplasts (periodicity 20 nm). A deep, microtubule-supported, ventral invagination appears to function as a gullet. A small number of mitotic stages observed in amoeboid and flagellate individuals suggests similarity in the division process in both stages: intranuclear mitotic apparatus, nucleolus persisting through mitosis, no centrioles or basal bodies functioning as centrioles, difficulty in resolving chromosomes. The text compares ultrastructures of several amoeboflagellate organisms and evaluates the phylogenetic significance of those features common to different species. On the basis of this study, Tetramitus most closely resembles Naegleria spp.

Animals↗
PubMedDate not supplied

Confusing selective feeding with differential digestion in bacterivorous nanoflagellates.

Food selectivity and the mechanisms of food selection were analyzed by video microscopy for three species (Spumella, Ochromonas, Cafeteria) of interception-feeding heterotrophic nanoflagellates. The fate of individual prey particles, either live bacteria and/or inert particles, was recorded during the different stages of the particle-flagellate-interaction, which included capture, ingestion, digestion, and egestion. The experiments revealed species-specific differences and new insights into the underlying mechanisms of particle selection by bacterivorous flagellates. When beads and bacteria were offered simultaneously, both particles were ingested unselectively at similar rates. However, the chrysomonads Spumella and Ochromonas egested the inert beads after a vacuole passage time of only 2-3 min, which resulted in an increasing proportion of bacteria in the food vacuoles. Vacuole passage time for starved flagellates was significantly longer compared to that of exponential-phase flagellates for Spumella and Ochromonas. The bicosoecid Cafeteria stored all ingested particles, beads as well as bacteria, in food vacuoles for more then 30 min. Therefore "selective digestion" is one main mechanism responsible for differential processing of prey particles. This selection mechanism may explain some discrepancies of former experiments using inert particles as bacterial surrogates for measuring bacterivory.

Animals↗
PubMed1997

Protistan communities in aquifers: a review.

Eukaryotic microorganisms (protists) are a very important component of microbial communities inhabiting groundwater aquifers. This is not unexpected when one considers that many protists feed heterotrophically, by means of either phagotrophy (bacterivory) or osmotrophy. Protistan numbers are usually low (< 10(2) per g dw of aquifer material) in pristine, uncontaminated aquifers but may increase by several orders of magnitude in aquifers subject to organic pollution. Small flagellates (typically 2-3(5) microns in size in situ) are by far the dominant protists in aquifers, although amoebae and occasionally ciliates may also be present in much lower numbers. Although a wealth of new taxonomic information is waiting to be brought to light, interest in the identity of aquifer protists is not exclusively academic. If verified, the following hypotheses may prove to be important towards our understanding of the functioning of microbial communities in aquifers: (1) Differences in swimming behavior between species of flagellates lead to feeding heterogeneity and niche differentiation, implying that bacterivorous flagellates graze on different subsets of the bacterial community, and therefore play different roles in controlling bacterial densities. (2) Bacterivorous flagellates grazing on bacteria capable of degrading organic compounds have an indirect effect on the overall rates of biodegradation.

Animals↗
PubMed1988

Rates of digestion of bacteria by marine phagotrophic protozoa: temperature dependence.

The effect of temperature on length of time for digestion of bacteria was evaluated, by using fluorescently labeled bacteria (FLB), for phagotrophic flagellates and ciliates isolated from coastal northwest Mediterranean waters. Accumulation of FLB in protozoan food vacuoles was followed until a plateau of FLB per cell occurred; then after a 1:10 dilution of FLB with unlabeled bacteria, disappearance of FLB in food vacuoles was monitored. For both 3- to 5-mum flagellates and 10- to 40-mum ciliates, the absolute linear slopes of FLB uptake and disappearance were nearly identical in individual experiments over a temperature range of 12 to 22 degrees C. We inferred from these results that the leveling off of the uptake curves resulted when equilibrium between ingestion and digestion of bacteria was attained. The time to leveling off then represented the average time needed for complete digestion of the bacteria ingested at the start of the experiment, and the inverse of this time represented a bacterial digestion rate. The digestion rate increased exponentially from 12 to 22 degrees C for both a mixed flagellate assemblage and the oligotrichous ciliate Strombidium sulcatum, with a Q(10) of 2.8 for the flagellates and 2.0 for the ciliate. Although bacterial ingestion rates varied greatly, depending on protozoan cell size, total bacterial abundance, and temperature, digestion times appeared to be significantly influenced only by protozoan cell size (or type of protozoan) and by temperature.

Journal Article↗
PubMed1997

Flow Cytometric Analysis of Characteristics of Hybridization of Species-Specific Fluorescent Oligonucleotide Probes to rRNA of Marine Nanoflagellates.

Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples. Flow cytometry also permits measurement of intensity of probe binding by cells. Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate. Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase; cell size changed in a comparable manner. Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved.

Journal Article↗
PubMed2003

Termite gut symbiotic archaezoa are becoming living metabolic fossils.

Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termite's cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.

Amino Acid Sequence↗
PubMed1972

Taxonomy of aerobic marine eubacteria.

Two hundred and eighteen strains of nonfermentative marine bacteria were submitted to an extensive morphological, physiological, and nutritional characterization. All the strains were gram-negative, straight or curved rods which were motile by means of polar or peritrichous flagella. A wide variety of organic substrates served as sole sources of carbon and energy. The strains differed extensively in their nutritional versatility, being able to utilize from 11 to 85 carbon compounds. Some strains had an extracellular amylase, gelatinase, lipase, or chitinase and were able to utilize n-hexadecane and to denitrify. None of the strains had a yellow, cell-associated pigment or a constitutive arginine dihydrolase system, nor were they able to hydrolyze cellulose or agar. The results of the physiological and nutritional characterization were submitted to a numerical analysis which clustered the strains into 22 groups on the basis of phenotypic similarities. The majority of these groups were separable by a large number of unrelated phenotypic traits. Analysis of the moles per cent guanine plus cytosine (GC) content in the deoxyribonucleic acid of representative strains indicated that the peritrichously flagellated groups had a GC content of 53.7 to 67.8 moles%; polarly flagellated strains had a GC content of 30.5 to 64.7 moles%. The peritrichously flagellated groups were assigned to the genus Alcaligenes. The polarly flagellated groups, which had a GC content of 43.2 to 48.0 moles%, were placed into a newly created genus, Alteromonas; groups which had a GC content of 57.8 to 64.7 moles% were placed into the genus Pseudomonas; and the remaining groups were left unassigned. Twelve groups were given the following designations: Alteromonas communis, A. vaga, A. macleodii, A. marinopraesens, Pseudomonas doudoroffi, P. marina, P. nautica, Alcaligenes pacificus, A. cupidus, A. venustus, and A. aestus. The problems of assigning species of aerobic marine bacteria to genera are discussed.

Alcaligenes↗
PubMedDate not supplied

Susceptibility of laboratory-reared female Lutzomyia longipalpis (Lutz & Neiva, 1912) to infection by different species and strains of Leishmania Ross, 1903.

A study was undertaken to compare the susceptibility of laboratory-reared female Lutzomyia longipalpis to infection by different species or strains of New World Leishmania. The sand flies proved to be highly susceptible to infection by a strain of Le. guyanensis, with flagellates developing in all (18/18) of the specimens examined. A lower infection rate of 37% (10/27) was recorded in flies exposed to infection by a strain of Le. amazonensis. Flagellates developed in 13% (6/46) of the sand flies that blood fed on dogs in the early stage of experimental infection with an old laboratory strain of Le. chagasi. In contrast, promastigotes did not develop in sand flies that blood fed on dogs with naturally acquired Le. chagasi. The naturally infected dogs were in an advanced stage of disease. Flagellates developed in 9% (3/32) of the sand flies that blood fed on lesions of hamsters infected with a strain of Le. braziliensis and in 9% (3/34) of those that fed on hamsters with lesions due to a parasite of the mexicana complex (strain MHOM/BR/73/BH121). Sand flies did not develop flagellate infections after blood feeding on hamsters bearing lesions induced by strain MHOM/BR/71/BR49. Factors influencing the susceptibility of Lu. longipalpis to infection by New World species of Leishmania are discussed.

Animals↗
PubMed1996

Functions of bacterial flagella.

Many bacterial species are motile by means of flagella. The structure and implantation of flagella seems related to the specific environments the cells live in. In some cases, the bacteria even adapt their flagellation pattern in response to the environmental conditions they encounter. Swarming cell differentiation is a remarkable example of this phenomenon. Flagella seem to have more functions than providing motility alone. For many pathogenic species, studies have been performed on the contribution of flagella to the virulence, but the result is not clear in all cases. Flagella are generally accepted as being important virulence factors, and expression and repression of flagellation and virulence have in several cases been shown to be linked. Providing motility is always an important feature of flagella of pathogenic bacteria, but adhesive and other properties also have been attributed to these flagella. In nonpathogenic bacterial colonization, flagella are important locomotive and adhesive organelles as well. In several cases where competition between several bacterial species exists, motility by means of flagella is shown to provide a specific advantage for a bacterium. This review gives an overview of studies that have been performed on the significance of flagellation in a wide variety of processes where flagellated bacteria are involved.

Animal Population Groups↗