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PubMed2005

Cellular identity of an 18S rRNA gene sequence clade within the class Kinetoplastea: the novel genus Actuariola gen. nov. (Neobodonida) with description of the type species Actuariola framvarensis sp. nov.

Environmental molecular surveys of microbial diversity have uncovered a vast number of novel taxonomic units in the eukaryotic tree of life that are exclusively known by their small-subunit (SSU) rRNA gene signatures. In this study, we reveal the cellular and taxonomic identity of a novel eukaryote SSU rRNA gene sequence clade within the Kinetoplastea. Kinetoplastea are ubiquitously distributed flagellated protists of high ecological and medical importance. We isolated an organism from the oxic-anoxic interface of the anoxic Framvaren Fjord (Norway), which branches within an unidentified kinetoplastean sequence clade. Ultrastructural studies revealed a typical cellular organization that characterized the flagellated isolate as a member of the order Neobodonida Vickerman 2004, which contains five genera. The isolate differed in several distinctive characters from Dimastigella, Cruzella, Rhynchobodo and Rhynchomonas. The arrangement of the microtubular rod that supports the apical cytostome and the cytopharynx differed from the diagnosis of the fifth described genus (Neobodo Vickerman 2004) within the order Neobodonida. On the basis of both molecular and microscopical data, a novel genus within the order Neobodonida, Actuariola gen. nov., is proposed. Here, we characterize its type species, Actuariola framvarensis sp. nov., and provide an in situ tool to access the organism in nature and study its ecology.

Animals↗
PubMed2004

Downregulation of the motA gene delays the escape of the obligate predator Bdellovibrio bacteriovorus 109J from bdelloplasts of bacterial prey cells.

Bdellovibrio bacteriovorus is a Gram-negative bacterium that preys on other Gram-negative bacteria. The lifecycle of B. bacteriovorus alternates between an extracellular flagellated and highly motile non-replicative attack-phase cell and a periplasmic non-flagellated growth-phase cell. The prey bacterium containing periplasmic bdellovibrios becomes spherical but osmotically stable, forming a structure known as the bdelloplast. After completing the growth phase, newly formed bdellovibrios regain their flagellum and escape the bdelloplast into the environment, where they encounter more prey bacteria. The obligate predatory nature of B. bacteriovorus imposes a major difficulty to introducing mutations in genes directly involved in predation, since these mutants could be non-viable. This work reports the cloning of the B. bacteriovorus 109J motAB operon, encoding proteins from the flagellar motor complex, and a genetic approach based on the expression of a motA antisense RNA fragment to downregulate motility. Periplasmic bdellovibrios carrying the plasmid expressing antisense RNA displayed a marked delay in escaping from bdelloplasts, while the released attack-phase cells showed altered motility. These observations suggest that a functionally intact flagellar motor is required for the predatory lifecycle of B. bacteriovorus. Also, the use of antisense RNA expression may be a useful genetic tool to study the Bdellovibrio developmental cycle.

Bacterial Proteins↗
PubMed2006

Low tyrosine content of growth media yields aflagellate Salmonella enterica serovar Typhimurium.

Identification of Salmonella serotypes is based on flagellar and somatic antigens. The absence of flagella may consequently affect complete identification of the serotype; here it is shown that Salmonella enterica serovar Typhimurium exhibits morphological differences dependent on the peptone constituents of the culture medium. Aflagellate salmonella were produced in certain media where the nutritional ingredient was casein-based peptone or gelatin-based peptone; in gelatin-based peptone, aggregates of salmonella were observed. However, in media containing soy-based peptone as the primary nutrient, salmonella displayed a normal flagellated morphology. Transfer of aflagellate salmonella from nutritionally poor media, with casein- or gelatin-based peptone, into rich nutrient broth allowed flagella synthesis, indicating that the aflagellate form is still able to produce flagella. Amino acid sequencing of the peptones producing aflagellate organisms showed a relatively low tyrosine concentration: only 0.03+/-0.01 g l(-1) for gelatin-based buffered peptone water, compared to 0.21+/-0.01 for soy-based buffered peptone water. Tyrosine is essential for flagellin, which is the subunit of the salmonella flagellar filament. The addition of 200 muM tyrosine to casein-based peptone media produced flagellate salmonella; 2 mM glucose was needed in addition to tyrosine to achieve a similar morphology in gelatin-based media. Therefore, culture media containing less than 1.20 g tyrosine l(-1), and of limited carbohydrate source, when used for serological testing of clinical isolates, may result in an incomplete serological identification.

Carbohydrates↗
PubMed2006

Antigen-43-mediated autoaggregation impairs motility in Escherichia coli.

Functional interaction between bacterial surface-displayed autoaggregation proteins such as antigen 43 (Ag43) of Escherichia coli and motility organelles such as flagella has not previously been described. Here, it has been demonstrated for the first time that Ag43-mediated aggregation can inhibit bacterial motility. Ag43 overexpression produces a dominant aggregation phenotype that overrides motility in the presence of low levels of flagella. In contrast, induction of an increased flagellation state prevents Ag43-mediated aggregation. This phenomenon was observed in naturally occurring subpopulations of E. coli as phase variants expressing and not expressing Ag43 revealed contrasting motility phenotypes. The effects were shown to be part of a general mechanism because other short adhesins capable of mediating autoaggregation (AIDA-I and TibA) also impaired motility. These novel insights into the function of bacterial autoaggregation proteins suggest that a balance between these two systems, i.e. autoaggregation and flagellation, influences motility.

Adhesins, Bacterial↗
PubMed1988

Role of pili in the pathogenesis of Pseudomonas aeruginosa burn infection.

The present study using three isogenic mutants (F+P-, F-P+, F-P-) of Pseudomonas aeruginosa indicates that the presence of pili enhances the virulence of the organisms in experimental P. aeruginosa burn infection of mice. The 50% lethal dose (LD50) value for burned mice inoculated with non-piliated (P-) mutant was at least ten times higher than those inoculated with piliated (P+) bacteria. Meanwhile the LD50 value for burned mice inoculated with non-flagellated (F-) mutant was at least 10(5) times higher than those inoculated with flagellated (F+) bacteria. At 24 hr after inoculation, the bacterial counts in burned skin of mice inoculated with P+ bacteria were ten times higher than those inoculated with P- bacteria; and at 48 hr the bacterial counts became a hundred times higher in the former mice than the latter. At 24 hr after inoculation, P+ bacteria were isolated from blood, liver (F+P+), lung (F+P+), and kidney, while P- bacteria were not present in these tissues. And at 48 hr after inoculation, P+ bacteria were isolated from all tissues, while P- bacteria were isolated from some sites only. These results suggested that pili and flagella each play an important role as virulence factors independently, and that pili-mediated enhancement of virulence of P. aeruginosa was attributed to pili-mediated enhanced colonization of the organisms at the burned skin surfaces.

Animals↗
PubMed1994

Differential elimination of enteric bacteria by protists in a freshwater system.

The short-term (1 h) and long-term (3 d) elimination of low and high densities of five enteric bacteria, Klebsiella pneumoniae, Aeromonas hydrophila, Escherichia coli, Enterococcus faecalis and Staphylococcus epidermidis, by flagellate and ciliate protists were measured in a freshwater system. In addition, the two processes, ingestion and digestion, which cause the disappearance of those enteric bacteria as time passes, were quantified. The results showed that the elimination of these enteric bacteria by protists depends on their initial density, which confirms that the lower the bacterial density the more difficult is their elimination. On the other hand, the short-term and long-term elimination rates of each enteric bacteria were different, and moreover, the order of priority for elimination in the two cases was not the same. Escherichia coli showed the highest elimination rate in short-term experiments, while Aer. hydrophila disappeared at highest rates in long-term experiments. This different order of priority in the elimination rates and the different digestion rates on the five enteric bacteria by phagotrophic protists indicated that the elimination in time is very much influenced by the digestive capacity on each enteric bacteria of those protists. Thus, the low digestion rates of Ent. faecalis and Staph. epidermidis by flagellates and ciliates as well as their low disappearance percentages in the long-term experiments confirm that enteric Gram-positive bacteria are eliminated from the aquatic systems at lower rates, because their digestion is difficult.

Aeromonas hydrophila↗
PubMed1991

Closely linked genetic loci required for swarm cell differentiation and multicellular migration by Proteus mirabilis.

The pathogenic bacterium Proteus mirabilis exhibits a form of multicellular behaviour called swarming migration. This involves the differentiation of vegetative cells at the colony margin into swarm cells which are long, aseptate, multinucleate, hyper-flagellated filaments able to undergo repeated cycles of co-ordinated population migration and consolidation (reversion to vegetative cells). Transposon mutagenesis of uropathogenic P. mirabilis strain U6450 with Tn5 generated 4860 chromosomal insertions and, of these, 75 (1.6%) caused visibly abnormal swarming behaviour, indicating that at least 45 genes are involved in directing motility, cell differentiation and multicellular behaviour. While about one fifth of the swarm-defective mutants lacked flagella and were non-motile non-swarming (NMNS) the majority were normally flagellated and motile but were unable to form swarm cells (motile non-swarming, MNS), or were motile and able to form swarm cells but displayed aberrant patterns of multicellular migration (dendritic swarming, DS) or consolidation (frequent and infrequent consolidation, FC and IC). Restriction enzyme mapping of representative mutant DNAs by Southern hybridization with transposon DNA probes identified eight different mutated genetic loci within the five phenotypic classes. Subsequent Southern analysis of large restriction fragments separated by pulsed-field electrophoresis showed that these eight mutated loci required for motility, cell differentiation and multicellular migration were clustered on a region of DNA spanning approximately 8% of the 4.2 mbp P. mirabilis chromosome. Further linkage analysis showed that the DS locus involved in the ordered migration of the swarm cell population mapped separately from two main clusters of swarm loci, one cluster containing, within 112 kbp, genetic determinants of motility (NMNS) and also differentiation into swarm cells (MNS1, MNS2), and a second within a neighbouring 95 kbp DNA sequence containing three loci involved in the control of consolidation (FC, IC1, IC2).

Base Sequence↗
PubMed2004

Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes.

Swarming is a specialized form of surface motility displayed by several flagellated bacterial genera, which shares features with other surface phenomenon such as biofilm formation and host invasion. Swarmer cells are generally more flagellated and longer than vegetative cells of the same species propagated in liquid media, and move within an encasement of polysaccharide 'slime'. Signals and signalling pathways controlling swarm cell differentiation are largely unknown. In order to test whether there is a genetic programme specific to swarming, we have determined global gene expression profiles of Salmonella typhimurium over an 8 h time course during swarming, and compared the microarray data with a similar time course of growth in liquid media as well as on harder agar where the bacteria do not swarm. Our data show that bacteria growing on the surface of agar have a markedly different physiology from those in broth, as judged by differential regulation of nearly one-third of the functional genome. The large number of genes showing surface-specific upregulation included those for lipopolysaccharide synthesis, iron metabolism and type III secretion. Although swarming-specific induction of flagellar gene expression was not generally apparent, genes for iron metabolism were strongly induced specifically on swarm agar. Surface-dependent regulation of many virulence genes suggests that growth on an agar surface could serve as a model for gene expression during the initial stages of host infection. Based on cluster analysis of distinctive expression patterns, we report here the identification of putative new genes involved in motility and virulence.

Bacterial Proteins↗
PubMed2004

Endocytosis, membrane recycling and sorting of GPI-anchored proteins: Trypanosoma brucei as a model system.

In the flagellated protozoon Trypanosoma brucei, endo- and exocytosis are restricted to a small area of the plasma membrane, the flagellar pocket. All endosomal compartments and the single Golgi complex are located within the posterior part of the cell between the flagellar pocket and the nucleus. The use of reverse genetic tools, including RNA interference, in combination with quantitative 3D-fluorescence and electron microscopic techniques has provided an insight into endosomal membrane traffic, which occurs at a very high rate and appears to exhibit a lower level of complexity than in mammalian cells. The flagellate is an excellent model system for studies on endocytosis, sorting and recycling of glycosylphosphatidylinositol-anchored glycoproteins, because 10(7) molecules of the variant surface glycoprotein form a dense coat at the cell's surface. Because the endocytic rate varies widely at different stages in the parasite's life cycle, trypanosomes may be used for investigating developmental aspects of their endocytic system.

Animals↗
PubMed1976

Comparative kinetics of arginine and lysine transport by epimastigotes and trypomastigotes from two strains of Trypanosoma cruzi.

Three-day-old cultures of Y and MR strains of Trypanosoma cruzi ahd a higher rate of lysine and arginine uptake than 10-day cultures. Amino acid uptake by cells of the MR strain was consistently higher than that of the Y strain. Flagellates separated on DEAE-cellulose columns have normal structure, motility, and infectivity; they have the higher rates of lysine and arginine uptake than the original 3- and 10-day cultures. In addition passage through DEAE-cellulose columns modified the kinetic behavior of amino acid transport systems in the flagellate membranes.

Animals↗
PubMedDate not supplied

Identification and characterization of variants of Crithidia luciliae with altered morphological and surface properties.

Variants of a cloned laboratory stock of the trypanosomatid parasite Crithidia luciliae have been distinguished from "parental type" organisms. These variants accumulated spontaneously over time as the protozoan was maintained by continuous passage in a chemically defined medium. Cloned lines of these variants have been isolated by plating on nutrient agar and partially characterized on the basis of their growth characteristics in culture, their colony and cellular morphology as well as their surface protein expression. One cloned line consisted of motile, flagellated forms which, unlike "parental type" organisms, did not adhere to the surface of culture flasks. Another cloned line was composed of non-adherent, nonmotile, amastigote-like forms which were further distinguished from "parental type" cells by virtue of their constitutive expression, in nutrient-replete medium, of high levels of a surface membrane associated 3'-nucleotidase/nuclease (3'-N'ase) activity. Both the motile, flagellated and amastigote-like variants, like the "parental type" organisms, exhibited elevated levels of the 3'-N'ase activity upon exposure to purine starvation conditions. The variants described are of potential importance in elucidating the mechanism of induction of the highly regulated 3'-N'ase activity as well as for understanding the cytoskeletal systems and the surface properties of these protozoa.

Animals↗
PubMedDate not supplied

Membrane-associated polysaccharides composition, nutritional requirements and cell differentiation in Herpetomonas roitmani: influence of the endosymbiont.

Herpetomonas roitmani, a trypanosomatid containing a bacterial endosymbiont, was cured by high doses of chloramphenicol. Wild-type and cured flagellates were compared as to polysaccharide composition, nutritional requirements and cellular differentiation. Fucose (18.0%), xylose (15.7%), mannose (38.9%), galactose (10.8%), glucose (16.4%) and inositol (< 1.0%) were identified as polysaccharide components of cured H. roitmani as assessed by gas-liquid chromatography. However, the wild-type strain displayed a markedly different sugar profile, in that xylose was absent and inositol preferentially synthesized, whereas the other monosaccharide components remained unchanged. Variations in nutritional pattern also occurred between both strains. The bacterial endosymbiont seems to provide the flagellates with nutritional factors, including usual amino acids, vitamins, purine (as adenine) and hemin. The process of cell differentiation was also significantly influenced by the endosymbiont. Opisthomastigote forms predominate (72.0%) in cured as compared with wild-type H. roitmani (37.0%).

Animals↗
PubMedDate not supplied

Trophic controls on stage transformations of a toxic ambush-predator dinoflagellate.

The toxic dinoflagellate, Pfiesteria piscicida, was recently implicated as the causative agent for about 50% of the major fish kills occurring over a three-year period in the Albemarle-Pamlico Estuarine System of the southeastern USA. Transformations between life-history stages of this dinoflagellate are controlled by the availability of fresh fish secretions or fish tissues, and secondarily influenced by the availability of alternate prey including bacteria, algae, microtauna, and mammalian tissues. Toxic zoospores of P. piscicida subdue fish by excreting lethal neurotoxins that narcotize the prey, disrupt its osmoregulatory system, and attack its nervous system. While prey are dying, the zoospores feed upon bits of fish tissue and complete the sexual phase of the dinoflagellate life cycle. Other stages in the complex life cycle of P. piscicida include cryptic forms of filose, rhizopodial, and lobose amoebae that can form within minutes from toxic zoospores, gametes, or planozygotes. These cryptic amoebae feed upon fish carcasses and other prey and, thus far, have proven less vulnerable to microbial predators than flagellated life-history stages. Lobose amoebae that develop from toxic zoospores and planozygotes during colder periods have also shown ambush behavior toward live fish. In the presence of abundant flagellated algal prey, amoeboid stages produce nontoxic zoospores that can become toxic and form gametes when they detect what is presumed to be a threshold level of a stimulatory substance(s) derived from live fish. The diverse amoeboid stages of this fish "ambush-predator" and at least one other Pfiesteria-like species are ubiquitous and abundant in brackish waters along the western Atlantic and Gulf Coasts, indicating a need to re-evaluate the role of dinoflagellates in the microbial food webs of turbid nutrient-enriched estuaries.

Animals↗
PubMedDate not supplied

The development of Trypanosoma cruzi (Trypanosomatidae) in the reduviid bug Triatoma infestans (Insecta): influence of starvation.

Fifth instars of Triatoma infestans with established Trypanosoma cruzi infections were dissected after different periods of starvation to determine the population density and the percentage of different developmental stages of T. cruzi in the small intestine and rectum of the bugs. After a short starvation period of 20 days, the population density in the small intestine was 20% (about 60,000) of the rectal population. The population in the small intestine was strongly reduced after an additional ten days of starvation, and no flagellates could be found there 60, 90 and 120 days after the last feeding. In the rectum, this reduction went down to 1% of the initial population, but a total elimination never occurred. Usually the remaining population contained more live than dead flagellates. Starvation also resulted in an increase in the rectum in the number and percentage of drop-like forms, intermediates between sphero- and epi- or trypomastigotes, from 1% initially to about 10% after 90 days of starvation. The percentage of spheromastigotes increased from 2% at 20 days after the last feeding to about 20% after an additional 40 and 70 days. Therefore, the spheromastigotes of T. cruzi seem to be induced by stress conditions.

Animals↗
PubMedDate not supplied

Phylogenetic identification of hypermastigotes, Pseudotrichonympha, Spirotrichonympha, Holomastigotoides, and parabasalian symbionts in the hindgut of termites.

The phylogenetic diversity of parabasalian flagellates was examined based on the sequences of small subunit ribosomal RNA genes amplified directly from the mixed population of flagellates in the hindgut of lower termites. In total, 33 representative sequences of parabasalids were recovered from eight termite species. Fluorescent-labeled oligonucleotide probes specific for certain sequences were designed and used for the in situ identification of parabasalian species by whole-cell hybridization. The hypermastigotes, Pseudotrichonympha grassii, Spirotrichonympha leidyi, and Holomastigotoides mirabile in the hindgut of Coptotermes formosanus, and Spirotrichonympha sp. and Trichonympha spp. in Hodotermopsis sjoestedti were identified. In the phylogenetic tree constructed, the sequences from the termites were dispersed within the groups of known members of parabasalids, reflecting the presence of diverse parabasalids in the hindgut of termites. There were three paraphyletic lineages of hypermastigotes represented by Pseudotrichonympha, Trichonympha, and Spirotrichonympha, in agreement with the morphology-based taxonomic groups. The analysis of the tree-root suggested that the Pseudotrichonympha group is the most probable ancient lineage of parabasalids and that the Trichonympha group is the secondly deep-branching lineage. The Spirotrichonympha group and the Trichomonadida may have emerged later.

Animals↗
PubMedDate not supplied

Isolation and characterization of glutathione reductase from Physarum polycephalum and stage-specific expression of the enzyme in life-cycle stages with different oxidation-reduction levels.

Physarum polycephalum has a life cycle with several distinct phases that have different oxidation-reduction requirements. To investigate the relationship between the life cycle and the oxidation-reduction state, we isolated glutathione reductase (GR; EC 1.6.4.2) from Physarum microplasmodia. The enzyme was found to be a homodimer with a subunit M(r) of 49,000, and K(m) values for oxidized glutathione and NADPH of 40 and 28.6 microM, respectively. We then constructed a cDNA library from microplasmodium mRNA and cloned GR cDNA from the library. The isolated cDNA consisted of 1,475 bp encoding a polypeptide of 452 amino acids. The amino acid sequence similarity was about 50% with GRs of other organisms, and several conserved sequence motifs thought to be necessary for activity are evident in the Physarum enzyme. Escherichia coli transformed with an expression vector containing the cDNA synthesized the active GR. Genomic Southern blot analysis indicated that the GR gene is present as a single copy in the Physarum genome. Immunoblot analysis and RT-PCR analysis detected GR mRNA expression in the microplasmodium, plasmodium, and sclerotium, but not in the spore or flagellate. GR activity was low in the spore and flagellate. These results suggest that the glutathione oxidation-reduction system relates to the Physarum life cycle.

Amino Acid Sequence↗
PubMedDate not supplied

Ellobiopsids of the genus Thalassomyces are alveolates.

Ellobiopsids are multinucleate protist parasites of aquatic crustaceans that possess a nutrient absorbing 'root' inside the host and reproductive structures that protrude through the carapace. Ellobiopsids have variously been affiliated with fungi, 'colorless algae', and dinoflagellates, although no morphological character has been identified that definitively allies them with any particular eukaryotic lineage. The arrangement of the trailing and circumferential flagella of the rarely observed bi-flagellated 'zoospore' is reminiscent of dinoflagellate flagellation, but a well-organized 'dinokaryotic nucleus' has never been observed. Using small subunit ribosomal RNA gene sequences from two species of Thalassomyces, phylogenetic analyses robustly place these ellobiopsid species among the alveolates (ciliates, apicomplexans, dinoflagellates and relatives) though without a clear affiliation to any established alveolate lineage. Our trees demonstrate that Thalassomyces fall within a dinoflagellate + apicomplexa + Perkinsidae + "marine alveolate group 1" clade, clustering most closely with dinoflagellates. However, the poor statistical support for branches within this region indicates that additional data will be needed to resolve relationships among these taxa.

Animals↗
PubMedDate not supplied

Growth-phase dependent substrate adhesion in Crithidia fasciculata.

Crithidia fasciculata is a trypanosomatid flagellate that parasitizes several species of mosquito. Within the alimentary tract of its host, C. fasciculata exists in two forms: one is a non-motile form, attached in clusters to the lining of the gut, the other a more elongated form swimming freely in the gut lumen. We have developed an in vitro culture system that reproduces the appearance of these two distinct morphological forms. Using two different cultivation methods, shaking and stationary incubations, we have demonstrated that adherence phenotypes are growth-phase dependent. Organisms in the logarithmic phase of growth possess the ability to adhere to substrates; this ability is lost when the organism enters a stationary growth phase. Parasite adherence was independent of cultivation method or substrate. Furthermore, adherent forms of Crithidia maintained their adhesive properties following their removal from substrates. Our data reveal a growth-phase-regulated process of cell attachment that may influence the transmission and dissemination of this parasitic flagellate.

Animals↗