Characterization of chitinases from the GH18 gene family in the myxomycete Physarum polycephalum.

BACKGROUND: Physarum polycephalum is an unusual macroscopic myxomycete expressing a large range of glycosyl hydrolases. Among them, enzymes from the GH18 family can hydrolyze chitin, an important structural component of the cell walls in fungi and in the exoskeleton of insects and crustaceans. METHODS: Low stringency sequence signature search in transcriptomes was used to identify GH18 sequences related to chitinases. Identified sequences were expressed in E. coli and corresponding structures modelled. Synthetic substrates and in some cases colloidal chitin were used to characterize activities. RESULTS: Catalytically functional hits were sorted and their predicted structures compared. All share the TIM barrel structure of the GH18 chitinase catalytic domain, optionally fused to binding motifs, such as CBM50, CBM18, and CBM14, involved in sugar recognition. Assessment of the enzymatic activities following deletion of the C-terminal CBM14 domain of the most active clone evidenced a significant contribution of this extension to the chitinase activity. A classification based on module organization, functional and structural criteria of characterized enzymes was proposed. CONCLUSIONS: Physarum polycephalum sequences encompassing a chitinase like GH18 signature share a modular structure involving a structurally conserved catalytic TIM barrels decorated or not by a chitin insertion domain and optionally surrounded by additional sugar binding domains. One of them plays a clear role in enhancing activities toward natural chitin. GENERAL SIGNIFICANCE: Myxomycete enzymes are currently poorly characterized and constitute a potential source for new catalysts. Among them glycosyl hydrolases have a strong potential for valorization of industrial waste as well as in therapeutic field.

Structural Organization of S516 Group I Introns in Myxomycetes.

Group I introns are mobile genetic elements encoding self-splicing ribozymes. Group I introns in nuclear genes are restricted to ribosomal DNA of eukaryotic microorganisms. For example, the myxomycetes, which represent a distinct protist phylum with a unique life strategy, are rich in nucleolar group I introns. We analyzed and compared 75 group I introns at position 516 in the small subunit ribosomal DNA from diverse and distantly related myxomycete taxa. A consensus secondary structure revealed a conserved group IC1 ribozyme core, but with a surprising RNA sequence complexity in the peripheral regions. Five S516 group I introns possess a twintron organization, where a His-Cys homing endonuclease gene insertion was interrupted by a small spliceosomal intron. Eleven S516 introns contained direct repeat arrays with varying lengths of the repeated motif, a varying copy number, and different structural organizations. Phylogenetic analyses of S516 introns and the corresponding host genes revealed a complex inheritance pattern, with both vertical and horizontal transfers. Finally, we reconstructed the evolutionary history of S516 nucleolar group I introns from insertion of mobile-type introns at unoccupied cognate sites, through homing endonuclease gene degradation and loss, and finally to the complete loss of introns. We conclude that myxomycete S516 introns represent a family of genetic elements with surprisingly dynamic structures despite a common function in RNA self-splicing.

Loss of the Polyketide Synthase StlB Results in Stalk Cell Overproduction in Polysphondylium violaceum.

Major phenotypic innovations in social amoeba evolution occurred at the transition between the Polysphondylia and group 4 Dictyostelia, which comprise the model organism Dictyostelium discoideum, such as the formation of a new structure, the basal disk. Basal disk differentiation and robust stalk formation require the morphogen DIF-1, synthesized by the polyketide synthase StlB, the des-methyl-DIF-1 methyltransferase DmtA, and the chlorinase ChlA, which are conserved throughout Dictyostelia. To understand how the basal disk and other innovations evolved in group 4, we sequenced and annotated the Polysphondylium violaceum (Pvio) genome, performed cell type-specific transcriptomics to identify cell-type marker genes, and developed transformation and gene knock-out procedures for Pvio. We used the novel methods to delete the Pvio stlB gene. The Pvio stlB- mutants formed misshapen curly sorogens with thick and irregular stalks. As fruiting body formation continued, the upper stalks became more regular, but structures contained 40% less spores. The stlB- sorogens overexpressed a stalk gene and underexpressed a (pre)spore gene. Normal fruiting body formation and sporulation were restored in Pvio stlB- by including DIF-1 in the supporting agar. These data indicate that, although conserved, stlB and its product(s) acquired both a novel role in the group 4 Dictyostelia and a role opposite to that in its sister group.

The Response of Litter-Associated Myxomycetes to Long-Term Nutrient Addition in a Lowland Tropical Forest.

Myxomycetes (plasmodial slime molds) are abundant protist predators that feed on bacteria and other microorganisms, thereby playing important roles in terrestrial nutrient cycling. Despite their significance, little is known about myxomycete communities and the extent to which they are affected by nutrient availability. We studied the influence of long-term addition of N, P, and K on the myxomycete community in a lowland forest in the Republic of Panama. In a previous study, microbial biomass increased with P but not N or K addition at this site. We hypothesized that myxomycetes would increase in abundance in response to P but that they would not respond to the sole addition of N or K. Moist chamber cultures of leaf litter and small woody debris were used to quantify myxomycete abundance. We generated the largest myxomycete dataset (3,381 records) for any single locality in the tropics comprised by 91 morphospecies. In line with our hypothesis, myxomycete abundance increased in response to P addition but did not respond to N or K. Community composition was unaffected by nutrient treatments. This work represents one of very few large-scale and long-term field studies to include a heterotrophic protist highlighting the feasibility and value in doing so.

Accumulation of Stable Full-Length Circular Group I Intron RNAs during Heat-Shock.

Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility.

Evaluating the potential use of myxomycetes as a source of lipids for biodiesel production.

The myxomycetes are a group of primitive phagotrophic eukaryotes characterized by a distinctive plasmodial stage that is well known for its rapid growth rate. In the present study, biomass and lipid production of several different species of myxomycetes were investigated. Physarum polycephalum was found to produce the highest amounts of both dry biomass (1.30g), and lipid (0.143g) per 20mL medium (equal to 65.0g biomass and 7.15g lipid per one liter of medium). Analysis of P. polycephalum lipids by thin layer chromatography (TLC) and fatty acid methyl esters (FAMES) by gas chromatography-mass spectrometry (GC-MS) techniques showed that the major lipid type is triglyceride (95.5%), followed by phospholipids (2.6%); diglyceride (0.92%) and monoglyceride (0.92%). Myxomycete lipids consist of three dominant fatty acids: oleic (20%), linoleic (33%), and palmitoleic (17%). These results suggest that P. polycephalum has considerable potential as a source of lipids for biodiesel production.

Organellar RNA editing.

RNA editing is a term used for a number of mechanistically different processes that alter the nucleotide sequence of RNA molecules to differ from the gene sequence. RNA editing occurs in a wide variety of organisms and is particularly frequent in organelle transcripts of eukaryotes. The discontiguous phylogenetic distribution of mRNA editing, the mechanistic differences observed in different organisms, and the nonhomologous editing machinery described in different taxonomic groups all suggest that RNA editing has appeared independently several times. This raises questions about the selection pressures acting to maintain editing that are yet to be completely resolved. Editing tends to be frequent in organisms with atypical organelle genomes and acts to correct the effect of DNA mutations that would otherwise compromise the synthesis of functional proteins. Additional functions of editing in generating protein diversity or regulating gene expression have been proposed but so far lack widespread experimental evidence, at least in organelles.

Use of sweet sorghum juice for lipid production by Schizochytrium limacinum SR21.

Stalk juice from sweet sorghum grown in Southern Illinois, USA, was examined for lipid production through microalgal fermentation. Juice concentrations at 100%, 75%, 50%, and 25% led to different biomass, lipid, and docosahexaenoic acid (DHA) production by Schizochytrium limacinum SR21. Biomass dry weight as 9.4g/l at 50% juice concentration was similar to that from pure glucose (10.9g/l). But with a 73.4% lipid content, this dose resulted in higher lipid and DHA production than those from pure glucose. Major fatty acids in cells grown on juice were identical to those fed by other substrates. Among the three sugars - glucose, fructose, and sucrose in sorghum juice, only glucose was utilized for growth. Spent medium after algal removal may be further processed for white sugar production in a traditional way since sucrose content remained the same throughout the algal fermentation process. Algal cells or lipids harvested can be utilized as fish meal, human nutrition supplements, or for biodiesel purpose.

LRRK2 in Parkinson's disease: in vivo models and approaches for understanding pathogenic roles.

The recent discovery of the genetic causes for Parkinson's disease (PD) is fruitful; however, the continuing revelation of PD-related genes is rapidly outpacing the functional characterization of the gene products. Although the discovery of multiple PD-related genes places PD as one of the most complex multigenetic diseases of the brain, it will undoubtedly facilitate the unfolding of a central pathogenic pathway and an understanding of the etiology of PD. Recent findings of pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2) (PARK8) that are linked to the most common familial forms and some sporadic forms of PD provide a unique opportunity to gain insight into the pathogenesis of PD. Despite rapid growth in biochemical, structural and in vitro cell culture studies of LRRK2, the in vivo characterizations of LRRK2 function generally fall short and are largely limited to invertebrates. The investigation of LRRK2 or homologs of LRRK2 in nonmammalian models provides important clues with respect to the cellular functions of LRRK2, but an elucidation of the physiology and pathophysiology of LRRK2 relevant to PD would still depend on mammalian models established by multiple genetic approaches, followed by rigorous examination of the models for pathological process. This minireview summarizes previous studies of genes for ROCO and LRRK2 homologs in slime mold, nematode worms and fruit flies. It also discusses the results obtained from available mouse models of LRRK2 that begin to provide information for understanding LRRK2-mediated pathogenesis in PD.

Secondary metabolites of slime molds (myxomycetes).

The compounds reported from the slime molds (myxomycetes) species are described. Almost 100 natural compounds including their chemical structures and biological activities are described in this review article. Only metabolites with a well-defined structure are included.

Evolution of hierarchical cytoplasmic inheritance in the plasmodial slime mold Physarum polycephalum.

A striking linear dominance relationship for uniparental mitochondrial transmission is known between many mating types of plasmodial slime mold Physarum polycephalum. We herein examine how such hierarchical cytoplasmic inheritance evolves in isogamous organisms with many self-incompatible mating types. We assume that a nuclear locus determines the mating type of gametes and that another nuclear locus controls the digestion of mitochondria DNAs (mtDNAs) of the recipient gamete after fusion. We then examine the coupled genetic dynamics for the evolution of self-incompatible mating types and biased mitochondrial transmission between them. In Physarum, a multiallelic nuclear locus matA controls both the mating type of the gametes and the selective elimination of the mtDNA in the zygotes. We theoretically examine two potential mechanisms that might be responsible for the preferential digestion of mitochondria in the zygote. In the first model, the preferential digestion of mitochondria is assumed to be the outcome of differential expression levels of a suppressor gene carried by each gamete (suppression-power model). In the second model (site-specific nuclease model), the digestion of mtDNAs is assumed to be due to their cleavage by a site-specific nuclease that cuts the mtDNA at unmethylated recognition sites. Also assumed is that the mtDNAs are methylated at the same recognition site prior to the fusion, thereby being protected against the nuclease of the same gamete, and that the suppressor alleles convey information for the recognition sequences of nuclease and methylase. In both models, we found that a linear dominance hierarchy evolves as a consequence of the buildup of a strong linkage disequilibrium between the mating-type locus and the suppressor locus, though it fails to evolve if the recombination rate between the two loci is larger than a threshold. This threshold recombination rate depends on the number of mating types and the degree of fitness reduction in the heteroplasmic zygotes. If the recombination rate is above the threshold, suppressor alleles are equally distributed in each mating type at evolutionary equilibrium. Based on the theoretical results of the site-specific nuclease model, we propose that a nested subsequence structure in the recognition sequence should underlie the linear dominance hierarchy of mitochondrial transmission.

A new labyrinthulid isolate, which solely produces n-6 docosapentaenoic acid.

A labyrinthulid strain, L59, was isolated from a leaf floating on seawater collected at the coastal area of Hokkaido Prefecture, Japan. Strain L59 contained only n-6 docosapentaenoic acid ( n-6 DPA) among all the long-chain polyunsaturated fatty acids. The proportion of n-6 DPA in the total fatty acids was 48.1% and the total fatty acids content in the cell dry weight was 26.6%. Many oil bodies were observed in the cell, mostly in the vicinity of cell membranes. The strain had spindle-shaped cell bodies and all cells were surrounded by ectoplasmic net elements. It was also clearly classified in the labyrinthulid group by phylogenetic analysis. In the optimum culture condition, using soybean oil and peptone as carbon and nitrogen sources, 0.53 g of n-6 DPA/l was produced at 20 degrees C in 7 days.

Plasmodium immobilization of Physarella oblonga (Berk. & Curt.) Morgan (Myxomycetes) using kaolinite as a matrix of entrapment.

The possibility of using kaolinite-immobilized plasmodium fragments of Physarella oblonga (Berk. & Curt.) Morgan to maintain their metabolic activity was examined. The immobilization process was carried out with 1 mg of plasmodium of P. oblonga entrapped in 10 g of kaolinite. Sodium acetate (1 mM) was used as a metabolic precursor. The collection of fractions was carried out during a one month period, and extracted with ether/ethyl acetate and chloroform/acetonitrile. The extractions from plasmodium in natura were accomplished with the same solvents. The extracts obtained were analyzed in a spectrophotometer at 266 nm and 310 nm, and by thin layer chromatography to assess the productivity of the immobilized plasmodium. The absorbances of the extracts in both wavelengths and the chromatographic tests showed the synthesis of compounds by the immobilized material. Three chromatographic spots were observed in the extracts obtained from the immobilized plasmodium. Two spots coincided with the R(f) values and coloration of the spots observed for the material in natura used as a reference. The kaolinite-immobilized plasmodium of P. oblonga can remain metabolically active for at least one month at room temperature and ambient light conditions.