A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont

Nukleär kodierte Plastidenproteine bei der Grünalge Chlamydomonas reinhardtii\textit {Chlamydomonas reinhardtii}

Im Rahmen dieser Arbeit wurde die $\it trans$-Spleißmutante TR72 der einzelligen Grünalge $\textit {Chlamydomonas reinhardtii}$ charakterisiert. Ausgehend von einem Cosmid-Klon wurde durch Subklonierungen zwei benachbarte Gene, $\it Rat1$ und $\it Rat2$, identifiziert, welche zusammen den Mutanten-Phänotyp komplementieren. Die Expression des $\it Rat1$-Gens wurde mit Hilfe von Primer-Extension-Analysen und RT-PCR untersucht. Ausgehend von der cDNA-Sequenz des $\it Rat1$-Gens wurden in der abgeleiteten Aminosäuresequenz des Polypeptids Homologien zu einer NAD$^{+}$-Bindedomäne nachgewiesen. Eine funktionelle Charakterisierung dieser Domäne wurde mittels ortsgerichteter Mutagenesen durchgeführt. Durch Epitop-Tagging und Western-Analysen konnte die plastidäre Lokalisation des $\it Rat1$-Polypeptides nachgewiesen werden. Ektopische Effekte der Mutation in TR72 auf die Genexpression des Zellkerns wurden mit Hilfe der DNA-Macroarray-Technik untersucht

Free-Living Amoebae as Hosts for and Vectors of Intracellular Microorganisms with Public Health Significance

Free-living amoebae (FLA) are parasites within both humans and animals causing a wide range of symptoms and act as hosts of, and vehicles for phylogenetically diverse microorganisms, called endocytobionts. The interaction of the FLA with sympatric microorganisms leads to an exceptional diversity within FLA. Some of these bacteria, viruses, and even eukaryotes, can live and replicate intracellularly within the FLA. This relationship provides protection to the microorganisms from external interventions and a dispersal mechanism across various habitats. Among those intracellularly-replicating or -residing organisms there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals and are therefore of interest to Public Health Authorities. Mimiviruses, Pandoraviruses, and Pithoviruses are examples for interesting viral endocytobionts within FLA. Future research is expected to reveal further endocytobionts within free-living amoebae and other protozoa through co-cultivation studies, genomic, transcriptomic, and proteomic analyses

Component Endoparasite Communities Mirror Life-History Specialization in Syntopic Reed Frogs (Hyperolius spp.)

Most of our knowledge on the processes structuring parasite communities in amphibians originate from temperate-zone taxa, whereas Afrotropical communities have been neglected so far. We found evidence that ecological fitting of the hosts and, probably, differential immune response may influence the variation in parasite species richness, prevalence, and infestation intensity of East African frogs Hyperolius kivuensis and H. viridiflavus. The most closely related host species share the same macrohabitat (that implies the same pool of potential parasites), but differ in microhabitat preference, so that a comparative analyses of syntopic and allopatric populations is expedient to reveal ecological fitting. We detected 11 parasite species (one annelid, four nematodes, five trematodes, one cestode) and two endocommensal species (protozoans). The component parasite communities included 4–5 helminth species in H. kivuensis and 6–8 in the more aquatic H. viridiflavus, supporting the hypothesis that trematode diversity increases with the amount of time spent in water. Five parasite species (Orneoascaris chrysanthemoides, Clinostomum chabaudi, an undetermined echinostomatid) and two protozoans (Nyctotheroides sp., and Protoopalina sp.) are shared among the syntopic amphibian populations. This finding indicates a similar susceptibility of these amphibians to infestation from the local parasite pool. Yet, the low prevalence of single- and multi-species infestations in H. kivuensis indicates that parasite clearing by its immune response is probably more effective and prominent than in H. viridiflavus. Therefore, H. viridiflavus suffered from significantly reduced short-term survival due to the infection. Thus, we conclude that the processes structuring component parasite communities in amphibians do not differ generally between temperate-zone and Afrotropical host species, but they do in the magnitude of ecological fitting

Component Endoparasite Communities Mirror Life-History Specialization in Syntopic Reed Frogs (<i>Hyperolius</i> spp.)

Most of our knowledge on the processes structuring parasite communities in amphibians originate from temperate-zone taxa, whereas Afrotropical communities have been neglected so far. We found evidence that ecological fitting of the hosts and, probably, differential immune response may influence the variation in parasite species richness, prevalence, and infestation intensity of East African frogs Hyperolius kivuensis and H. viridiflavus. The most closely related host species share the same macrohabitat (that implies the same pool of potential parasites), but differ in microhabitat preference, so that a comparative analyses of syntopic and allopatric populations is expedient to reveal ecological fitting. We detected 11 parasite species (one annelid, four nematodes, five trematodes, one cestode) and two endocommensal species (protozoans). The component parasite communities included 4–5 helminth species in H. kivuensis and 6–8 in the more aquatic H. viridiflavus, supporting the hypothesis that trematode diversity increases with the amount of time spent in water. Five parasite species (Orneoascaris chrysanthemoides, Clinostomum chabaudi, an undetermined echinostomatid) and two protozoans (Nyctotheroides sp., and Protoopalina sp.) are shared among the syntopic amphibian populations. This finding indicates a similar susceptibility of these amphibians to infestation from the local parasite pool. Yet, the low prevalence of single- and multi-species infestations in H. kivuensis indicates that parasite clearing by its immune response is probably more effective and prominent than in H. viridiflavus. Therefore, H. viridiflavus suffered from significantly reduced short-term survival due to the infection. Thus, we conclude that the processes structuring component parasite communities in amphibians do not differ generally between temperate-zone and Afrotropical host species, but they do in the magnitude of ecological fitting

Alternative Development Strategies of Clinostomum chabaudi (Digenea) Metacercariae in Frog Hosts (Hyperolius spp.)

Clinostomum metacercariae are common endoparasites of fish and frogs. In this study, we examine taxonomic identity and developmental strategy of Clinostomum metacercariae infesting reed frogs Hyperolius kivuensis and H. viridiflavus in Rwanda. Moreover, we evaluate the impact of infestation on demographic and morphological life-history traits of the hosts. Morphological and molecular features, particularly genital morphology and COX1 sequences, provided evidence that the metacercariae belong to C. chabaudi Vercammen-Grandjean, 1960. Depending on the host’s defensive behavior and the availability of resources, metacercariae develop either as sedentary “yellow grubs” encysted in the lymphatic sacs or mouth of the host or as initially encysted, but later free-ranging individuals invading the host´s body cavity. Nutrition on lymphatic fluid within the cyst leads to yellow-colored gut content, feeding on blood or host tissue, to brownish green gut content in free-ranging individuals. Almost all metacercariae opted for the first developmental strategy in H. kivuensis, whereas the second strategy dominated in metacercariae infesting H. viridiflavus. Hyperolius kivuensis suffered significant morphological modifications, when infested with encysted metacercariae. Both developmental modes permitted a coexistence with the host of less than one year. We hypothesize that the presence of alternative development modes is an adaptation of C. chabaudi to cope with resource limitation within host-produced cysts

Molecular Diversity of Nematode Parasites in Afrotropical Reed Frogs (Hyperolius spp.)

The diversity of nematodes infecting amphibians is understudied in tropical Africa and unknown in Rwanda. Diversity assessment is hampered by the fact that species descriptions refer mostly to morphological features that are unlinked to DNA sequences of marker genes available in public databases. In this paper, we explore the abundance and diversity of parasitic nematodes in reed frogs Hyperolius kivuensis (n = 115), H. parallelus (n = 45) and H. viridiflavus (n = 100) collected in Rwanda. Five nematode species were identified morphologically as Orneoascaris chrysanthemoides, O. schoutedeni, Gendria leberrei, Aplectana chamaeleonis and Rhabdias collaris. Corresponding DNA sequences of 18S and COI genes were determined and subsequently deposited in GenBank. Aplectana chamaeleonis showed the highest prevalence (8.7%), but O. chrysanthemoides the highest mean intensity of infection (6.0) and largest number (24) of individuals in H. kivuensis. To the best of our knowledge, all amphibian hosts are new records for these nematode species, which are known to infect a wide range of amphibian and reptile species. Our findings suggest that nematode diversity is probably lower than previously assumed due to low host specificity. As morphological species identification is often challenging, our data facilitate molecular identification of adult and specifically larval nematodes found in amphibians of Sub-Saharan Africa

Chlamydomonas reinhardtii

binds to chloroplast group II intron RNA i