Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids.     

The Bacterium Endosymbiont of Crithidia deanei Undergoes Coordinated Division with the Host Cell Nucleus

In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three-dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells.     

Reduction of Tubulin Expression in Angomonas deanei by RNAi Modifies the Ultrastructure of the Trypanosomatid Protozoan and Impairs Division of Its Endosymbiotic Bacterium

In the last two decades, RNA interference pathways have been employed as a useful tool for reverse genetics in trypanosomatids. Angomonas deanei is a nonpathogenic trypanosomatid that maintains an obligatory endosymbiosis with a bacterium related to the Alcaligenaceae family. Studies of this symbiosis can help us to understand the origin of eukaryotic organelles. The recent elucidation of both the A. deanei and the bacterium symbiont genomes revealed that the host protozoan codes for the enzymes necessary for RNAi activity in trypanosomatids. Here, we tested the functionality of the RNAi machinery by transfecting cells with dsRNA to a reporter gene (green fluorescent protein), which had been previously expressed in the parasite and to α‐tubulin, an endogenous gene. In both cases, protein expression was reduced by the presence of specific dsRNA, inducing, respectively, a decreased GFP fluorescence and the formation of enlarged cells with modified arrangement of subpellicular microtubules. Furthermore, symbiont division was impaired. These results indicate that the RNAi system is active in A. deanei and can be used to further explore gene function in symbiont‐containing trypanosomatids and to clarify important aspects of symbiosis and cell evolution.     

Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.     

Electrophoretic Analysis of Endonuclease-Generated Fragments of k-DNA,of Esterase Isoenzymes,and of Surface Proteins as Aids for Species Identification of Insect Trypanosomatids1

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

Characterization of a venom gland-associated rhabdovirus in the parasitoid wasp Diachasmimorpha longicaudata

Parasitoid wasps reproduce by laying their eggs on or inside of a host insect, which triggers a defense response in the host insect that kills the developing wasp. To counteract the host’s lethal response, some parasitoid wasps are associated with symbiotic viruses that alter host metabolism and development to promote successful development of the wasp embryo. These symbiotic viruses display a number of characteristics that differ from those of pathogenic viruses, but are poorly understood with the exception of one group, the polydnaviruses. Here, we characterize the genome of a non-polydnavirus associated with parasitoid wasps, Diachasmimorpha longicaudata rhabdovirus (DlRhV), and assess its role as a potential mutualistic virus. Our results show that the DlRhV genome contains six open reading frames (ORFs). Three ORFs show sequence homology to known viral genes and one ORF encodes a previously identified protein, called parasitism-specific protein 24 (PSP24), that has been hypothesized to play a role in promoting successful parasitism by D. longicaudata. We constructed a phylogeny that shows that DlRhV is most closely related to other insect-infecting rhabdoviruses. Finally, we report that DlRhV infection does not occur in all populations of D. longicaudata, and is not required for successful parasitism.     

Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium

Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.     

Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

Background

Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels.

Methodology/Principal Findings

The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases ({"type":"entrez-protein","attrs":{"text":"ORF01222","term_id":"1178790230","term_text":"ORF01222"}}ORF01222, {"type":"entrez-protein","attrs":{"text":"ORF03896","term_id":"1178792974","term_text":"ORF03896"}}ORF03896, and {"type":"entrez-protein","attrs":{"text":"ORF01315","term_id":"1178790325","term_text":"ORF01315"}}ORF01315) exhibited the highest levels of up-regulation.

Conclusions/Significance

The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.     

Trypanosomatid protozoa: survey of acetylornithinase and ornithine acetyltransferase

Trypanosomatid protozoa (Crithidia deanei, C. deanei aposymbiotic, C. oncopelti, C. fasciculata, C. acanthocephali, Leptomonas seymouri, L. collosoma, L. samueli, Herpetomonas samuelpessoai, H. sp., H. megaseliae, H. muscarum muscarum, Leishmania donovani, L. braziliensis, Trypanosoma cruzi, T. conorhini and T. mega) were examined for the presence of acetylornithinase (EC 3.5.1.16) and ornithine acetyltransferase (EC 2.3.1.35). As a rule, species of the genus Crithidia presented one of the two enzymes for the conversion of acetylornithine into ornithine. Crithidia fasciculata and C. acanthocephali presented acetylornithinase, while C. deanei and C. oncopelti, species harboring symbionts, presented ornithine acetyltransferase. The enzyme was absent in the aposymbiotic strain of C. deanei, which suggests that the enzyme belongs to the symbiont. Among the other trypanosomatids examined only Herpetomonas samuelpessoai presented acetylomithinase. The participation of acetylornithinase and ornithine acetyltransferase in the metabolism of trypanosomatids is discussed in the light of their nutritional requirements and possession of enzymes of the arginineornithine metabolism.     

Arbuscular mycorrhizal phosphate transport under monoxenic conditions using radio-labelled inorganic and organic phosphate

Compartmented monoxenic cultures of Ri T-DNA transformed carrot roots and a symbiotic arbuscular mycorrhizal fungus demonstrated for the first time that phosphate in an organic form (³²P-labelled AMP) may be hydrolysed by extra-radical mycorrhizal hyphae in the absence of other organisms, and subsequently utilized as a mineral nutrient source by the host plant after fungal transport.     

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, β-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for β-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.     

Development of a Functional Genomics Platform for Sinorhizobium meliloti: Construction of an ORFeome

The nitrogen-fixing, symbiotic bacterium Sinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of the S. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis of S. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis of S. meliloti, such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and the S. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.     

Directed Construction and Analysis of a Sinorhizobium meliloti pSymA Deletion Mutant Library

Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a medium-throughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.     

Cloning and genetic analyses of the bacteriocin 41 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI14: a novel bacteriocin complemented by two extracellular components (lysin and activator)

The conjugative plasmid pYI14 (61 kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E. faecalis. Physical mapping of pYI14 showed that it consisted of EcoRI fragments A to P. The clone pHT1100, containing EcoRI fragments A (12.6 kbp) and H (3.5 kbp), conferred the bacteriocin activity on E. faecalis strains. Genetic analysis showed that the determinant was located in a 6.6-kbp region within the EcoRI AH fragments. Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL₁) ORF8 (bacL₂), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL₁, bacL₂, bacA, and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL₁ and -L₂ and bacA mutants. bacL₁ and -L₂ and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, providing the host with resistance to its own bacteriocin activity. The bacL₁-encoded protein had significant homology with lytic enzymes that attack the gram-positive bacterial cell wall. Sequence data for the deduced bacL₁-encoded protein suggested that it has a domain structure consisting of an N-terminal signal peptide, a second domain with the enzymatic activity, and a third domain with a three-repeat structure directing the proenzyme to its cell surface receptor.     

Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea

The production of erythromycin A by Saccharopolysporaerythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-α-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV?), ORF17 (eryCIV?) and ORF7 (eryBII?) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.