Martin P. Vazquez

Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi Genome Project

a Escola Paul&a de Medicina, Rua Botucatu, 862, CEP 04023-062, Stio Pa&o, Brazil b Institute de Quimica da USP, Sao Paula, Brazil ’ Institute de Inuestigaciones en Engenharia Genetica y Biologia Molecular, Buenos Aires. Argentina ’ Instituto de Parasitologia y Biomedicina, Granada, Spain e FIOCRUZ, Rio de Janeiro, Brazil f Instituto de Biofisica, UFRJ, Rio de Janeiro, Brazil g Centro de Biologia Celular, XV, Caracas, Venezuela h Centro de Biologia Molecular, IJAM_ Madrid, Spain

Functional analysis of the intergenic regions of TcP2β gene loci allowed the construction of an improved Trypanosoma cruzi expression vector

TcP2beta ribosomal protein genes in Trypanosoma cruzi are encoded by four different loci, H6.4, H1.8, H1.5 and H1.3. All loci have a similar organization, except for H1.8 that harbors two TcP2beta genes arranged in tandem and separated by a short repetitive sequence, named SIRE (short interspersed repetitive element), which is also found upstream of the first gene of the tandem and downstream of the second. In this locus the trans-splicing signal of TcP2beta is located within the SIRE element, while in the other loci it is positioned within the first 50bases upstream of the AUG with an AG acceptor site at position -12 respective to the initiation codon. Transient transfection experiments were used to evaluate the efficiency of these two different trans-splicing regions to drive CAT activity. The region named HX1 located upstream the TcP2beta H1. 8 gene was clearly more efficient than the SIRE sequence contained in the region named HX2. Therefore, we decided to use the HX1 region to ameliorate the performance of the cryptic trans-splicing signal present in the T. cruzi expression vector pRIBOTEX (Martinez-Calvillo, S., López, I., Hernandez, H., 1997. pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene 199, 71-76). By insertion of the region HX1 downstream of the ribosomal promoter of pRIBOTEX, we constructed pRHX1CAT40 that, in stable transfected cells, was able to drive CAT activity 2760 times more efficiently than the control plasmids. Based on this, a novel plasmid vector was conceived, named pTREX-n, which retains the neo gene of pRIBOTEX as a positive selectable marker and replaces the CAT-SV40 cassette in pRHX1CAT40 by a multiple cloning site.

Structure, Composition and Metagenomic Profile of Soil Microbiomes Associated to Agricultural Land Use and Tillage Systems in Argentine Pampas

Agriculture is facing a major challenge nowadays: to increase crop production for food and energy while preserving ecosystem functioning and soil quality. Argentine Pampas is one of the main world producers of crops and one of the main adopters of conservation agriculture. Changes in soil chemical and physical properties of Pampas soils due to different tillage systems have been deeply studied. Still, not much evidence has been reported on the effects of agricultural practices on Pampas soil microbiomes. The aim of our study was to investigate the effects of agricultural land use on community structure, composition and metabolic profiles on soil microbiomes of Argentine Pampas. We also compared the effects associated to conventional practices with the effects of no-tillage systems. Our results confirmed the impact on microbiome structure and composition due to agricultural practices. The phyla Verrucomicrobia, Plactomycetes, Actinobacteria, and Chloroflexi were more abundant in non cultivated soils while Gemmatimonadetes, Nitrospirae and WS3 were more abundant in cultivated soils. Effects on metabolic metagenomic profiles were also observed. The relative abundance of genes assigned to transcription, protein modification, nucleotide transport and metabolism, wall and membrane biogenesis and intracellular trafficking and secretion were higher in cultivated fertilized soils than in non cultivated soils. We also observed significant differences in microbiome structure and taxonomic composition between soils under conventional and no- tillage systems. Overall, our results suggest that agronomical land use and the type of tillage system have induced microbiomes to shift their life-history strategies. Microbiomes of cultivated fertilized soils (i.e. higher nutrient amendment) presented tendencies to copiotrophy while microbiomes of non cultivated homogenous soils appeared to have a more oligotrophic life-style. Additionally, we propose that conventional tillage systems may promote copiotrophy more than no-tillage systems by decreasing soil organic matter stability and therefore increasing nutrient availability.

The discovery of stromatolites developing at 3570 m above sea level in a high-altitude volcanic lake Socompa, Argentinean Andes.

We describe stromatolites forming at an altitude of 3570 m at the shore of a volcanic lake Socompa, Argentinean Andes. The water at the site of stromatolites formation is alkaline, hypersaline, rich in inorganic nutrients, very rich in arsenic, and warm (20–24°C) due to a hydrothermal input. The stromatolites do not lithify, but form broad, rounded and low-domed bioherms dominated by diatom frustules and aragonite micro-crystals agglutinated by extracellular substances. In comparison to other modern stromatolites, they harbour an atypical microbial community characterized by highly abundant representatives of Deinococcus-Thermus, Rhodobacteraceae, Desulfobacterales and Spirochaetes. Additionally, a high proportion of the sequences that could not be classified at phylum level showed less than 80% identity to the best hit in the NCBI database, suggesting the presence of novel distant lineages. The primary production in the stromatolites is generally high and likely dominated by Microcoleus sp. Through negative phototaxis, the location of these cyanobacteria in the stromatolites is controlled by UV light, which greatly influences their photosynthetic activity. Diatoms, dominated by Amphora sp., are abundant in the anoxic, sulfidic and essentially dark parts of the stromatolites. Although their origin in the stromatolites is unclear, they are possibly an important source of anaerobically degraded organic matter that induces in situ aragonite precipitation. To the best of our knowledge, this is so far the highest altitude with documented actively forming stromatolites. Their generally rich, diverse and to a large extent novel microbial community likely harbours valuable genetic and proteomic reserves, and thus deserves active protection. Furthermore, since the stromatolites flourish in an environment characterized by a multitude of extremes, including high exposure to UV radiation, they can be an excellent model system for studying microbial adaptations under conditions that, at least in part, resemble those during the early phase of life evolution on Earth.

Genome and transcriptome analysis of the Mesoamerican common bean and the role of gene duplications in establishing tissue and temporal specialization of genes

BackgroundLegumes are the third largest family of angiosperms and the second most important crop class. Legume genomes have been shaped by extensive large-scale gene duplications, including an approximately 58 million year old whole genome duplication shared by most crop legumes.ResultsWe report the genome and the transcription atlas of coding and non-coding genes of a Mesoamerican genotype of common bean (Phaseolus vulgaris L., BAT93). Using a comprehensive phylogenomics analysis, we assessed the past and recent evolution of common bean, and traced the diversification of patterns of gene expression following duplication. We find that successive rounds of gene duplications in legumes have shaped tissue and developmental expression, leading to increased levels of specialization in larger gene families. We also find that many long non-coding RNAs are preferentially expressed in germ-line-related tissues (pods and seeds), suggesting that they play a significant role in fruit development. Our results also suggest that most bean-specific gene family expansions, including resistance gene clusters, predate the split of the Mesoamerican and Andean gene pools.ConclusionsThe genome and transcriptome data herein generated for a Mesoamerican genotype represent a counterpart to the genomic resources already available for the Andean gene pool. Altogether, this information will allow the genetic dissection of the characters involved in the domestication and adaptation of the crop, and their further implementation in breeding strategies for this important crop.

Expression of Fluorescent Genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): Its Application to Parasite-Vector Biology

Abstract Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green fluorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of fluorescent proteins. Tagged flagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its fluorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasites.

Integration of expression vectors into the ribosomal locus of Trypanosoma cruzi.

The expression vectors of the protozoan parasite Trypanosoma cruzi pRIBOTEX and pTREX harbor a ribosomal promoter that improves gene expression and clone selection. Interestingly, the solely presence of this 810 bp long sequence leads to the integration of these vectors into the ribosomal locus, even though circular plasmids are poorly recombinogenic. Initially, it was suggested that a 174 bp long ribosomal-specific repeat element present in the ribosomal promoter region could be responsible for the genetic exchange. On the contrary, we demonstrate that recombination of pTREX occurs within a 86 bp long region located 120 bp downstream the transcription start point (tsp1) of the ribosomal promoter, and it does not depend on the presence of the ribosomal repeat. We also determined that a 291 bp segment encompassing the tsp1 and the 86 bp long recombination region contains all necessary signals to drive transcription and complete recombination into the rRNA locus. Finally, we demonstrate that the integration of pTREX derived plasmids into the nuclear genome occurs within the first 5 h post-transfection, and that non-integrated copies are rapidly degraded.

Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea

Arsenic metabolism is proposed to be an ancient mechanism in microbial life. Different bacteria and archaea use detoxification processes to grow under high arsenic concentration. Some of them are also able to use arsenic as a bioenergetic substrate in either anaerobic arsenate respiration or chemolithotrophic growth on arsenite. However, among the archaea, bioenergetic arsenic metabolism has only been found in the Crenarchaeota phylum. Here we report the discovery of haloarchaea (Euryarchaeota phylum) biofilms forming under the extreme environmental conditions such as high salinity, pH and arsenic concentration at 4589 m above sea level inside a volcano crater in Diamante Lake, Argentina. Metagenomic analyses revealed a surprisingly high abundance of genes used for arsenite oxidation (aioBA) and respiratory arsenate reduction (arrCBA) suggesting that these haloarchaea use arsenic compounds as bioenergetics substrates. We showed that several haloarchaea species, not only from this study, have all genes required for these bioenergetic processes. The phylogenetic analysis of aioA showed that haloarchaea sequences cluster in a novel and monophyletic group, suggesting that the origin of arsenic metabolism in haloarchaea is ancient. Our results also suggest that arsenite chemolithotrophy likely emerged within the archaeal lineage. Our results give a broad new perspective on the haloarchaea metabolism and shed light on the evolutionary history of arsenic bioenergetics.

Whole Genome Sequencing Reveals a De Novo SHANK3 Mutation in Familial Autism Spectrum Disorder

Introduction Clinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD). Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS) for the diagnostic approach to ASD. Methods We identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents. Results Three male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon (NM_033517:c.3259_3259delC, p.Ser1088Profs*6). Conclusions We reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder.

Genome Sequence of Sphingomonas sp. S17, Isolated from an Alkaline, Hyperarsenic, and Hypersaline Volcano-Associated Lake at High Altitude in the Argentinean Puna

The high-altitude Andean lakes (HAAL) in the Argentinean Puna-high Andes region represent an almost unexplored ecosystem exposed to extreme conditions (high UV irradiation, hypersalinity, drastic temperature changes, desiccation, and high pH). Here we present the first genome sequence, a Sphingomonas sp., isolated from this extreme environment.