Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

<p>Abstract</p> <p>Background</p> <p>Group II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP) assists the splicing of the intron <it>in vivo </it>and remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP) that promotes intron mobility. Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA)] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC))] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ') in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron.</p> <p>Results</p> <p>In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 <it>in vivo</it>. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision <it>in vivo.</it></p> <p>Conclusions</p> <p>The excision <it>in vivo </it>of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.</p

Has cupper ion any effect on Sinorhizobium Melitoili bacteria?

Proceedings of the I Congress PIIISA celebrado en la Estación Experimental del Zaidín, Granada, el 26 de abril de 2012.Sinorhizobium meliloti is a soil bacterium that in symbiosis with Alfalfa leguminous plants fixes N2 to ammonia. This process occurs after an intimate communication between host and microsimbiont in the rhizosphere niche. This soil bacterium may suffer biotic and abiotic stresses in this complex environment. Resistance to any of this stresses can be an advantage for this type of bacteria. In this work we test the effects of Cupper ion in the bacterium Sinorhizobium meliloti. We found an effect on pigmentation and this effect is specific of each isolate (i.e. it is very pronounced for the isolate AK83 from Aral sea in Russia). On the other hand, comparison of Cupper ion resistance phenotype between isolates 1021 and GR4 suggests that the resistance of GR4 could be due to a set of 10 genes found in the cryptic plasmid pRmeGR4a of this strain. However, this resistance phenotype also appears in other isolates, and it could be explained by an alternative phenotype as Exopolysaccaride production (EPS+).It was supported mainly by research project MICINN Consolider‐Ingenio 2010. CSD2009‐00006; Scientific research by Young Students in Science (PIIISA2012; http://emc2astronomy.blogspot.com/).Peer reviewe

Complete genome sequence of Mesorhizobium ciceri strain R30, a Rhizobium used as a commercial inoculant for Chickpea in Argentina

We report the complete genome sequence of Mesorhizobium ciceri strain R30, a rhizobium strain recommended and used as a commercial inoculant for chickpea in Argentina. The genome consists of almost 7 Mb, distributed into two circular replicons: a chromosome of 6.49 Mb and a plasmid of 0.46 Mb.This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (PID2020-113207GBI00 funded by MCIN/AEI/10.13039/501100011033); by “ERDF: A Way of Making Europe” (P20_0047), funded by the Junta de Andalucía PAIDI/FEDER/EU; and by the Biotechnology and Biosciences Research Council (BBSRC). We are grateful to Plateforme de Microbiologie Mutualisée (P2M) and the Pasteur International Bioresources network (PIBnet) and to Institut Pasteur Paris for providing the resources for Illumina sequencing. We thank O.G.C. at the Wellcome Centre for Human Genetics for the sequencing data and B.M.R.C. for processing (supported by Wellcome Trust Core Award grant 203141/Z/16/Z and the NIHR Oxford BRC). We are also grateful to Vincent Enouf from Unité de Génétique Moléculaire des Virus à ARN-UMR3569 CNRS, Université de Paris, Centre National de Référence Virus des Infections Respiratoires (dont la grippe) and to F. Sgarlatta for proofreading the manuscript

Characterisation of symbiotically efficient alfalfa-nodulating rhizobia isolated from acid soils of Argentina and Uruguay

The diversity, growth and symbiotic behaviour of symbiotically efficient alfalfa-nodulating rhizobia isolated from acid soils of Argentina and Uruguay were analysed. Partial sequencing of the 16S rDNA indicated that these isolates belong to Sinorhizobium meliloti species. IS-fingerprinting analysis revealed a high diversity among the isolates but some of them appear related to inoculant strains currently used in the region. The S. meliloti isolates showed a decreased growth rate with increasing acidity. They were, however, able to nodulate alfalfa at pH 5.6, but showed a delayed nodulation and decreased nodule number typical of S. meliloti strains. The impaired nodulation of S. meliloti at pH 5.6 did not result in a reduction of alfalfa dry matter production or nitrogen content. However, significant differences were observed for the relative symbiotic effectiveness of the strains analysed. LPU63 (Argentina) was the most effective among the isolates and exhibited a high nodulation competitiveness at both neutral and acidic pH. These results suggest that the isolate LPU63 may be a potential efficient inoculant for alfalfa in acid soils.Instituto de Biotecnología y Biología Molecula

Identification and characterization of a nodH ortholog from the alfalfa-nodulating Or191-like rhizobia

Nodulation of Medicago sativa (alfalfa) is known to be restricted to Sinorhizobium meliloti and a few other rhizobia that include the poorly characterized isolates related to Rhizobium sp. strain Or191. Distinctive features of the symbiosis between alfalfa and S. meliloti are the marked specificity from the plant to the bacteria and the strict requirement for the presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its reducing end. Here, we present evidence of the presence of a functional nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene, present in single copy, maps to a high molecular weight megaplasmid. As in S. meliloti, a nodF homolog was identified immediately upstream of nodH that was transcribed in the opposite direction (local synteny). This novel nodH ortholog was cloned and shown to restore both NF sulfation and the Nif +Fix+ phenotypes when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH disruption in the Or191-like bacteria did not abolish their ability to nodulate alfalfa, resulting instead in a severely delayed nodulation. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor.Instituto de Biotecnologia y Biologia Molecula

GPD1 and ADH3 Natural Variants Underlie Glycerol Yield Differences in Wine Fermentation

Glycerol is one of the most important by-products of alcohol fermentation, and depending on its concentration it can contribute to wine flavor intensity and aroma volatility. Here, we evaluated the potential of utilizing the natural genetic variation of non-coding regions in budding yeast to identify allelic variants that could modulate glycerol phenotype during wine fermentation. For this we utilized four Saccharomyces cerevisiae strains (WE - Wine/European, SA – Sake, NA – North American, and WA – West African), which were previously profiled for genome-wide Allele Specific Expression (ASE) levels. The glycerol yields under Synthetic Wine Must (SWM) fermentations differed significantly between strains; WA produced the highest glycerol yields while SA produced the lowest yields. Subsequently, from our ASE database, we identified two candidate genes involved in alcoholic fermentation pathways, ADH3 and GPD1, exhibiting significant expression differences between strains. A reciprocal hemizygosity assay demonstrated that hemizygotes expressing GPD1WA, GPD1SA, ADH3WA and ADH3SA alleles had significantly greater glycerol yields compared to GPD1WE and ADH3WE. We further analyzed the gene expression profiles for each GPD1 variant under SWM, demonstrating that the expression of GPD1WE occurred earlier and was greater compared to the other alleles. This result indicates that the level, timing, and condition of expression differ between regulatory regions in the various genetic backgrounds. Furthermore, promoter allele swapping demonstrated that these allele expression patterns were transposable across genetic backgrounds; however, glycerol yields did not differ between wild type and modified strains, suggesting a strong trans effect on GPD1 gene expression. In this line, Gpd1 protein levels in parental strains, particularly Gpd1pWE, did not necessarily correlate with gene expression differences, but rather with glycerol yield where low Gpd1pWE levels were detected. This suggests that GPD1WE is influenced by recessive negative post-transcriptional regulation which is absent in the other genetic backgrounds. This dissection of regulatory mechanisms in GPD1 allelic variants demonstrates the potential to exploit natural alleles to improve glycerol production in wine fermentation and highlights the difficulties of trait improvement due to alternative trans-regulation and gene-gene interactions in the different genetic background

Localization of a bacterial group II intron-encoded protein in human cells

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.This work was supported by research grants CSD 2009–0006 from the Consolider-Ingenio, BIO2011-24401 and BIO2014-51953-P from the Spanish Ministerio de Economía y Competitividad all including ERDF (European Regional Development Funds). We thank Dr. Antonio Barrientos Durán for technical advice. MRC was supported by an FPI Ph.D grant. J.L.G.P´s laboratory is supported by CICE-FEDER-P09-CTS-4980, CICE-FEDER-P12-CTS-2256, Plan Nacional de I+D+I 2008–2011 and 2013–2016 (FIS-FEDER-PI11/01489 and FIS-FEDER-PI14/02152), PCIN-2014-115-ERA-NET NEURON II, the European Research Council (ERC-Consolidator ERC-STG-2012-233764) and by an International Early Career Scientist grant from the Howard Hughes Medical Institute (IECS-55007420).Peer Reviewe

Six-year follow-up of slaughterhouse surveillance (2008-2013): the Catalan Slaughterhouse Support Network (SESC)

Meat inspection has the ultimate objective of declaring the meat and offal obtained from carcasses of slaughtered animals fit or unfit for human consumption. This safeguards the health of consumers by ensuring that the food coming from these establishments poses no risk to public health. Concomitantly, it contributes to animal disease surveillance. The Catalan Public Health Protection Agency (Generalitat de Catalunya) identified the need to provide its meat inspectors with a support structure to improve diagnostic capacity: the Slaughterhouse Support Network (SESC). The main goal of the SESC was to offer continuing education to meat inspectors to improve the diagnostic capacity for lesions observed in slaughterhouses. With this aim, a web-based application was designed that allowed meat inspectors to submit their inquiries, images of the lesions, and samples for laboratory analysis. This commentary reviews the cases from the first 6 years of SESC operation (2008–2013). The program not only provides continuing education to inspectors but also contributes to the collection of useful information on animal health and welfare. Therefore, SESC complements animal disease surveillance programs, such as those for tuberculosis, bovine cysticercosis, and porcine trichinellosis, and is a powerful tool for early detection of emerging animal diseases and zoonoses

A Model for the Development of the Rhizobial and Arbuscular Mycorrhizal Symbioses in Legumes and Its Use to Understand the Roles of Ethylene in the Establishment of these two Symbioses

We propose a model depicting the development of nodulation and arbuscular mycorrhizae. Both processes are dissected into many steps, using Pisum sativum L. nodulation mutants as a guideline. For nodulation, we distinguish two main developmental programs, one epidermal and one cortical. Whereas Nod factors alone affect the cortical program, bacteria are required to trigger the epidermal events. We propose that the two programs of the rhizobial symbiosis evolved separately and that, over time, they came to function together. The distinction between these two programs does not exist for arbuscular mycorrhizae development despite events occurring in both root tissues. Mutations that affect both symbioses are restricted to the epidermal program. We propose here sites of action and potential roles for ethylene during the formation of the two symbioses with a specific hypothesis for nodule organogenesis. Assuming the epidermis does not make ethylene, the microsymbionts probably first encounter a regulatory level of ethylene at the epidermis–outermost cortical cell layer interface. Depending on the hormone concentrations there, infection will either progress or be blocked. In the former case, ethylene affects the cortex cytoskeleton, allowing reorganization that facilitates infection; in the latter case, ethylene acts on several enzymes that interfere with infection thread growth, causing it to abort. Throughout this review, the difficulty of generalizing the roles of ethylene is emphasized and numerous examples are given to demonstrate the diversity that exists in plants

Identification and characterization of a nodH ortholog from the alfalfa-nodulating Or191-like rhizobia

Nodulation of Medicago sativa (alfalfa) is known to be restricted to Sinorhizobium meliloti and a few other rhizobia that include the poorly characterized isolates related to Rhizobium sp. strain Or191. Distinctive features of the symbiosis between alfalfa and S. meliloti are the marked specificity from the plant to the bacteria and the strict requirement for the presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its reducing end. Here, we present evidence of the presence of a functional nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene, present in single copy, maps to a high molecular weight megaplasmid. As in S. meliloti, a nodF homolog was identified immediately upstream of nodH that was transcribed in the opposite direction (local synteny). This novel nodH ortholog was cloned and shown to restore both NF sulfation and the Nif +Fix+ phenotypes when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH disruption in the Or191-like bacteria did not abolish their ability to nodulate alfalfa, resulting instead in a severely delayed nodulation. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor.Instituto de Biotecnologia y Biologia Molecula