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1. Synthetic multi-antibiotic resistant plasmids in plant-associated bacteria from agricultural soils

Author

Silvina Brambilla, Romina Frare, Margarita Stritzler, Gabriela Soto, Carolina Berini, Cintia Jozefkowicz, and Nicolás Ayub

Subjects
Recombinant, GMO, GMM, Antibiotic resistance, Multiple cloning sites, β-galactosidase, Microbiology, QR1-502
Abstract

Objectives: Unlike higher organisms such as domestic animals and cultivated plants, which display a robust reproductive isolation and limited dispersal ability, microbes exhibit an extremely promiscuous gene flow and can rapidly disperse across the planet by multiple ways. Thus, microbial plasmids, including synthetic replicons, containing antibiotic resistance genes are a serious risk to public health. In this short communication, we explored the presence of synthetic elements in alfalfa symbionts (Ensifer meliloti strains) from agricultural soils. Methods: A total of 148 E. meliloti isolates from alfalfa plants growing under field conditions were collected from January 2015 to June 2019. Antimicrobial susceptibility testing was performed under laboratory conditions. We identified five kanamycin-resistant E. meliloti strains (named K1-K5). Whole genome sequencing analysis and conjugations were used to identify and study the plasmids of K strains. Results: We found that the genomes of K strains contain ampicillin, kanamycin and tetracycline resistance genes, the reporter gene lacZ from Escherichia coli and multiple cloning sites. These sequences were found within

Published
2020
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2. Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa

Author

Cintia Jozefkowicz, Cristina Gómez, Ariel Odorizzi, Anelia Iantcheva, Pascal Ratet, Nicolás Ayub, and Gabriela Soto

Subjects
transformation, Tnt1, dominant mutation, polyploidy, alfalfa, Plant culture, SB1-1110
Abstract

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

Published
2021
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3. Loop A is critical for the functional interaction of two Beta vulgaris PIP aquaporins.

Author

Cintia Jozefkowicz, Pablo Rosi, Lorena Sigaut, Gabriela Soto, Lía Isabel Pietrasanta, Gabriela Amodeo, and Karina Alleva

Subjects
Medicine, Science
Abstract

Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties.

Published
2013
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4. Maximizing the expression of transgenic traits into elite alfalfa germplasm using a supertransgene configuration in heterozygous conditions

Author

Cintia Jozefkowicz, Elba Pagano, Ariel Odorizzi, Gabriela Soto, Nicolás Daniel Ayub, Romina Fox, Romina Frare, Daniel Horacio Basigalup, and Valeria Arolfo

Subjects
Crops, Agricultural, 0106 biological sciences, 0301 basic medicine, Germplasm, Heterozygote, Transgene, Introgression, ALFALFA, Outcrossing, Forage, Biology, 01 natural sciences, Ciencias Biológicas, 03 medical and health sciences, Polyploid, Genetics, Transgenes, Cultivar, Legume, fungi, food and beverages, General Medicine, Bioquímica y Biología Molecular, Plants, Genetically Modified, 030104 developmental biology, Agronomy, GERMPLASM, TRANSGENIC, Genetic Engineering, Agronomy and Crop Science, CIENCIAS NATURALES Y EXACTAS, Herbicide Resistance, Medicago sativa, 010606 plant biology & botany, Biotechnology
Abstract

Numerous species of legumes, including alfalfa, are critical factors for agroecosystems due to their ability to grow without nitrogen fertilizers derived from non-renewable fossil fuels, their contribution of organic nitrogen to the soil, and their increased nutritional value. Alfalfa is the main source of vegetable proteins in meat and milk production systems worldwide. Despite the economic and ecological importance of this autotetraploid and allogamous forage crop, little progress has been made in the incorporation of transgenic traits into commercial alfalfa. This is mainly due to the unusually strong transgene silencing and complex reproductive behavior of alfalfa, which limit the production of events with high transgene expression and the introgression of selected events within heterogeneous synthetic populations, respectively. In this report, we describe a novel procedure, called supertransgene process, where a glufosinate-tolerant alfalfa variety was developed using a single event containing the BAR transgene associated with an inversion. This approach can be used to maximize the expression of transgenic traits into elite alfalfa germplasm and to reduce the cost of production of transgenic alfalfa cultivars, contributing to the public improvement of this legume forage and other polyploid and outcrossing crop species. Fil: Jozefkowicz, Cintia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Frare, Romina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Odorizzi, Ariel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Manfredi; Argentina Fil: Arolfo, Valeria. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Manfredi; Argentina Fil: Pagano, Elba. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Basigalup, Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Manfredi; Argentina Fil: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Published
2018
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5. Stable symbiotic nitrogen fixation under water-deficit field conditions by a stress-tolerant alfalfa microsymbiont and its complete genome sequence

Author

Silvina Maricel Brambilla, Margarita Stritzler, Carlos Fabian Piccinetti, Gabriela Soto, Cintia Jozefkowicz, Carolina Berini, Nicolás Daniel Ayub, Romina Frare, Mariana Puente, and Pedro Reyes Pérez

Subjects
0301 basic medicine, SINORHIZOBIUM MELILOTI, 030106 microbiology, Biotecnología del Medio Ambiente, ALFALFA, Bioengineering, INGENIERÍAS Y TECNOLOGÍAS, Applied Microbiology and Biotechnology, Genome, GLYCINE BETAINE, 03 medical and health sciences, chemistry.chemical_compound, Betaine, Biosynthesis, Nitrogen Fixation, Botany, Symbiosis, Bioremediación, Diagnóstico Biotecnológico en Gestión Medioambiental, Sinorhizobium meliloti, biology, COMPLETE GENOME, food and beverages, Genomics, General Medicine, biology.organism_classification, Adaptation, Physiological, Droughts, 030104 developmental biology, chemistry, Biochemistry, Glycine, Nitrogen fixation, Osmoprotectant, Genome, Bacterial, Bacteria, Medicago sativa, Biotechnology
Abstract

We here characterized the stress-tolerant alfalfa microsymbiont Sinorhizobium meliloti B401. B401-treated plants showed high nitrogen fixation rates under humid and semiarid environments. The production of glycine betaine in isolated bacteroids positively correlated with low precipitation levels, suggesting that this compound acts as a critical osmoprotectant under field conditions. Genome analysis revealed that strain B401 contains alternative pathways for the biosynthesis and uptake of glycine betaine and its precursors. Such genomic information will offer substantial insight into the environmental physiology of this biotechnologically valuable nitrogen-fixingbacterium. Fil: Jozefkowicz, Cintia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Brambilla, Silvina Maricel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Frare, Romina Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Stritzler, Margarita. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Piccinetti, Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Puente, Mariana L. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Berini, Carolina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Pérez, Pedro Reyes. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Published
2017

6. Prediction of aquaporin function by integrating evolutionary and functional analyses

Author

Cintia Jozefkowicz, Juliana Andrea Pérez Di Giorgio, Karina Alleva, Gabriela Soto, Gabriela Amodeo, Jorge P Muschietti, and Nicolás Daniel Ayub

Subjects
Transport water, CIENCIAS MÉDICAS Y DE LA SALUD, Protein Conformation, Physiology, Biophysics, Aquaporin, Computational biology, Biology, Aquaporins, Bioinformatics, Fisiología, AQUAPORIN, Evolution, Molecular, Ciencias Biológicas, Phylogenetics, FUNCTIONAL ANALYSIS, Animals, Humans, Protein Interaction Domains and Motifs, Gene, Phylogeny, Bacteria, Phylogenetic tree, Eukaryota, Biological Transport, Cell Biology, Human physiology, Plants, Bioquímica y Biología Molecular, Medicina Básica, Multigene Family, Community perception, FUNCTION PREDICTION, EVOLUTIONARY ANALYSIS, Function (biology), CIENCIAS NATURALES Y EXACTAS
Abstract

Aquaporins (AQPs) are a family of channel proteins that transport water and/or small solutes across cell membranes. Aquaporins are present in Bacteria, Eukarya and Archaea. The classical AQP evolution 47 paradigm explains the inconsistent phylogenetic trees by multiple transfer events and emphasizes that the assignment of orthologous AQPs is not possible, making it difficult to integrate functional information. Recently, a novel phylogenetic framework of eukaryotic AQP evolution showed congruence between eukaryotic AQPs and organismal trees identifying 32 orthologous clusters in plants and animals (Gene 2012 503:165-176). In this article, we discuss in depth the methodological strength, the ability to predict functionality and the aquaporin community perception about the different paradigms of AQP evolution. Moreover, we show an updated review of AQPs transport functions in association with phylogenetic analyses. Finally, we discuss the possible effect of aquaporin data integration in the understanding of water and solute transport in eukaryotic cells. Fil: Pérez, Juliana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina Fil: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Alleva, Karina Edith. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biologia Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Jozefkowicz, Cintia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biologia Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Amodeo, Gabriela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biologia Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Muschietti, Jorge Prometeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Ayub, Nicolas Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Published
2013
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7. Intracellular pH sensing is altered by plasma membrane PIP aquaporin co-expression

Author

Gabriela Amodeo, Karina Alleva, Cintia Jozefkowicz, Gabriela Soto, Victoria Andrea Vitali, and Jorge Bellati

Subjects
Intracellular Fluid, Whole membrane, Cell Membrane Permeability, DNA, Plant, Intracellular pH, Molecular Sequence Data, Gene Expression, Aquaporin, Plant Science, Gating, In Vitro Techniques, Biology, Aquaporins, Genes, Plant, Plant Roots, AQUAPORIN GATING, Ciencias Biológicas, Xenopus laevis, Genetics, Animals, Amino Acid Sequence, pH SENSING, Phylogeny, DNA Primers, Plant Proteins, Water transport, Base Sequence, Sequence Homology, Amino Acid, PLASMA MEMBRANE INTRINSIC PROTEIN, CYTOSOLIC ACIDIFICATION, Water, General Medicine, Hydrogen-Ion Concentration, Permeation, Biofísica, Recombinant Proteins, Cytosol, Membrane, Biochemistry, Oocytes, Biophysics, Female, Beta vulgaris, Agronomy and Crop Science, CIENCIAS NATURALES Y EXACTAS
Abstract

The plant plasma membrane barrier can express aquaporins (PIP1 and PIP2) that show two intriguing aspects: (1) the potential of modulating whole membrane water permeability by co-expression of both types, which have recently been distinguished for showing a different capacity to reach the plasma membrane; and (2) the faculty to reduce water permeation through the pore after cytosolic acidification, as a consequence of a gating process. Our working hypothesis is that these two key features might enhance plasticity of the membrane water transport capacity if they jointly trigger any cooperative interaction. In previous work, we proved by biophysical approaches that the plasma membrane of the halophyte Beta vulgaris storage root presents highly permeable aquaporins that can be shut down by acidic pH. Root Beta vulgaris PIPs were therefore subcloned and expressed in Xenopus oocytes. Co-expression of BvPIP1;1 and BvPIP2;2 not only enhances oocyte plasma membrane water permeability synergistically but also reinforces pH inhibitory response from partial to complete shut down after cytosolic pH acidifi- cation. This pH dependent behavior shows that PIP1–PIP2 co-expression accounts for a different pH sensitivity in comparison with PIP2 expression. These results prove for the first time that PIP co-expression modulates the membrane water permeability through a pH regulatory response, enhancing in this way membrane versatility to adjust its water transfer capacity. Fil: Bellati, Jorge. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alleva, Karina Edith. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular "dr. Hector N Torres"; Argentina Fil: Vitali, Victoria Andrea. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jozefkowicz, Cintia. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Amodeo, Gabriela. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Published
2010
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