Marcos J. Maldonado. 1. , Gabriela M. Ferrer. 1. , Virginia H. Albarracin. 1,2. , Marta F. Gorriti. 1. , Fabiano Thompson. 3. , Nicolás Rascovan. 4. , Santiago Revale.
IDENTIFICATION OF GENES AND PROTEINS INVOLVED IN THE RESPONSE TO ARSENIC STRESS IN SEQUENCED STRAINS FROM HIGH ALTITUDE ANDEAN LAKES (HAAL) Omar F. Ordoñez1, Daniel G. Kurth1, Marcos J. Maldonado1, Gabriela M. Ferrer1, Virginia H. Albarracin1,2, Marta F. Gorriti1, Fabiano Thompson3, Nicolás Rascovan4, Santiago Revale4, Martin P. Vazquez4, Esteban Lanzarotti5,6, Adrian G. Turjanski5,6, María E. Farías1 and Néstor Cortez7. 1
Laboratorio de Investigaciones Microbiológicas de Lagunas Andinas (LIMLA), Planta Piloto de Procesos Industriales Microbiológicos (PROIMI), CCT-CONICET, Tucumán, Argentina. 2 Facultad de Ciencias Naturales e Instituto Miguel Lillo, Universidad Nacional de Tucumán, Tucumán, Argentina. 3 Laboratorio de Microbiología, Instituto de Biología. Universidad Federal de Rio de Janeiro (UFRJ), Rio de Janeiro, Brasil. 4 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina. Instituto de Agrobiotecnología Rosario (INDEAR)-CONICET, Ocampo 210 bis, CCT-CONICET, Rosario, Argentina. 5 Departamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires, C1428EHA, Argentina. 6 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires, C1428EHA, Argentina. 7 Instituto de Biología. Molecular y Celular de Rosario (IBR-CONICET), Facultad de Cs. Bioquímicas y Farmacéuticas (Universidad Nacional de Rosario), Suipacha 531, S2002LRK-Rosario, Argentina.
Abstract Arsenic (As) is one of the most important global environmental pollutants and a persistent bioaccumulative carcinogen. As an ubiquitous toxic metalloid it was released in the environment mainly by volcanic activity. The ars gene system can be chromosomal or plasmid-borne and provides arsenic resistance to a variety of microorganisms. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is scavenged from the cell by extrusion pumps such as the arsB gene. The less studied gene ACR3 codify for an extrusion pump (ACR3) which ejects specifically arsenite out of the cell, providing high resistance towards As[III] to the microorganisms expressing it. The high concentration of arsenic present in HAAL is strongly limiting not only for human life but also for growth of many microorganisms, and favored evolution of arsenic tolerant bacteria. Maximal arsenic concentration observed in these environments was 33.81 mg/L. Genomes of several microorganisms isolated from the HAAL have been sequenced recently. We present here the identification of genes involved in the response to arsenic stress in these HAAL strains. The effect of As [V] and As [III] during isolates growth was also evaluated. The strains Acinetobacter sp. Ver3, Exiguobacterium sp. N30 and S17, Salinivibrio sp N34, N35 and S10B, Nesterenkonia sp Act20, and Halorubrum sp. AJ67 were selected for this study. Genome sequences were obtained using a whole-genome shotgun strategy with a 454 GS Titanium pyrosequencer (INDEAR, Argentina) and Ion Torrent-based genome sequencing (UFRJ, Brazil). Final genome sequences were annotated and analyzed in the RAST annotation server. PSI-BLAST and ClustalW were used to compare and align sequences, and phylogenetic trees were built using Mega5. Genetic analysis and physiological characterization of resistance to arsenic was performed and the presence of the genes encoding the arsenite detoxification machinery (ars genes) was
observed in all sequenced strains. Although these microorganisms showed high tolerance to As (V), the response to As (III) was more diverse, as Exiguobacterium sp. S17 was the only strain able to grow at As concentrations above 5 mM. Two types of arsenic extrusion pumps were observed: ACR3 and ArsB. ACR3 gene was more widely distributed in the genomes under study. HAAL sequenced strains show enhanced resistance compared to other bacteria carrying the ars operon. This could be explained by the presence of additional genes related to this function, including extra copies of the ars operon or supplementary extrusion pumps. Besides basic knowledge about structure and molecular mechanisms of metal extrusion pumps, the analysis of sequenced genomes of microorganisms displaying high tolerance to arsenic, could give information useful for studies on bioremediation of metals and metalloids, a methodology considered of low cost and environmentally friendly.
