For instance, MP (L5C1) cross-reacted with anti-CKB (L6C1), both members of the Rhodocyclales family, and also with the antibody produced against the sp. method based on graph theory to discriminate between specific signals and cross-reactions from related microorganisms. We validated the system by analyzing multiple bacterial isolates, crude extracts from contaminated reactors and salt-rich natural samples from the high Arctic. The PRB detecting chip (PRBCHIP) allowed us to detect and classify environmental isolates as well as to detect similar strains by using crude extracts obtained from 0.5 g even from soils with low organic-matter levels (<103 cells/g of soil). Our results demonstrated that PRBCHIP is a valuable tool for sensitive and reliable detection of perchlorate-reducing bacteria for research purposes, environmental monitoring and planetary exploration. Keywords: perchlorate, perchlorate-reducing bacteria, antibody microarrays, biochip, life detection, planetary exploration, Mars, graph theory Introduction Perchlorate is a stable and soluble toxic anion that is found in the environment from natural and anthropogenic sources (Kounaves et al., 2010). As it accumulates in the food chain (Kirk et al., 2005; Snchez et al., 2005), it represents a health risk to humans by interfering with thyroid function JNJ-10397049 upon ingestion (Lawrence et al., 2009). On the other hand, natural perchlorate salts have been detected on distinct arid regions as well as on the surface of Mars at relatively high (0.4C0.6 wt%) concentrations (Klein, 1974; Hoffman et al., 2008; Hecht et al., 2009; Glavin et al., 2013; Freissinet et al., 2015). The study of perchlorate metabolism has advanced significantly in a short time, revealing that perchlorate respiration is environmentally ubiquitous and widespread in bacteria and archaea domains (Coates and Achenbach, 2004). Perchlorate-reducing bacteria (PRB) are a phylogenetically diverse group of microorganisms capable of growth ILKAP antibody by respiring perchlorate as the sole electron acceptor (Youngblut et al., 2016). These dissimilatory perchlorate-reducing bacteria reduce perchlorate to chlorate which is further reduced to chlorite by means of the perchlorate reductase enzyme in the periplasmic compartment. Then, a chlorite dismutase converts chlorite completely to chloride ion with the release of molecular oxygen that can be utilized by a membrane-bound respiratory oxidase (Youngblut et al., 2016). In fact, the removal of perchlorate by means of microbial reduction has been identified as the most efficient method of removing JNJ-10397049 this harmful substance from contaminated environments (Coates and Achenbach, 2004; Hatzinger, 2005; Gu and Coates, 2006; Coates and Jackson, 2009). Bacterial bioremediation of perchlorate-contaminated water is a viable treatment option, which has spurred both applied (Urbanskya and Schock, 1999; Hatzinger, 2005) and basic (Coates and Achenbach, 2004) science research. As a consequence, PRB have been isolated from a variety of habitats ranging from pristine areas to anthropogenic-contaminated sites (Coates et al., 1999). Phenotypic characterization studies have demonstrated that the known perchlorate-reducing bacteria exhibit a broad range of metabolic capabilities and can thrive in adverse environments such as the Atacama desert, the high Artic and the cold deserts in Antarctica (Wynn-Williams and Edwards, 2000; Parro et al., 2011a; Lay et al., 2013; Youngblut et al., 2016). Bender et al. (2004) developed two degenerate primer sets targeting the chlorite dismutase (for 16 min at 4C. The supernatant was JNJ-10397049 discharged and the resulting pellet suspended in 0.4 mL phosphate buffer (pH = 7.2) and transferred to a 2 mL tube. One mL of isopropanol was added to re-precipitate and concentrate the EPS fraction. Tubes were stored at ?20C for 10 days. The EPS collected by centrifugation at 6,000 for 17 min at 4C and then dried under a flow of nitrogen. TABLE 1 List of the strains and immunogens used to produce the antibodies. PSBeta-A/21<103<103L2C1VDYAlpha-A/21<102<102L3C1LT-1Beta-A/4<103<104L5C1MPBeta-A/21<103<103L6C1CKBBeta-A/5<103<10L7C1sp. WDAlpha-A/4<105<105L8C1sp. ZAPBeta-A/21<103<103L9C1NSSGamma-B/21<104<105L11C1sp. PS EPSBeta-A/21<104<105L2S2VDY EPSAlpha-A/21CCL3S2EPSBeta-A/21CCL4S2LT-1 EPSBeta-A/4<103<104L6S2CKB EPSBeta-A/5<102<10L8S2sp. ZAP EPSBeta-A/21<103<103L11S2EPSBeta-A/4<105<105L12S2sp. EPSEpsilon-C/78<106C Open in a separate window nodes (number of antibodies represented in the microarray) and links (number of cross-reactions) and has an associated matrix of size The methodology to calculate the antibody graph associated with the PRBCHIP from the cross-reactivity matrix obtained after testing one by one all the pairs antibody-immunogen is described in Rivas et al. (2011). The information embedded in the antibody graph.