Cd 2+ resistance mechanisms in Methanosarcina acetivorans involve the increase in the coenzyme M content and induction of biofilm synthesis Elizabeth Lira-Silva, 1 M. Geovanni Santiago-Martínez, 1 Rodolfo García-Contreras, 1 Armando Zepeda-Rodríguez, 2 Alvaro Marín-Hernández, 1 Rafael Moreno-Sánchez 1 and Ricardo Jasso-Chávez 1 * 1 Departamento de Bioquímica, Instituto Nacional de Cardiología, Mexico City, Mexico. 2 Facultad de Medicina, UNAM, Mexico City, Mexico. Summary To assess what defence mechanisms are triggered by Cd 2+ stress in Methanosarcina acetivorans, cells were cultured at different cadmium concentrations. In the presence of 100 μM CdCl2, the intracellular contents of cysteine, sulfide and coenzyme M increased, respec- tively, 8, 27 and 7 times versus control. Cells incubated for 24 h in medium with less cysteine and sulfide removed up to 80% of Cd 2+ added, whereas their cysteine and coenzyme M contents increased 160 and 84 times respectively. Cadmium accumulation (5.2 μmol/10–15 mg protein) resulted in an increase in methane synthesis of 4.5 times in cells grown on acetate. Total phosphate also increased under high (0.5 mM) Cd 2+ stress. On the other hand, cells preadapted to 54 μM CdCl2 and further exposed to > 0.63 mM CdCl2 developed the formation of a biofilm with an extracellular matrix constituted by carbohy- drates, DNA and proteins. Biofilm cells were able to synthesize methane. The data suggested that increased intracellular contents of thiol molecules and total phosphate, and biofilm formation, are all involved in the cadmium resistance mechanisms in this marine archaeon. Introduction Organisms respond to heavy metal toxicity through differ- ent biochemical mechanisms, such as decreased uptake, active expulsion, chelation and compartmentalization of metal ions (Cervantes et al., 2006; Haferburg and Kothe, 2007). Thus, control of uptake, trafficking and detoxifica- tion of heavy metals are important steps in the complex cellular mechanisms that deal with heavy metal toxicity and confer resistance. Chelators contribute to metal detoxifica- tion by buffering cytosolic metal ions. In plants, physiologi- cal metal chelators include thiol-containing compounds, such as cysteine (Cys), glutathione (GSH), phytochelatins (PCs) and metallothioneins, and organic acids and amino acids (Clemens, 2001). Cadmium is a highly toxic non-essential heavy metal that has become a serious contaminant of soils and water bodies, mainly due to anthropogenic activity and inappro- priate methods of disposal (Simpson, 1981). This metal strongly interacts with S, O, and N atoms in biomolecules (Sillen and Martell, 1964). A widespread mechanism for Cd 2+ detoxification in plants and yeast is based on the sequestration of Cd 2+ by PCs and the subsequent compartmentalization into the vacuole of the PCs-Cd 2+ complexes (Cobbett, 2000). Synthesis of PCs, and enhanced synthesis of their metabolic precursors Cys, γ-glutamylcysteine (γ-EC), GSH and sulfide (S 2− ), is a commonly used cellular response against Cd 2+ stress in higher plants, some yeast and algae (for review, see Mendoza-Cózatl et al., 2005). In the Archaea domain, progress has been made on the mechanisms of resistance to heavy metals at the gene expression level (Villafane et al., 2011; Maezato et al., 2012; Orell et al., 2013), but studies on the characterization of the enzymatic activities and biochemical processes involved are scarce. Some gene sequences encoding heavy metal-exporting proteins putatively involved in the efflux-mediated heavy metal resistance, and many P-type ATPases, have been found in archaeal genomes (Dopson et al., 2003), although no activity of these proteins has been determined as yet. In contrast, activities of P-type Ag + and Cu 2+ ATPases (Mandal et al., 2002) and the formation of biofilms con- stituted mainly by carbohydrates, DNA and proteins induced by Cr (VI) and Cu 2+ have been reported for the extremophile Archaeoglobus fulgidus (Lapaglia and Hartzell, 1997). In acetoclastic methanogens, studies on Cu 2+ toxicity have been carried out for consortia in digesters or eco- logical niches (Karri et al., 2006), and the formation of Received 8 April, 2013; accepted 22 June, 2013. *For correspond- ence. E-mail rjass_cardiol@yahoo.com.mx; Tel. (+52) 55 5573 2911; Fax (+52) 55 5573 0994. Environmental Microbiology Reports (2013) 5(6), 799–808 doi:10.1111/1758-2229.12080 © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology