Recent analysis of a set of reductive dehalogenases revealed that sequence similarity and substrate specificity are not correlated, making functional prediction from sequences difficult (1). To this respect, several enrichment cultures were investigated for their distinct dechlorination potential of tetrachloroethene (PCE). Indeed, SL2-PCEb, an enrichment culture dominated by Sulfurospirillum spp., was shown to stepwise dechlorinate PCE to TCE and cis-DCE, suggesting the successive involvement of two dechlorinating populations (2). Two subcultures were derived from SL2-PCEb showing distinct dechlorination patterns: SL2-PCEc which dechlorinates PCE to TCE only, and SL2-TCE which was selected on TCE but kept the potential to dechlorinate both PCE and TCE. A T-RFLP method dedicated to Sulfurospirillum reductive dehalogenase (rdhA) genes was designed to distinguish the populations and allowed the identification of new RdhA enzymes, named PceADCE and PceATCE. The culture harboring PceATCE showed a higher PCE dechlorination activity and is involved in the dechlorination of PCE to TCE exclusively (3). The sequence of PceATCE showed a high value of 92% identity compared to the PceA of S. multivorans, which is known to dechlorinate both PCE and TCE (4). Purification and biochemical characterization of PceATCE are ongoing to study its kinetic parameters. Preliminary data suggest that the corrinoid cofactor present in PceATCE is a norpseudovitamin B12 as in PceA of S. multivorans (5). Currently attempts are made to isolate pure Sulfurospirillum strains from SL2 consortia to allow further physiological and metabolic characterization. This study emphasizes the difficulty of substrate specificity prediction for reductive dehalogenases by showing that small changes in the sequence are responsible for differences in their substrates. Moreover, the identification of a new PCE reductive dehalogenase restricted to PCE as substrate along with the indication that it displays higher dechlorination activity suggests that recent evolution likely promoted new catalytic properties that help individual populations to compete for the same substrate. References (1) Hug et al. (2013), Philos Trans R Soc Lond B Biol Sci 368 :20120322. (2) Maillard et al. (2011), Biodegradation 22:949. (3) Buttet et al. (2013), Appl Environ Microbiol 79:6941. (4) Neumann et al. (1998), J Bacteriol 180:4140. (5) Kräutler et al. (2003), Helv Chim Acta 86:3698.