Background Several enrichment cultures were investigated for their bioremediation potential of tetrachloroethene (PCE). The underlying process (organohalide respiration, OHR) is an anaerobic bacterial respiration during which the chlorinated compounds are used as electron acceptors. The key enzyme in OHR is the reductive dehalogenase (RdhA). 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 at least two dechlorinating populations (Maillard et al.,2011). Objectives The aim of this work is to obtain an evaluation of the functional diversity of Sulfurospirillum rdhA genes in mixed cultures, and of their interplay during PCE dechlorination, as well as the characterization of the inherent dechlorinating activity. Methods As genotyping based on rRNA genes and internal transcribed spacer was not possible here to distinguish Sulfurospirillum populations, a T-RFLP method dedicated to distinguish the functional genes was developed and successfully applied. Rapid sub-cultivation of SL2-PCEb bacterial consortium allowed selecting for a sub-culture (SL2-PCEc) with a restricted PCE-to-TCE dechlorinating activity. Quantitative PCR, Western blot analysis and enzymatic assays were also applied to characterize this sub-culture. Conclusions A new reductive dehalogenase (PceATCE) was identified. It shows 92% sequence identity to S. multivorans PceA (Neumann et al.,1998), catalyzes however exclusively the first step in PCE dechlorination with a 5-fold higher rate as measured in crude extracts. The SL2-PCEc sub-culture and the new enzyme is now the focus of several new investigation lines, which will be presented. References (1) Maillard et al. (2011), Biodegradation 22:949. (2) Buttet et al. (2013), Appl Environ Microbiol, submitted. (3) Neumann et al. (1998), J Bacteriol 180 :4140