Organohalide respiration (OHR) is a bacterial anaerobic respiratory metabolism dedicated to use halogenated compounds as terminal electron acceptors. OHR bacteria are strictly anaerobic microorganisms which use reductive dehalogenases (RdhA) as key enzymes in this respiration process (1,2). Dehalobacter restrictus, an obligate OHR bacterium has been shown to use exclusively tetra- (PCE) and trichloroethene (TCE) as electron acceptors. The PCE RdhA (so called PceA) has been characterized previously (3). D. restrictus genome is currently being sequenced (in collaboration with JGI) revealing an unexpected high number of rdhA genes. Aim: The overall aim of this study is to characterize the functional diversity of rdhA genes in D. restrictus. Specific attention will be given to the use made by this bacterium of such redundant genetic information, and to the possible evolution mechanisms by which these numerous gene copies have emerged. Methods: The rdh gene clusters will be analyzed using bioinformatics comparing their diversity and gene composition to the well-characterized cpr (2) and pce (4) gene clusters in OHR isolates. Although D. restrictus is characterized by a restricted metabolism, different culture conditions will be tested either by replacing PCE with other organohalides, or by spiking organohalides in a culture growing on PCE. The level of transcription of the rdhA genes in D. restrictus will be measured by a targeted approach using optimized RNA extraction, RT, PCR and qPCR protocols. A special focus will also be given to the operon structure of the active pceABCT gene cluster of D. restrictus. Results: So far the analysis of the 2.9 Mb draft genome of D. restrictus revealed the presence of 20 different rdh gene clusters. While most of them are scattered in the available genome contigs, an array of 4 consecutive rdhAB operons is interspersed with regulatory genes suggesting a tight transcription regulation. When compared to the vast family of RdhA sequences in databases, D. restrictus RdhAs reflect the diversity observed in Firmicutes and are fairly distant to Dehalococcoides-type RdhAs. Preliminary transcription analysis of the 20 rdhA genes in standard growth conditions suggests that only the known pceA gene is significantly used in PCE/TCE dechlorination. Proteomic data will be obtained to confirm this view. More transcriptional analysis from cells subjected to other organohalides will be also presented. Conclusion: Based on genome analysis, D. restrictus seems to adopt an intermediate position between the facultative OHR bacteria such as Desulfitobacteria, and the obligate ones like Dehalococcoides members. The high rdhA gene redundancy appears as an evolutionary strategy to grow on many organohalides. This hypothesis still needs to be proven experimentally. References: (1) Holliger et al., Eds. Häggblom & Bossert, Kluwer Academic, 2003, p115. (2) Smidt & de Vos, Annu. Rev. Microbiol., 2004, 58:43. (3) Maillard et al., Appl. Environ. Microbiol., 2003, 69:4628. (4) Maillard et al., Environ. Microbiol., 2005, 7:107
