a b c d e f

1. 120 100 80 + 2e- + 2Cl- Cl Cl A B 60 DCA (% initial value) 40 Dichloro-elimination 20 0 0 20 40 60 80 time (days) + DCA Salts Lactate Control FormiateAcetate Cheese whey dcaA tnpA dcaB C B a b c d e f A Catabolic gene redundancy in Contaminated Groundwater Annalisa Balloi, Massimo Marzorati1, Francesca de Ferra2, Sara Borin1, Elena Allifranchini2 ,Giovanna Carpani2, Luca Serbolisca2 ,Willy Verstraete3 and Daniele Daffonchio1. The phylogentic and functional gene diversity of the bacterial communities of a double layer aquifer contaminated with 1,2-dichloroethane (1,2-DCA) was examined in anaerobic water microcosms. Biotransformation of 1,2-dichloroethane to ethene in a single step via reductive dihaloelimination was enhanced by 5 mM lactate addition as electron donor. Bacterial and archaeal diversity were analysed by sequencing 16S rRNA gene clone libraries prepared prior and after a biostimulation treatment. Upper aquifer was characterized by microrganisms belonging to low G + C gram positive bacteria, e and b Proteobacteria. The clone library following the treatment was dominated by Clostridiaceae such as Desulfitobacterium or Dehalobacter, all phylotypes known to be active dechlorinators. A rather different microbial community characterized lower aquifer. Archaeal nested PCR allowed to obtain 16S rRNA amplicons and the clone libraries of bacterial 16S rRNA gene showed a population characterized by a wide bacterial diversity (b-, d-, e-Proteobacteria and a phylotype referred to Trichlorobacter thiogenes) that was substituted by a less diverse microflora predominated by a distinct phylotype most similar to an uncultured Clostridiaceae. Hence, it appeared that very different microbial populations could lead to the same complete degradation of DCA. Functional redundancy was evaluated through gene libraries of the catabolic genes associated with the key steps of the detoxification process using primers previously developed for reductive dehalogenases. Two major groups of RD genes have been identified: one with a high sequence homology to the RD identified in Desulfitobacterium dichloroeliminans strain DCA1, a microorganism that can couple 1,2-DCA dechlorination to ethene with energy production; a second group with sequences that differed in two small regions both from the previous one and from the PCE specific RDs. Catabolic gene redundancy in a contaminated site is a key aspect, index of the ability of different metabioses to adapt to environmental changes and potentially, of their ability in degrading different xenobiotic compounds. Pump&Treat San Donato Milanese 1,2 DCA dechlorinating culture named 6Vs Laboratory forMicrobial Ecology and Technology (LabMET), Ghent University Microbial characterisation before and after the treatment in the well A Microbial characterisation before and after the treatment in the well B . , . Uncult Clostridiaceae Desulfitobacterium dichloroeliminans Dehalobacter sp. E1 Desulfitobacterium hafniense Uncult. Clostridium D.restrictus Uncult. archaeon In well B lactate addition stimulates the enrichment of a clostridium clone. The interaction between Archaea and Bacteria can play a major role in the DCA degradation. In well A lactate addition stimulates the increase of well known degraders of chlorinated alkanes such as D. restrictus, D. dichloeliminans, D. hafniense and many others. No Archaea can be identified. Uncult. Crenarchaeota RDsèèuu Characterisation of the RD from 6vs culture (DcA RD-54) Expression of dcaA gene Alignment of the complete gene cluster sequence, named RD-54, with known gene clusters encoding emzyme complexes active in reductive dechlorination of clorinated aliphatics, showed that all of the genes except dcaA were >98% identical, at the nucleotide level, to the corresponding genes of Dehalobecter restictus strain DSMZ 9455t and of Desulfobacteium dihloroeliminans DCA1. M nc M DNA RNA cDNA M 1 2 3 DNase RNA 1 2 3 4 5 6 7 8 9 10 tpnA = Transposase; dcaA = Catalytic subunit ; dcaB = Anchor protein;dcaC = Membrane bound regulatory protein dcaT = Trigger factor; Orf1 = Unknown function 1500 bp 400bp Aminoacidic alignment of DcaA RD-54 Vs Other A subunits of well known RDs proteins 180bp A No changes in N-terminal sequence, restriction site and signal peptide B Rectangles 1 and 2 = a double Arg and Pro is typical of consensus of TAT System • Rectangles 3 and 4 = two iron-sulfur cluster binding motifs: • FeS cluster I is totally conserved • Some changes in FeS cluster II • Cys conserved in both FeS clusters, in the fixed position typical of RDases Light gray areas A and B indicate the two amino acid stretches were resides about 53% of the diversity between the RD-54 from 6VS culture and the other RDs. Black areas indicate the different positions between PcaA and DcaA, but conserved between DcaA of RD-54 and RD-DCA1. Transciption of dcaA and dcaB in culture 6VS during dechlorination of 1,2-DCA. PCR experiment using: A) specific primers for the dcaA gene (DHL F1, DHL F2 and DHL R1) to assess the transcription of dcaA; PCR on genomic DNA (lanes 1, 2), total RNA before the treatment with DNAse (lanes 3, 4), total extracted RNA following the treatment with DNAse (lanes 5, 6) and on the cDNA synthesized from the pure RNA (lanes 7, 8). Negative control (lane 9). B) Primer DcaB Rev (specific for dcaB) coupled with DHL F1; PCR on genomic DNA (lane 1), total extracted RNA following the treatment with RNAse (lane 2) and on the cDNA synthesized from the pure RNA (lane 3). M = marker (band size is given in bp) The genes dcaA and dcaBare co-transcipted About 53% of the diversity between the RD-54 from 6VS culture and the other RDs was localized in two regions (A and B ) that represented only the 19% of the total Dca residues. Southern blot hybridization analysis performed to survey the RD genes in the genome of strain DCA1 • Southern blot analyses of gel in panel A using Probe A specific for dcaA gene of strain DCA1. • Southern blot analyses of gel in panel A using Probe B universal for gene B known up to date (dcaB, pceB). • The Ds. Dichloroeliminans strain DCA1 harbors two identical or highly similar copies of the same RD cluster The two DcaA proteins (RD-54 and RD-DCA1) clustered in a phylogenetic brnch divergent from the other corresponding RD proteins active on chlorinated ethens and chlorophenol Phylogenetic relationship between DcaA of the 6VS culture (DcaA RD-54) and D.dichloroeliminans strain DCA1 with other A subunits of genetically characterized RDases.The numbers at each branch point represent bootstrap percentage calculated from 1000 replicate trees. The scale bar represents the sequence divergence. • Following the lactate treatment a new dehalogenase gene cluster composed of 6 genes is enriched • The dehalogenase (gene dcaA) is expressed during the incubation with lactate • Gene dcaA can be used as a molecular tool during the bioremediation treatment Strain DCA1 has the same unique gene cluster • CONCLUSIONS • The two aquifers are characterized by different microbial communities • The levels of known degraders of halogenated alkanes increased during the biostimulation treatment