Functional genotyping of Sulfurospirillum spp. in mixed cultures allowed the identification of a new tetrachloroethene reductive dehalogenase

Abstract

Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceATCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed.

Fig 1

Sequence likelihood analysis showing the relationships of newly identified RdhA proteins from SL2 consortia to both Sulfurospirillum RdhA types. The neighbor-joining method of ClustalX was used to build the tree, including 100× bootstrap values. All sequences used in the alignment had similar lengths. The following sequences were used: S. multivorans PceA (GenBank accession no. AAC60788), S. halorespirans PceA (GenBank accession no. AAG46194), RdhA1 from an uncultured bacterium (GenBank accession no. BAJ09319), and RdhA2 from an uncultured bacterium (GenBank accession no. BAJ09321). The tree was rooted with PceA of Dehalobacter restrictus (GenBank accession no. CAD28790). The scale bar represents 2% sequence divergence.

Fig 2

Sequence alignment of Sul-rdhA gene fragments for rdhA-specific T-RFLP analysis. Sequences were aligned with ClustalX. The Sul-rdhA primers used in the dedicated T-RFLP analysis match the sequences of all 3 rdhA genes present in Sulfurospirillum spp. and are indicated above the alignment. Positions indicated by dashes represent a 12-nucleotide deletion in the sequence of pceA_TCE. Positions indicated by dots are identical to the sequence of pceA_TCE. A box indicates the position of the TaqI restriction site.

Fig 3

Transformation of chloroethenes and interplay of rdhA genes in the SL2-PCEb culture along the dechlorination of PCE to cis-DCE. Chloroethenes are represented by diamonds for PCE, triangles for TCE, and squares for DCE. Dark gray bars represent the relative abundances of pceA_TCE, light gray bars represent those of pceA_DCE, and white bars represent those of rdhA₂. The figure was obtained by averaging the data obtained from two independent batch cultures.