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The genomes of two novel Dehalococcoides mccartyi strains, DCMB5 and BTF08, enriched from the heavily organohalide-contaminated megasite around Bitterfeld (Germany), were fully sequenced and annotated. Although overall similar, the genome sequences of the two strains reveal remarkable differences in their genetic content, reflecting a specific adaptation to the contaminants at the field sites from which they were enriched. The genome of strain BTF08 encodes for 20 reductive dehalogenases, and is the first example of a genome containing all three enzymes that are necessary to couple the complete reductive dechlorination of PCE to ethene to growth. The genes encoding trichloroethene and vinyl chloride reductive dehalogenases, tceA and vcrA, are located within mobile genetic elements, suggesting their recent horizontal acquisition. The genome of strain DCMB5 contains 23 reductive dehalogenase genes, including cbrA, which encodes a chlorobenzene reductive dehalogenase, and a gene cluster encoding arsenic resistance proteins, both corresponding to typical pollutants at its isolation site.
Dehalococcoides mccartyi, classified in the phylum Chloroflexi, is able to transform a wide range of highly persistent aliphatic and aromatic organohalides (Löffler et al., 2013). It is a key player in the natural attenuation of organohalides, which are often toxic remnants of industrialisation. All known Dehalococcoides strains depend on organohalide respiration for energy conservation and growth (Löffler et al., 2013), and this lifestyle is reflected by the presence of multiple nonidentical reductive dehalogenase homologous protein (Rdh)-encoding genes in their genomes. For several Rdh proteins, the biochemical function has been elucidated, for example, for tetrachloroethene, trichloroethene, vinyl chloride and chlorobenzene reductive dehalogenase, encoded by the genes pceA, tceA, vcrA and cbrA, respectively (Hug et al., 2013).
Here, we report the genome sequences of two novel strains of D. mccartyi, enriched from the highly contaminated megasite around Bitterfeld, Germany. Strain DCMB5 was isolated from sediment of the creek Spittelwasser, which contains high loads of chlorinated contaminants, including polychlorinated dibenzo-p-dioxins and heavy metals such as arsenic (Bunge et al., 2007). The enrichment was achieved through 15 successive transfers using sequentially 1,2,3,4-tetra- and 1,2,4-trichlorodibenzo-p-dioxin and 1,2,3-trichlorobenzene as electron acceptors. The pure culture DCMB5 was finally obtained from a serial dilution in agar shake cultures supplemented with 1,2,3-trichlorobenzene (Bunge et al., 2008). Strain BTF08 was highly enriched (> 98% of total cells) from a groundwater well polluted with tetrachloroethene (PCE), trichloroethene (TCE) and high concentrations of cis-dichloroethene (DCE; Cichocka et al., 2010). Minor populations were closely related to Sulfurospirillum spp. or microorganisms that have been found to be associated with dehalogenating consortia, previously (Cichocka et al., 2010). After one additional transfer of the enrichment culture to medium with PCE as electron acceptor, the resulting cells of the culture were subjected to DNA extraction.
Whole-genome sequencing was performed using FLX Titanium pyrosequencing (Margulies et al., 2005), and de novo assembly of contigs was conducted with Newbler (Version 2.3; 454 Life Sciences). For strain DCMB5, a total of 48 842 777 bases (34-fold coverage) were determined by paired-end sequencing. In total, 21 contigs were produced in 1 scaffold through de novo assembly. The contigs obtained for the BTF08 culture were compared using blastn against the NCBI nr database and subjected to a filtering process in which small contigs with a size below 2000 bp with no hit against D. mccartyi genomes were discarded. The remaining contigs assembled from reads with a total number of 45 152 987 bases (31-fold coverage) were assembled into eight contigs with a length between 1367 and 798 655 bp. The contigs were arranged according to the reference genomes of D. mccartyi strains 195 (Seshadri et al., 2005) and CBDB1 (Kube et al., 2005). Arrangements were confirmed, and gaps were closed by PCR and Sanger sequencing. The fact that the whole genome could be unambiguously assembled indicates that only one D. mccartyi strain was present in the BTF08 culture. Initial annotation was performed with RAST (Aziz et al., 2008) and then refined using published D. mccartyi genomes as a reference. Manual curation was performed with ARTEMIS (Rutherford et al., 2000) as an annotation platform, and the InterPro (Apweiler et al., 2001), the Rfam (Gardner et al., 2009), TCDB (Saier et al., 2009) and Swiss-Prot (Boeckmann et al., 2003) databases as well as the TMHMM server (Krogh et al., 2001).
Both strains DCMB5 and BTF08 contain one circular chromosome with a length of 1 431 902 and 1 452 335 bases, a G+C content of 47.1% and 47.3% and a number of 1477 and 1529 predicted CDSs, respectively, and 46 tRNA genes. The overall structure of the two genomes is similar to those of the described D. mccartyi genomes, as the majority of the genome is conserved with the exception of two high plasticity regions (HPR) on both sides of the origin of replication (Kube et al., 2005; McMurdie et al., 2009). An intact prophage was found in the BTF08 genome by PHAST analysis (Zhou et al., 2011; Fig. 1). Screening of the phage-flanking regions using REPuter (Kurtz et al., 2001) indicated a 22-bp direct repeat supposed to be the attL/R sites of phage integration. While BTF08 is devoid of a CRISPR locus, DCMB5 contains two different CRISPR regions with adjacent cas genes, one in each HPR (Fig. 1). Another remarkable feature of the DCMB5 genome is the presence of a gene cluster, which codes for arsenic resistance proteins, including an As (III) efflux pump. This might allow strain DCMB5 to grow in the arsenic-polluted environment from which it was isolated.
Figure 1. Schematic representation of the two HPRs in the genomes of the Dehalococcoides mccartyi strains DCMB5 and BTF08. The HPRs are flanked by tRNA-Ala genes (HPR 1) or by tRNA-Val/Arg genes (HPR 2) (McMurdie et al., 2009), the borders are shown as nucleotide positions of the respective genomes. The HPRs surround the core region, which is shown in a compressed manner. The conserved ori core is not shown. Genes coding for reductive dehalogenases (rdhA) are shown in blue; rdhA orthologues [bidirectional blast hits with more than 90% amino acid sequence identity (Hug et al., 2013)] between the two strains are marked in green and connected with lines. rdhA genes without orthologues in other D. mccartyi genomes are shown in darker blue. All rdhA genes are labelled with the corresponding locus_tag numbers. The orthologues of pceA, tceA, vcrA and cbrA encoding functionally characterised tetrachloroethene, trichloroethene, vinyl chloride and chlorobenzene reductases, respectively, are labelled in red. The positions of the CRISPR regions (orange), the intact prophage (brown), the attachment sites (attL/R) of phage integration (arrows) and a gene cluster putatively coding for arsenic resistance proteins (red) are indicated. The region coding for proteins presumably functioning in mobilisation of rdhA genes in D. mccartyi (integration module; McMurdie et al., 2011) is shown in dark green, ssrA (the gene coding for tmRNA) at the base positions 1 258 194 for BTF08 and 1 277 831 for DCMB5 are shown as purple tick marks.
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The numerous rdhA and rdhB gene clusters encoding the catalytic subunit and the putative membrane anchor of RDHs are enriched in the two HPRs (Fig. 1). The DCMB5 genome comprises 23 rdhAB genes. One rdhA gene, dcmb_86, is the first known orthologue of cbrA of strain CBDB1. This correlates with the remarkable property of DCMB5 to couple reductive dehalogenation of chlorinated benzenes to growth (Bunge et al., 2008). Eight of the rdhAs found in the DCMB5 genome have no orthologues in other sequenced D. mccartyi genomes. BTF08 contains 20 rdhAB gene clusters, of which only five are orthologous to those of strain DCMB5 (Fig. 1), and these represent five recently classified orthologous groups of reductive dehalogenases, which are well conserved throughout cultivated D. mccartyi strains (Hug et al., 2013). Of the 20 rdhA genes in BTF08, four have no orthologues in other D. mccartyi genomes. Interestingly, the other rdhA genes of BTF08 include orthologues of pceA, tceA and vcrA. To our knowledge, this is the first example of genes encoding dehalogenases for all steps of the reductive dehalogenation of PCE, TCE, DCE and the toxic vinyl chloride being assembled in one genome. This result is in accordance with the unique capability of strain BTF08 to couple all reductive dehalogenation steps from PCE to ethene to growth (Cichocka et al., 2010; Kaufhold et al., 2013). Notably, the tceA orthologue is located within the proposed phage attachment sites, and the vcrA orthologue was found to be part of a genomic island located downstream of ssrA (the gene coding for tmRNA), suggesting the recent horizontal acquisition of both genes (for details, see Fig. 1). A large number of ssrA-specific genomic islands was found in many Dehalococcoides strains (McMurdie et al., 2011), and DCMB5 is no exception either, with genomic islands including rdhAs downstream of ssrA.
Taken together, the annotated genomes of the D. mccartyi strains DCMB5 and BTF08 (Genbank accession nos. CP004079 and CP004080) reveal a remarkable genetic adaptation of the organisms to site-specific pollutants. Their further exploration by comparative genomics, linked to proteome and transcriptome studies, will help to elucidate the molecular basis of this obligate organohalide-dependent lifestyle.