Draft genome sequence and characterization of Desulfitobacterium hafniense PCE-S
© Goris et al.; licensee BioMed Central. 2015
Received: 26 September 2014
Accepted: 4 December 2014
Published: 24 February 2015
This genome report describes the draft genome and the physiological characteristics of Desulfitobacterium hafniense PCE-S, a Gram-positive bacterium known to dechlorinate tetrachloroethene (PCE) to dichloroethene by a PCE reductive dehalogenase. The draft genome has a size of 5,666,696 bp with a G + C content of 47.3%. The genome is very similar to the already sequenced Desulfitobacterium hafniense Y51 and the type strain DCB-2. We identified two complete reductive dehalogenase (rdh) genes in the genome of D. hafniense PCE-S, one of which encodes PceA, the PCE reductive dehalogenase, and is located on a transposon. Interestingly, this transposon structure differs from the PceA-containing transposon of D. hafniense Y51. The second rdh encodes an unknown reductive dehalogenase, highly similar to rdhA 7 found in D. hafniense DCB-2, in which the corresponding gene is disrupted. This reductive dehalogenase might be responsible for the reductive dechlorination of 2,4,5-trichlorophenol and pentachlorophenol, which is mediated by D. hafniense PCE-S in addition to the reductive dechlorination of PCE.
KeywordsAnaerobic respiration Organohalide respiration Reductive dechlorination Chlorinated ethenes Chlorinated phenols Bioremediation Reductive dehalogenase
Desulfitobacterium spp. are anaerobic Gram-positive bacteria belonging to the phylum Firmicutes. Desulfitobacteria are metabolically versatile bacteria capable of utilizing a wide range of electron donors and acceptors, the latter also including organohalides. Previously, the genome sequences of Desulfitobacterium hafniense Y51 and DCB-2 have been published [1, 2], and further genomes of various desulfitobacteria are expected to be published in the near future as the result of ongoing sequencing projects (Kruse et al, unpublished results). The genomes of Desulfitobacterium hafniense DCB-2 and Y51 are relatively large (5.3 and 5.7 Mbp, respectively) and are characterized by a high number of genes related to energy metabolism. In both genomes, at least one gene encoding a reductive dehalogenase was found. D. hafniense DCB-2 contains seven rdh genes, two of which are likely non-functional due to either a transposase insertion or a frameshift mutation. The D. hafniense Y51 genome harbours one reductive dehalogenase gene, encoding a PCE reductive dehalogenase . Despite the great interest in the potential application of Desulfitobacterium spp. and other organohalide-respiring bacteria for bioremediation, only a few reductive dehalogenases have been biochemically characterized. One example of a well-studied reductive dehalogenase is the tetrachloroethene reductase, PceA, from D. hafniense PCE-S [3–6].
Here, we describe the isolation and characterization of D. hafniense PCE-S together with its draft genome sequence. The organism is capable of dechlorinating PCE via TCE to cis-DCE as well as of several chlorophenols. The draft genome is 5,666,696 bp in size and is compared to the genome sequences of D. hafniense Y51 and DCB-2. In addition, some morphological and physiological characteristics of strain PCE-S are given and compared to those of other members of the Desulfitobacterium genus.
Characterization and features
Classification and general features of Desulfitobacterium hafniense PCE-S
Evidence code a
Species Desulfitobacterium hafniense
+ (only exponentially growing cells)
20 – 45°C
pH range; Optimum
Soil contaminated with chlorinated ethenes
Sample collection time
With PCE as electron acceptor (20 mM, supplied from a hexadecane phase), pyruvate was oxidized to acetate and PCE was dechlorinated to cis,1,2-dichloroethene as the main dechlorination product (≥95%) and minor amounts of trichloroethene (≤5%). The chlorinated ethenes were determined gas chromatographically with N2 as carrier gas using two bonded-phase fused silica capillary columns.
The generation time of growth with pyruvate as electron donor and PCE as electron acceptor was 10 h without and 8 h with 0.1% yeast extract at 30°C. Fumarate as electron acceptor plus yeast extract led to a slightly shorter generation time (7 h) than with PCE/yeast extract.
The ability of D. hafniense PCE-S to dechlorinate polychlorinated phenols was investigated with pyruvate as electron donor and 0.1% yeast extract. Chlorophenols were analysed by HPLC using an RP-18 (5 μm) LiChrospher 100 column (Merck, Darmstadt, Germany). Pentachlorophenol and 2,4,5-trichlorophenol at a concentration of 20 μmol l-1 in mineral medium were dechlorinated. 2,4,5-trichlorophenol was partially dechlorinated to 3,4-dichlorophenol, pentachlorophenol was partially dechlorinated to 3,4-dichlorophenol and an unidentified tetrachlorophenol. 2,6-dichlorophenol, 3,5-dichlorophenol, and 2,4-dichlorophenol were not dechlorinated by D. hafniense PCE-S.
Genome sequencing information
Genome project history
Improved high quality draft
One Illumina Miseq paired end library
Illumina MiSeq Personal Sequencer
Ray version 2.3, Edena version 3.130110
Gene calling method
Prodigal version 2.5
EMBL Date of Release
September 31, 2014
Source Material Identifier
Growth conditions and DNA preparation
D. hafniense PCE-S was cultivated under anoxic conditions as described by Scholz-Muramatsu et al.  and Reinhold et al. . For isolation of genomic DNA, D. hafniense PCE-S was cultivated for one subculture with fumarate after regularly being cultivated in the presence of PCE. The isolation was carried out as described by Reinhold et al. Approximately 12 μg of genomic DNA were used for genome sequencing. The genome sequence of Desulfitobacterium hafniense PCE-S has been deposited in the EMBL database under accession numbers LK996017-LK996040.
Genome sequencing and annotation
DNA was sequenced at GATC Biotech (Konstanz, Germany) on an Illumina MiSeq Personal Sequencer, generating 1,242,269 paired end reads with a length of 250 bp.
Genome size was estimated prior to assembly using kmer spectrumanalyzer .
The assembly was done in parallel with two different assemblers. One assembly was performed with Edena , with standard parameters, the second assembly with Ray, using a kmer-value of 125 . Afterwards both assemblies were merged with Zorro with one of the paired end files supplied . Next, this hybrid assembly was scaffolded with opera version 1.2 , which was set up to use Bowtie version 0.12.7 for mapping . As last step, Pilon version 1.4 was used for quality assurance on the assembly . Reads were mapped with Bowtie2 version 2.0.6 , further converted with Samtools version 0.1.18 (r982:295)  , and then provided to Pilon as input data.
All steps were done using standard parameters, unless stated otherwise. Before annotation, the genome was blasted  against itself with an e-value of 0.0001. All contigs with a length of less than 500 bp were discarded, as well as those with less than 1,000 bp which matched onto another genomic location with 100% identity.
After annotation, a check for technical duplications was performed. Contigs, which were determined to be such duplications, were manually removed from the initial assembly and replaced with contigs from the second assembler. The assembly workflow was repeated until no more technical duplications were found.
The assembly was then further scaffolded with CONTIGuator version 2.7.4  and the genome of Desulfitobacterium hafniense Y51 as reference . Disagreements with the reference genome were examined with Mauve  and Tablet , and in case of considerable drops of coverage, the contigs and related reads were isolated, and a re-assembly was performed with Edena. This re-assembly was again scaffolded with CONTIGuator using Y51 as reference genome. Non-scaffolded contigs were included as single contigs in the final result, unless they had a blast hit of more than 90% of their length with a minimum sequence identity of 90% to the scaffold result from CONTIGuator.
The annotation was carried out with an in-house pipeline. In short, this pipeline includes Prodigal version 2.5 for open reading frame identification , InterproScan version 5RC7 for protein annotation , tRNAscan SE 1.3.1 for tRNA identification  and rnammer 1.2 for the prediction of rRNAs . Additional protein function predictions were derived via BLAST  UniRef50 and  Swissprot databases (downloaded August 2013) . After the annotation process, EC numbers were added with PRIAM version March 06, 2013 . COG assignments were created via blastp best bidirectional hit assignments .
Nucleotide content and gene count levels of the genome
% of total a
Genome size (bp)
DNA G + C (bp)
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with the 25 general COG functional categories
% of total a
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Insights from the genome sequence
Orthologs to other Desulfitobacterium species were determined via bidirectional BLAST hits  with at least 70% sequence identity and similar size of both sequences (+/- 5%).
Despite the different organization of this transposon, D. hafniense PCE-S also loses the ability to dechlorinate PCE after prolonged cultivation in the absence of PCE . The second reductive dehalogenase gene (DPCES_1664) has no ortholog in Y51. A truncated ortholog is encoded in DCB-2 (Dhaf_2620). In D. hafniense DCB-2, the corresponding rdhA gene is truncated n-terminally (50 amino acids) due to the insertion of a stop codon through a frameshift mutation. It seems likely that the gene product of DPCES_1664 is responsible for the partial dechlorination of pentachlorophenol and 2,4,5-trichlorophenol by D. hafniense PCE-S.
Of the 5,417 protein coding sequences found in the genome of D. hafniense PCE-S, 4,402 are orthologous to proteins encoded in either Y51 or DCB-2. D. hafniense PCE-S harbours six putative phage regions, of which one was classified as a complete prophage, as detected by PHAST . This is opposed to D. hafniense DCB-2 or Y51, where four (DCB-2) and three (Y51) prophages were identified by PHAST as incomplete or questionable, but none as complete. The complete prophage found in D. hafniense PCE-S shows highest similarities to Vibrio phage X29 (NCBI RefSeq accession no. NC_024369). Several enzymes, of which orthologs fulfill a catabolic function, are not encoded in D. hafniense Y51 and DCB-2, but found on the genome of D. hafniense PCE-S: An ethanolamine ammonia lyase system (PCES_2016-2020), three molybdopterin oxidoreductase gene clusters (DPCES_4294-6, DPCES_4565-7, DPCES_4582-4), together with a molybdopterin import cluster (DPCES_0024-6), and a protein annotated as cellulose synthase (DPCES_2599). A cluster encoding polysaccharide synthesis enzymes (DPCES_3251 to 3245) might be responsible for the biosynthesis of the slime sacculus of PCE-S.
Five CRISPR regions with a length from 958 to 3415 bp and 14 to 51 spacers were identified in the genome of D. hafniense PCE-S with CRISPR finder . This is similar to the situation in DCB-2, where five CRISPR regions with a length of 7 to 60 spacers were found, and in Y51, where five CRISPR regions with a length of 12 to 47 spacers were found. The CRISPR regions in all Desulfitobacterium spp. genomes are located in close proximity to each other, separated by not more than 30 kb which are to a large extent covered by CRISPR associated (CAS) proteins.
Taken together, the genome sequence of Desulfitobacterium hafniense PCE-S expands our view on these environmentally interesting microorganisms. The genome sequence gives us insight into the putative chlorophenol dechlorinating activity of a reductive dehalogenase not studied before and might aid bioremediation of chlorinated phenols in the future.
Perchloroethylene or tetrachloroethene
We would like to thank Heidrun Scholz-Muramatsu and Silke Granzow for initial work on isolation and characterization of D. hafniense PCE-S. The work was funded by the German Research Foundation (DFG research unit FOR 1530). Work of HS and TK was financially supported by the EcoLinc Project of the Netherlands Genomics Initiative, as well as the European Community program FP7 (grants KBBE-211684; BACSIN, and KBBE-222625; METAEXPLORE). BH is supported by Wageningen University and the Wageningen Institute for Environment and Climate Research (WIMEK) through the IP/OP program Systems Biology (project KB-17-003.02-023).
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