Draft genome sequence of Desulfoplanes formicivorans Pf12BT, a sulfate-reducing bacterium of the family Desulfomicrobiaceae

Desulfoplanes formicivorans strain Pf12BT is the type strain of the type species in the genus Desulfoplanes, which is the one of the genera in the family Desulfomicrobiaceae within the order Desulfovibrionales. This deltaproteobacterium was isolated from a blackish meromictic lake sediment. D. formicivorans strain Pf12BT is a Gram-negative, motile and sulfate-reducing bacterium. Cells of strain Pf12BT are characterized by possession of vibroid morphology and red fluorescent pigment. Here we describe the features, draft genome sequence and annotation of this organism, the sole species of the genus Desulfoplanes. The genome comprised 3,000,979 bp, 2,657 protein-coding genes and 58 RNA genes.


Introduction
Strain Pf12B T (= NBRC 110391 T = DSM 28890 T ) is the type strain of Desulfoplanes formicivorans, which is the type species of the genus Desulfoplanes in the family Desulfomicrobiaceae. The family Desulfomicrobiaceae was proposed by Kuever et al. (2006) and contained only one genus, Desulfomicrobium. The genus Desulfoplanes was later added to this family because of the phylogenetic position [1]. All members of the family Desulfomicrobiaceae including D. formicivorans are sulfate reducers and incomplete oxidizers, which are unable to completely oxidize organic matters to CO 2 . All known strains of the genus Desulfomicrobium have rod-or ellipsoidal-shaped morphology and they all lack desulfoviridin, which is a red fluorescent pigment [2][3][4]. In contrast, D. formicivorans strain Pf12B T was characterized by vibroid morphology and possession of red fluorescent pigment.
In this study we summarize the features of D. formicivorans strain Pf12B T and provide an overview of the draft genome sequence and annotation of this strain.

Organism Information
Classification and features D. formicivorans strain Pf12B T was isolated from the anaerobic sediments of a meromictic lake [1,5]. Cells of this strain are Gram-negative, motile, non-spore-forming and vibroids (Fig 1, Table 1). Under UV illumination, cell lysate of the strain exhibited red fluorescence suggesting the presence of desulfoviridin. Temperature range for growth is 13-50°C, with an optimum temperature at 42-45°C. NaCl concentration for growth is 0.5-8% (w/v) and optimal concentration is 1-4% (w/v). This bacterium is strictly anaerobic and is capable of respiration and fermentation. Sulfate, thiosulfate and sulfite are used as electron acceptors for growth. Nitrate is not used for respiration. Pyruvate, malate and fumarate are used for fermentative growth.
Phylogenetic relationship of D. formicivorans strain Pf12B T and all members of the family Desulfomicrobiaceae are shown in the 16S rRNA gene phylogenetic tree (Fig. 2). D. formicivorans strain Pf12B T is assigned to the family Desulfomicrobiaceae but forms a well-separated branch among other cultivated relatives of the same family.

Genome sequencing information
Genome project history D. formicivorans strain Pf12B T was selected for genome sequencing on the basis of its 16S rRNA gene-based phylogenetic position in the family Desulfomicrobiaceae (Fig. 2). A summary of the genome sequencing project information and its association with MIGS version 2.0 compliance [6] are shown in Table 2. The genome consists of 26 contigs, which has been deposited at DDBJ/EMBL/ GenBank under accession number BDFE00000000.
Growth conditions and genomic DNA preparation D. formicivorans strain Pf12B T (DSM 28890) was grown on bicarbonate-buffered sulfide-reduced medium [7] containing 28 mM sulfate, 10 mM formate and 0.5 g l -1 yeast extract at 45°C. Genomic DNA was extracted from collected cells using Wizard® genomic DNA purification kit (Promega).

Genome sequencing and assembly
The genome of strain Pf12B T was sequenced using pairedend Illumina sequencing at Hokkaido System Science Co., Ltd. (Japan). From a library with 350 bp inserts, the 10,511,386 reads were generated. After trimming of the reads, a total of 9,393,309 high-quality filtered paired end reads with a hash length of 95 bp were obtained. Reads were assembled de novo using Velvet version 1.2.08 into 26 high quality scaffolds. Gap closing analysis in these scaffolds was performed using Platanus version 1.2.1.

Genome annotation
Draft genome sequences were automatically annotated using the MiGAP [8]. In the pipeline, RNAmmer [9] and tRNAscan-SE [10] were used to identify rRNA and tRNA genes, respectively. MetaGene Annotator [11] was used to predict ORFs likely to encode proteins (CDSs), and functional annotation was performed based on  Phylum Proteobacteria TAS [18] Class Deltaproteobacteria TAS [19,20] Order Desulfovibrionales TAS [20,21] Family Desulfomicrobiaceae TAS [4,20] Genus reference databases, including RefSeq, TrEMBL, and COGs. Manual annotation was performed using IMC-GE software (In Silico Biology; Yokohama, Japan). Putative CDSs were confirmed again by a sequence similarity search using the BLASTP tool. Putative CDSs possessing BLASTP matches with more than 70% coverage and 35% identity and E-values less than 1 × e −5 were considered potentially functional genes. When these cut-off values were not satisfied, the CDSs were annotated as hypothetical proteins. Transcription start sites of predicted proteins were corrected based on multiple sequence alignments. If the distance between CDSs was larger than 500 bp, further ORF extraction for coding genes was performed. The protein-coding genes in the genome were also subjected to analysis on WebMGA [12] for the COGs and Protein family (Pfam) annotations. Transmembrane helices and signal peptide prediction were analyzed using Phobius [13]. CRISPR loci were distinguished using the CRISPR Recognition Tool [14].

Genome properties
The total genome of strain D. formicivorans strain Pf12B T was 3,000,979 bp in size with a GC content of 49.81% (Table 3). It was predicted to contain 2,715 genes including 2,657 protein-coding genes and 58 RNA genes (for tRNA and rRNA). Approximately 83% of the predicted genes were assigned to COG functional categories. The distribution of genes into COGs functional categories is presented in Table 4.

Insights from the genome sequence
The draft genome provides interesting phylogenetic and metabolic information, including phylogeny of dsr genes, which are essential for dissimilatory sulfate reduction. The dsrAB genes are frequently used as marker genes to evaluate phylogenetic relationship of sulfate-reducing bacteria, as well as to reveal their diversity and distribution in environments. Phylogenetic analysis based on DsrAB amino acid sequence was performed to disclose the phylogenetic position of D. formicivorans strain Pf12B T among sulfate reducers belonging to the families Desulfovibrionales and Desulfobacterales (Fig. 3). In the resulting phylogenetic tree, D. formicivorans strain Pf12B T was clearly separated from all members of the family Desulfomicrobiaceae. This result partially conflicts with the 16S rRNA gene phylogeny, and this contradiction may represent a new case of lateral gene transfer event which frequently has been found among dissimilatory sulfate-reducing and sulfuroxidizing bacteria [15].

Conclusions
Draft genome sequence of D. formicivorans strain Pf12B T described here is the first published genome sequence of a member of the genus Desulfoplanes, which is a newly proposed taxon in the family Desulfomicrobiaceae. The genome of the strain Pf12B T consists of 2,657 protein-coding genes and 58 RNA genes. DsrAB phylogenetic tree shows the strain Pf12B T is located in the independent position, which is distant from a cluster of Desulfomicrobium species.