Complete genome sequence of the sulfur-oxidizing chemolithoautotrophic Sulfurovum lithotrophicum 42BKTT

A sulfur-oxidizing chemolithoautotrophic bacterium, Sulfurovum lithotrophicum 42BKTT, isolated from hydrothermal sediments in Okinawa, Japan, has been used industrially for CO2 bio-mitigation owing to its ability to convert CO2 into C5H8NO4 − at a high rate of specific mitigation (0.42 g CO2/cell/h). The genome of S. lithotrophicum 42BKTT comprised of a single chromosome of 2217,891 bp with 2217 genes, including 2146 protein-coding genes and 54 RNA genes. Here, we present its complete genome-sequence information, including information about the genes encoding enzymes involved in CO2 fixation and sulfur oxidation.


Introduction
Epsilonproteobacteria are well-known chemolithoautotrophic bacteria found in deep-sea hydrothermal fields that play significant roles in sulfur, nitrogen, and hydrogen flux [1,2].
Sulfurovum lithotrophicum 42BKT T is a sulfur-oxidizing member of Epsilonproteobacteria that was isolated from deep-sea hydrothermal sediments in Okinawa, Japan [3]. Strain 42BKT T is a Gram-negative, non-motile, and coccoid-to-short-rod-shaped bacterium that utilizes CO 2 as a carbon source, S or S 2 O 3 2− as electron donors, and O 2 and NO 3 − as electron acceptors [3,4]. Recent studies have focused on its potential industrial applications for CO 2 bio-mitigation, reporting that this strain could convert CO 2 into C 5 H 8 NO 4 − at a high specific mitigation rate of 0.42 g CO 2 /cell/h [4]. The CO 2 -bio-mitigation ability of S. lithotrophicum can be improved and optimized through genetic engineering; however, the present lack of genetic knowledge of S. lithotrophicum renders the genetic engineering of this strain difficult. Here, we presented a preliminary description and the general features of S. lithotrophicum 42BKT T , along with its genome-sequence annotations and interactions with other Sulfurovum species. This information would be helpful for improving the use of chemolithoautotrophic bacteria, including Sulfurovum species, in industrial applications in CO 2 bio-mitigation.

Classification and features
A representative 16S rRNA gene of S. lithotrophicum 42BKT T was compared with that of other species using NCBI BLAST [5]. Figure 1 shows the phylogenetic tree with S. lithotrophicum 42BKT T , constructed based on the 16S rRNA sequence. This strain shared 99.1% (1393/ 1406 bp) and 95.1% (1312/1379) sequence identity with the 16S rRNA genes of Sulfurovum sp. NBC37-1 [6] and Sulfurovum aggregans Monchim33 T , respectively.
S. lithotrophicum 42BKT T is a Gram-negative, nonmotile, coccoid-to-short-rod-shaped bacterium that is 0.5-1.2 μm in length and 0.4-0.8 μm in width (Fig. 2). The 42BKT T strain is a mesophilic, facultative anaerobe that requires sea salt to grow and can use NH 4 Cl as a nitrogen source. Normal growth occurs at a temperature of 10-40°C, pH of 5.0-9.0, and salinity of 5-60 g/l [3]. The basic details of its genome sequence are shown in Table 1.

Genome sequencing information
Genome project history S. lithotrophicum 42BKT T was selected for sequencing based on its ability to convert CO 2 into C 5 H 8 NO 4 − , which can be industrially used for CO 2 bio-mitigation. The draft sequencing and annotation were performed by ChunLab, Inc. (Seoul, Korea). The genome project was deposited in the Genomes OnLine Database [9] under the accession number Gp0118364. The complete genome sequence was also deposited in GenBank [10] under the accession number CP011308. Table 2 contains the details of the project and its association with MIGS version 2.0 compliance [11].
Growth conditions and genomic DNA preparation S. lithotrophicum 42BKT T was grown in a 125-mL serum bottle (Wheaton Industries, Millville, NJ, USA) with 20 mL of MJ basal medium and filled with a CO 2 / N 2 gas mixture. The bottle was incubated at 29°C while shaking at 120 rpm (Green Shaker, Vision Scientific Co., Daejeon, Korea) [4]. Genomic DNA was isolated using a QIAmp DNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions.

Genome sequencing and assembly
The genomic library was sequenced using an Illumina MiSeq PE 300 and PacBio 10 K with the Illumina 300-bp paired-end library (Illumina, San Diego, CA, USA) and the PacBio 20 K library (Pacific Biosciences, Menlo Park, CA, USA), respectively. The generated paired-end sequencing Fig. 1 Phylogenetic tree showing the relative position of Sulfurovum lithotrophicum 42BKT T , based 16S rRNA gene sequence. All sites were informative and free of gaps. Evolutionary history was inferred using the neighbor-joining method [35]. The tree was built using the maximum composite-likelihood method [36]. The percentage of replicate trees with the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the corresponding branches [37]. Evolutionary analyses were conducted in MEGA6 [38]. Corresponding GenBank accession numbers are shown in brackets next to the strain name

Genome annotation
The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline [12], which was designed to annotate bacterial genomes. Genome annotation was performed by predicting protein-coding, rRNA, tRNA, ncRNA, and pseudo genes. Phobius [13] was used to predict signal-peptide genes, and TMHMM Server version 2.0 [14] was used to predict transmembrane helix genes [15,16]. Protein families [17] were investigated using Pfam 29.0 [18], and GeneMarkS+ [19], which uses alignment data for gene prediction, was used as an annotation tool [20].

Genome properties
The genome of S. lithotrophicum 42BKT T comprised a single circular chromosome of 2217,891 bp with a GC content of 44.26%. Among the 2217 genes predicted, 2146 (96.80%) were protein-coding DNA sequences, 17 of which were pseudogenes. Among the CDSs, 89.66% were grouped into cluster of orthologous group functional categories. The genome contained a CRISPR array and 54 RNA genes, including 44 tRNAs, 9 rRNAs, and one ncRNA. The properties and statistics of the genome are summarized in Fig. 3 and Tables 3 and 4, 5.
Insights from the genome sequence S. lithotrophicum 42BKT T is a sulfur-oxidizing bacterium that can fix CO 2 through the reductive TCA cycle. Here, we focused on investigating its abilities for CO 2 fixation and sulfur oxidation (sox), based on its genome sequence.
So far, six pathways have been associated with CO 2 fixation: the Calvin-Benson-Bassham or reductive pentose pathway, the reductive TCA cycle or reverse citric acid cycle, the reductive acetyl CoA or Wood-Ljungdahl pathway, the 3-hydroxypropionate pathway  a Evidence codes -TAS Traceable Author Statement (i.e., a direct report exists in the literature); NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species or anecdotal evidence). These evidence codes are from the Gene Ontology project [34] or malyl CoA pathway, the 3-hydroxypropionate/4hydroxy-butyrate cycle, and the dicarboxylate/4hydroxybutyrate cycle [21,22]. Similar to the majority of Epsilonproteobacteria, S. lithotrophicum 42BKT T can also grow chemoautotrophically through its adenosine triphosphate citrate lyase, 2oxoglutarate:ferredoxin oxidoreductase, and pyruvate:ferredoxin oxidoreductase via the reductive TCA cycle [23][24][25]. We annotated these three key enzymes, as well as other relevant enzymes such as malate dehydrogenase, fumarate hydratase, fumarate reductase, isocitrate dehydrogenase, aconitate hydratase, PEP synthase, and PEP carboxylase, in the genome sequence of 42BKT T . Notably, Sulfurovum sp. NBC37-1 and Candidatus Sulfurovum sediminum AR could also assimilate CO 2 via the reductive TCA cycle [6,26]. S. lithotrophicum 42BKT T is known to oxidize or S 2 S O 3 2− via a sox system using SoxB, SoxXA, SoxYZ, and Sox(CD) 2 periplasmic proteins [27]. These enzymes catalyze the oxidation of S or S 2 O 3 2− using horse cytochrome c as the final electron acceptor [28]. Here, we confirmed the presence of SoxA, SoxB, SoxZ, SoxY, and SoxX genes in the 42BKT T genome.

Conclusions
To the best of our knowledge, this is the first report describing the genome sequence of S. lithotrophicum 42BKT T , which comprised a circular chromosome of 2217,891 bp (44.26% GC content) with 2217 genes, among which 2146 were CDSs, 17 were pseudogenes, and 54 were RNA genes. S. lithotrophicum 42BKT T assimilates CO 2 via the reductive TCA cycle and oxidizes S or S 2 O 3 2− via the sox system. The details of the genome sequence of this strain could provide potential strategies to enhance the industrial application of such bacteria for CO 2 bio-mitigation.