High-quality-draft genome sequence of the multiple heavy metal resistant bacterium Pseudaminobacter manganicus JH-7T

Pseudaminobacter manganicus JH-7T (= KCTC 52258T = CCTCC AB 2016107T) is a Gram-staining-negative, aerobic and non-motile strain that was isolated from a manganese mine. The strain JH-7T shows multiple heavy metal resistance and can effectively remove Mn2+ and Cd2+. In addition, it is able to produce exopolysaccharides (EPS), which may contribute to metal remove/adsorption. Thus, strain JH-7T shows a great potential in bioremediation of heavy metal-contaminated environment. In this study, we report the draft genomic sequence of P. manganicus JH-7T and compare it to related genomes. Strain JH-7T has a 4,842,937 bp genome size with a G + C content of 61.2%, containing 4504 protein-coding genes and 71 RNA genes. A large number of putative genes associated with heavy metal resistance and EPS synthesis are found in the genome. Electronic supplementary material The online version of this article (10.1186/s40793-018-0330-2) contains supplementary material, which is available to authorized users.

P. manganicus JH-7 T was isolated from a sludge sample of a wastewater ditch in Dalong manganese mine in 2015 [2]. It shows multiple heavy metal resistance and can effectively remove Mn 2+ and Cd 2+ . In addition, the strain produces EPS, which may facilitate heavy metal resistance and adsorption [4][5][6]. These features show great interests because of its potential applications in bioremediation of heavy metal contaminated environments. So far, only the genome of an atypical Pseudaminobacter strain Pseudaminobacter salicylatoxidans KCT001 has been sequenced [7]. Strain KCT001 can utilize tetrathionate as the substrate for sulfur-oxidizing chemolithotrophic growth [8]. For better understanding the mechanism of bacterial resistance and removal of heavy metals, here we analyze the genome of P. manganicus JH-7 T .

Classification and features
The phylogenetic relationship of P. manganicus JH-7 T to the related members is shown in a 16S rRNA gene based neighbor-joining tree. Strain JH-7 T is closely related to P. salicylatoxidans BN12 T , P. defluvii THI 051 T and P. salicylatoxidans KCT001 (Fig. 1). Strain JH-7 T is Gram-staining-negative, aerobic, non-motile and rod-shaped (0.3-0.8 × 1-2 μm) (Fig. 2). The colonies are white, circular, entire, slightly raised and smooth on LB agar plates. It is positive for oxidase and catalase activities and hydrolysis of casein [2]. The major fatty acids are C 18:1 ω7c, C 19:0 cyclo ω8c and C 16:0 and the G + C content is 61.2 mol% [2]. The major polyamine is sym-homospermidine and the respiratory quinone is ubiquinone-10. The polar lipids are phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, two aminolipids and two lipids [2]. Table 1 shows the general features of P. manganicus JH-7 T .
The resistant levels of P. manganicus JH-7 T to multiple metal(loid)s were tested with the MIC on LB agar plates incubated at 28°C for 7 days. The MICs for MnCl 2 , CdCl 2, PbCl 2 , CuCl 2 , ZnSO 4 and NiSO 4 are100, 2, 10, 5, 5 and 5 mmol/L respectively. The MICs for K 2 CrO 4 and Na 3 AsO 3 are both 0.1 mmol/L that are lower than the above six metals. Specifically, strain JH-7 T could remove nearly 60% of 5 mmol/L Mn 2+ and nearly 80% of 0.1 mmol/L Cd 2+ (Fig. 3), respectively. In addition, strain JH-7 T could produce EPS based on the aniline blue reaction incubated on LB agar in 3-7 days [9] (data not shown). This phenomenon is consistent with the cell image observed by TEM (Fig. 2). A lay of shadow around the strain was similar to the EPS observed in strain Bifidobacterium longum 35,624 [10].

Genome project history
This organism was selected for sequencing particularly due to its multiple heavy metals resistance and heavy metal removal ability. Genome sequencing was performed by Wuhan Bio-Broad Co., Ltd., Wuhan, China in 2016. The draft genome sequence of strain P. manganicus JH-7 T has been deposited at DDBJ/EMBL/GenBank under accession number MDET00000000. The project information is summarized in Table 2.
Growth conditions and genomic DNA preparation P. manganicus JH-7 T was grown under aerobic conditions in LB medium at 28°C for 40 h. DNA extraction

Genome sequencing and assembly
The genome of strain JH-7 T was sequenced on Illumina Hiseq2000 [11] and assembled by Bio-broad Technogoly Co., Ltd., Wuhan using SOAPdenovo v2.04 [12]. An Illumina standard shotgun library was constructed and sequenced, which generated 19,404,755 reads totaling 2,885,684,230 bp and average of 625 times genome coverage. The total size of the genome is 4,842,937 bp and a total of 60 scaffolds were obtained after arranging 68 contigs together. The part gaps of assembly were filled and the error bases were revised using GapCloser v1.12 [13].

Genome annotation
The draft genome was annotated through the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and  The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome b Also includes 110 pseudogenes, 54 tRNA genes, 12 rRNAs and 5 ncRNA The total is based on the total number of protein coding genes in the annotated genome genes were identified using the gene caller GeneMarkS + with the similarity-based gene detection approach [14]. The predicted CDSs were translated and were submitted to the Pfam protein family database [15] and KEGG database [16]. The genes in internal clusters were performed by OrthoMCL [17,18]. The protein function classification, transmembrane helices and signal peptides were predicted by WebMGA [19], TMHMM v. 2.0 [20] and SignalP 4.1 [21], respectively. In addition, the CRISPRfinder program [22] was used to predict CRISPRs in the genome.

Genome properties
The draft genome size of strain JH-7 T is 4,842,937 bp with 61.2 mol% G + C content and contains 60 scaffolds. The genome properties and statistics are shown in Table 3. From a total of 4685 genes, 4504 (96.2%) are protein coding genes, 110 (2.3%) are pseudo genes and the rest are 71 predicted RNA genes, including 54 tRNA, 12 rRNAs and 5 ncRNA. In addition, 3729 (82.8%) protein coding genes are distributed into COG functional categories (Table 4).
EPS are long-chain polysaccharides consisting of branched, repeating units of sugars or sugar derivatives [35]. Stain JH-7 T could produce EPS and all essential proteins for EPS production are found in the genome.  Table S2) including the syntheses of UDP-glucose, UDP-galactose, UDP-GlcNAc and GDP-D-mannose (Fig. 4a). EPS assembly gene clusters were also found in the genome of strain JH-7 T [36] (Additional file 1: Table S3, Fig. 4b). Based on gene analysis, it is suggested that the EPS assembly in strain JH-7 T might belong to Wzx/Wzy-dependent pathway [37], e.g., repeat units are assembled by glycosyltransferases (EpsI) and translocated across the cytoplasmic membrane to periplasm by flippase (Wzx) [37] and WbaP [38]. Next, Wzy (RfaL), polysaccharide co-polymerase (GumC) and the outer membrane polysaccharide exporter (GumB) transports the polymerized repeat units to cell surface [37,39]. EPS has been reported to contribute to heavy metal removal/adsorption in bacteria [3][4][5][6]. Hence, the ability of EPS may contribute to Mn 2+ and Cd 2+ removal.
To gain more insight, the genomic features of strain JH-7 T is compared with the available genome P. salicylatoxidans KCT001 [7]. Strain JH-7 T has similar genome size (4.84 Mbp) and G + C content (61.2 mol%) compared to strain KCT001 (4.61 Mbp; 62.8 mol%). A total of 2408 core proteins are shared between the two strains. Strain JH-7 T has 1724 strain-specific CDSs. Figure 5 shows the genome comparison results of strain JH-7 T and strain KCT001 using CGview comparison tool [40]. Comparing to P. salicylatoxidans KCT001, strain JH-7 T was unable to utilize tetrathionate for chemolithoautotrophy (data not shown). However, it harbors high quantitative and diverse heavy metal resistance genes.

Conclusions
To the best of our knowledge, this study provides the first typical strain genomic information of the genus Pseudaminobacter and revealed a consistency of important characters between genotypes and phenotypes. Strain JH-7 T is resistant to multiple heavy metals and capable of removal Mn 2+ /Cd 2+ . Genome analysis reveal various genes responsible for multiple heavy metal resistance, which provides the genomic basis for this strain to adapt the harmful environment. Fig. 5 A graphical circular map of the comparison between strain P. manganicus JH-7 T and P. salicylatoxidans KCT001. From outside to center, rings 1, 4 show protein-coding genes colored by COG categories on forward/reverse strand; rings 2, 3 denote genes on forward/reverse strand; rings 5 show the CDS vs CDS BLAST results of strain JH-7 T with strain KCT001; ring 6 shows G + C % content plot and the innermost ring shows GC skew