- Short genome report
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High-quality-draft genome sequence of the multiple heavy metal resistant bacterium Pseudaminobacter manganicus JH-7T
Standards in Genomic Sciences volume 13, Article number: 29 (2018)
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.
Genus Pseudaminobacter was established by Kämpfer et al. in 1999 and contains three species represented by Pseudaminobacter salicylatoxidans BN12T (type species) , Pseudaminobacter defluvii THI 051T  and Pseudaminobacter manganicus JH-7T . The common characteristics of Pseudaminobacter strains are Gram-staining-negative, rod-shaped and aerobic [1, 2]. P. salicylatoxidans BN12T contains a peculiar ring-fission dioxygenase with the ability to cleave salicylate in 1, 2-position to 2-oxohepta-3, 5-dienedioic acid .
P. manganicus JH-7T was isolated from a sludge sample of a wastewater ditch in Dalong manganese mine in 2015 . It shows multiple heavy metal resistance and can effectively remove Mn2+ and Cd2+. 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 . Strain KCT001 can utilize tetrathionate as the substrate for sulfur-oxidizing chemolithotrophic growth . For better understanding the mechanism of bacterial resistance and removal of heavy metals, here we analyze the genome of P. manganicus JH-7T.
Classification and features
The phylogenetic relationship of P. manganicus JH-7T to the related members is shown in a 16S rRNA gene based neighbor-joining tree. Strain JH-7T is closely related to P. salicylatoxidans BN12T, P. defluvii THI 051T and P. salicylatoxidans KCT001 (Fig. 1). Strain JH-7T 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 . The major fatty acids are C18:1 ω7c, C19:0 cyclo ω8c and C16:0 and the G + C content is 61.2 mol% . 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 . Table 1 shows the general features of P. manganicus JH-7T.
The resistant levels of P. manganicus JH-7T to multiple metal(loid)s were tested with the MIC on LB agar plates incubated at 28 °C for 7 days. The MICs for MnCl2, CdCl2, PbCl2, CuCl2, ZnSO4 and NiSO4 are100, 2, 10, 5, 5 and 5 mmol/L respectively. The MICs for K2CrO4 and Na3AsO3 are both 0.1 mmol/L that are lower than the above six metals. Specifically, strain JH-7T could remove nearly 60% of 5 mmol/L Mn2+ and nearly 80% of 0.1 mmol/L Cd2+ (Fig. 3), respectively. In addition, strain JH-7T could produce EPS based on the aniline blue reaction incubated on LB agar in 3–7 days  (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 .
Genome sequencing information
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-7T 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-7T was grown under aerobic conditions in LB medium at 28 °C for 40 h. DNA extraction was performed using the QiAamp kit (Qiagen, Germany) as the manufacturer’s instructions. A NanoDrop Spectrophotometer 2000 was used to determine the quality and quantity of the DNA. Seven microgram of DNA was sent to Bio-broad Technogoly Co., Ltd., Wuhan, China for sequencing.
Genome sequencing and assembly
The genome of strain JH-7T was sequenced on Illumina Hiseq2000  and assembled by Bio-broad Technogoly Co., Ltd., Wuhan using SOAPdenovo v2.04 . 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 .
The draft genome was annotated through the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and genes were identified using the gene caller GeneMarkS+ with the similarity-based gene detection approach . The predicted CDSs were translated and were submitted to the Pfam protein family database  and KEGG database . The genes in internal clusters were performed by OrthoMCL [17, 18]. The protein function classification, transmembrane helices and signal peptides were predicted by WebMGA , TMHMM v. 2.0  and SignalP 4.1 , respectively. In addition, the CRISPRfinder program  was used to predict CRISPRs in the genome.
The draft genome size of strain JH-7T 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).
Insights from the genome sequence
Strain JH-7T could tolerant multiple heavy metals (Mn2+, Cd2+, Pb2+, Cu2+, Zn2+ and Ni2+) and remove Mn2+ and Cd2+, suggesting that it has developed a number of evolutionary strategies to adapt the mine environment. According to the genome annotation results, strain JH-7T harbors various putative proteins related to heavy metal(loid)s resistance including transporters, resistance proteins and metal reductases (Additional file 1: Table S1). MntH  and metal ABC transport system  are involved in cation uptake. Heavy metal-transporting ATPase is responsible for the efflux of Pb2+, Zn2+, Cd2+ and Ni2+ [25,26,27,28]. The genome contains Cu2+ efflux system CopABC , mercuric reductase MerA and regulator MerR . Athough the MICs for Cr6+ and As3+ are not high, the Cr6+ efflux protein ChrA [27, 31] and As3+ resistant proteins (ArsRHC and ACR3) [32,33,34] are present.
EPS are long-chain polysaccharides consisting of branched, repeating units of sugars or sugar derivatives . Stain JH-7T could produce EPS and all essential proteins for EPS production are found in the genome. Four complete nucleotide sugar synthesis (EPS precursor) pathways are identified based on KEGG analysis (Additional file 1: 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-7T  (Additional file 1: Table S3, Fig. 4b). Based on gene analysis, it is suggested that the EPS assembly in strain JH-7T might belong to Wzx/Wzy-dependent pathway , e.g., repeat units are assembled by glycosyltransferases (EpsI) and translocated across the cytoplasmic membrane to periplasm by flippase (Wzx)  and WbaP . 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 Mn2+ and Cd2+ removal.
To gain more insight, the genomic features of strain JH-7T is compared with the available genome P. salicylatoxidans KCT001 . Strain JH-7T 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-7T has 1724 strain-specific CDSs. Figure 5 shows the genome comparison results of strain JH-7T and strain KCT001 using CGview comparison tool . Comparing to P. salicylatoxidans KCT001, strain JH-7T was unable to utilize tetrathionate for chemolithoautotrophy (data not shown). However, it harbors high quantitative and diverse heavy metal resistance genes.
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-7T is resistant to multiple heavy metals and capable of removal Mn2+/Cd2+. Genome analysis reveal various genes responsible for multiple heavy metal resistance, which provides the genomic basis for this strain to adapt the harmful environment.
Minimal inhibition concentration
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This study was supported by National key research and development program of China (2016YFD0800702).
The authors declare that they have no competing interests.
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Table S1. Putative heavy metal(loid)s resistance proteins. Table S2. Putative nucleotide sugars biosynthesis proteins for EPS production. Table S3. Putative proteins for EPS production. (XLSX 11 kb)