Draft genomic sequence of a chromate- and sulfate-reducing Alishewanella strain with the ability to bioremediate Cr and Cd contamination

Alishewanella sp. WH16-1 (= CCTCC M201507) is a facultative anaerobic, motile, Gram-negative, rod-shaped bacterium isolated from soil of a copper and iron mine. This strain efficiently reduces chromate (Cr6+) to the much less toxic Cr3+. In addition, it reduces sulfate (SO42−) to S2−. The S2− could react with Cd2+ to generate precipitated CdS. Thus, strain WH16-1 shows a great potential to bioremediate Cr and Cd contaimination. Here we describe the features of this organism, together with the draft genome and comparative genomic results among strain WH16-1 and other Alishewanella strains. The genome comprises 3,488,867 bp, 50.4 % G + C content, 3,132 protein-coding genes and 80 RNA genes. Both putative chromate- and sulfate-reducing genes are identified.

Alishewanella sp. WH16-1 was isolated from mining soil in 2009. This strain could resist to multiple heavy metals. During cultivation, it could efficiently reduce the toxic chromate (Cr 6+ ) to the much less toxic and less bioavaliable Cr 3+ . It could also reduce sulfate (SO 4 2− ) to S 2− . When Cd 2+ was present, the S 2− reacted with Cd 2+ and precipitated as CdS. These characteristics made strain WH16-1 a great potential for bioremediate Cr and Cd contamination. In pot experiments of rice, tobacco and Chinese cabbage, with the addition of the bacterial culture, the amount of Cr and Cd in the plants decreased significantly [14]. Sequencing the genome of WH16-1 and comparing its attributes with the other Alishewanella genomes would provide a means of establishing the molecular determinants required for chromate/sulfate reduction, heavy metal resistance and pectin degradation, and for better application of these strains. Here we report the high quality draft genomic information of strain WH16-1 and compare it to the three sequenced Alishewanella genomes.
Interestingly, the strain could reduce 1 mmol/L Cr 6+ (added as K 2 CrO 4 ) in 36 h and remove 60 μmol/L Cd 2+ (added as CdCl 2 ) in 60 h (by the production of precipitated CdS [20] in LB liquid medium) (Fig. 3). In addition, this strain is tolerant to multi-metal(loids). The minimal inhibition concentration tests for different heavy metals were carried out on LB agar plates and   These evidence codes are from the Gene Ontology project [56] IDA Inferred from Direct Assay, 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) a Evidence codes Fig. 3 Cr 6+ and Cd 2+ removed by Alishewanella sp. WH16-1. Control stands for null LB medium. Strain WH16-1 was incubated until OD 600 reach 1.0, and then amended with K 2 CrO 4 (1 mmol/L) and CdCl 2 (0.06 mmol/L), respectively. The cultures were removed at 12 h intervals. After centrifuging at 12,000 rpm for 2 min, the supernatant was used to determine the residual concentration of Cr 6+ and Cd 2+ . The concentration of Cr 6+ and Cd 2+ were measured by the UV spectrophotometer (DU800, Beckman, CA, USA) with the colorimetric diphenylcarbazide (DPC) method [46] and the atomic absorption spectrometry AAS, respectively incubated at 37°C for 2 days. The MICs for K 2 CrO 4 , CdCl 2 , PbCl 2 , CuCl 2 and Na 3 AsO 3 are 45, 0.08, 10, 1 and 1 mmol/L, respectively.

Genome sequencing information
Genome project history Strain WH16-1 was selected for genome sequencing based on its ability to reduce Cr 6+ and SO 4 2− and preliminary application for soil Cr and Cd bioremediation. Since 2009, this strain has been used in both basic and bioremediation studies and the results are very promising. It was sequenced by Majorbio Bio-pharm Technology Co., Ltd, Shanghai, China. The genome sequencing and assembly information of the project is given in

Growth conditions and genomic DNA preparation
A single colony of strain WH16-1 was incubated into 50 ml LB medium and grown aerobically at 37°C for 36 h with 150 rpm shaking. The cells were collected by centrifugation. The DNA was extracted, concentrated and purified using the QiAamp kit (Qiagen, Germany). A NanoDrop Spectrophotometer 2000 was used to determine the quality and quantity of the DNA. Six micrograms of DNA was sent to Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China) for sequencing.

Genome sequencing and assembly
The genome sequencing of strain WH16-1 was performed on an Illumina Hiseq2000 [21] and assembled by Majorbio Bio-pharm Technology Co., Ltd, Shanghai, China. An Illumina standard shotgun library was constructed and sequenced, which generated 12,683,662 reads totaling 1,281,049,862 bp. All original sequence data can be found at the NCBI Sequence Read Archive [22]. The following steps were performed for removing low quality reads: (1) removed the adapter o reads; (2) cut the 5′ end bases which were not A, T, G, C; (3) filtered the reads which have a quality score lower than 20; (4) filtered the reads which contained N more than 10 %; and (5) removed the reads which have the length less than 25 bp after processed by the previous four steps. The reads were assembled into 156 contigs using SOAPdenovo v1.05 [23]. A total of 149 contigs were obtained after removing the contigs < 200 bp. The total size of the genome is 3,488,867 bp and the final assembly is based on 1,205 Mbp of Illumina data which provides a coverage of 345.3 × .

Genome annotation
The draft genome of WH16-1 was annotated through the NCBI PGAP, which combines the gene caller GeneMarkS + [24] with the similarity-based gene detection approach. Protein function classification was performed by WebMGA [25] with E-value cutoff of 1-e10. The transmembrane helices were predicted by TMHMM v. 2.0 [26]. Signal peptides in the genome were predicted by SignalP 4.1 [27]. The translations of the predicted CDSs were also used to search against the Pfam protein family database with E-value cutoff of 1-e5 [28] and the KEGG database [29]. Internal gene clustering was performed by OrthoMCL using Match cutoff of 50 % and E-value Exponent cutoff of 1-e5 [30,31].

Genome properties
The whole genome of strain WH16-1 is 3,488,867 bp in length, with an average G + C content of 50.4 %, and is distributed in 149 contigs (>200 bp). The genome properties and statistics are summarized in Table 3. There are 80 predicted RNA including 73 tRNA, 5 rRNAs and 2 ncRNA. In addition, a total of 3,132 protein-coding  Table 4.

Insights from the genome sequence
Strain WH16-1 has the genes for a complete SO 4 2− reduction pathway according to the KEGG analysis, including CysPUWA, CysN, CysD, CysC, CysH and CysIJ (Additional file 1: Figure S2; Additional file 2: Table S1). This pathway contained several steps: 1) the SO 4 2− is uptaken by the putative CysPUWA into the cell [32]; 2) the intracellular SO 4 2− is acetylated to adenylylsulphate (APS) by sulfate adenylyltransferases CysN and CysD [33]; 3) the APS is phosphorylated to phosphoadenylylsulphate (PAPS) by APS kinase CysC and, 4) the PAPS is reduced to sulfite (SO 3 2− ) by PAPS reductase CysH [33] and, 5) the SO 3 2− is finally reduced to sulfide (S 2− ) by sulfite reductase CysIJ [33]. Strain WH16-1 was able to remove Cd 2+ most probably due to the reaction between S 2− and Cd 2+ to form the precipitated CdS [20]. For Cr 6+ reduction, a putative chromate reductase YieF was found (Additional file 2: Table S1). YieF was reported to responsible for the reduction of Cr 6+ in cytoplasm [34]. An individual chromate transport gene chrA and a chromate resistance cluster including chrBAC, hp1, chrF, lppy/lpqo, hp2 and ABC transport permease gene are found in the genome (Additional file 2: Table S2) [35,36]. Currently, we have disrupted the chrA (AAY72_02075) and the ABC transport permease genes, respectively. The chromate resistance levels were both decreased significantly in the chrA and ABC transport permease gene mutant strains (data not shown).
In addition, various heavy metal transformation and resistance determinants are identified in the genome of strain WH16-1 Several transporters (MntH, CzcA and ZntA) that might be involved in the efflux of Cd 2+ , Pb 2+ and Zn 2+ are found [37][38][39]. Cu 2+ , As 3+ and Hg 2+ resistance determinants are also present, such as Cu transporter ATPase [40], Cu 2+ resistance system CopABCD [41], Ars [42] and Pst [43] systems for arsenic resistance and MerT-PADE system for mercury resistance [44] (Additional file 2:  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 73 pseudogenes, 73 tRNA genes, 5 rRNAs and 2 ncRNA  (Fig. 4). The G + C content of strain WH16-1 (50.4 %) is also consistent with the other Alishewanella strains (A. jeotgali KCTC 22429 T , 50.7 %, A. aestuarii B11 T , 51 % and A. agri BL06 T , 50.6 %). Strain WH16-1 shares 2,474 proteins with the other three Alishewanella genomes and has 217 strain-specific proteins (Fig. 5). The 2,474 core genes include yieF, chrA, the ten genes in the whole sulfate reduction pathway and most of the heavy metal resistance genes (Additional file 2: Table S1-S2). Strain WH16-1 possesses the higher number of chromatin resistance genes compared to the other three strains.
In addition, A. agri BL06 T , A. jeotgali KCTC 22429 T and A. aestuarii B11 T were all reported to have the ability of degrading pectin and possess pectin degradation genes [8][9][10][11]. However, unlike strains BL06 T , KCTC 22429 T and B11 T , strain WH16-1 was unable to degrade pectin and the pectin degradation genes are not found in its genome. Since strain WH16-1 was isolated from a heavy metal rich environment, it may be more relevant for bioremediation of heavy metal contamination. The pectin degradation genes may be lost during the evolution.

Conclusions
The genomic results of Alishewanella sp. WH16-1 reveal correlation between the gene types and some phenotypes. The strain harbors various genes responsible for sulfate transport and reduction, chromate reduction and resistance of multi-heavy metals. These observations provide insights into understand the molecular mechanisms of heavy metals. In addition, all of the analyzed Alishewanella genomes have putative sulfate and chromate reduction genes, which indicates that sulfate and chromate reduction may be the important characters of the Alishewanella strains. Thus, these strains have a great potential for application in bioremediation of heavy metal or other industrial wastes.

Acknowledgments
This work was supported by the National Natural Science Foundation of China (31470226).
Authors' contributions XX carried out biochemical tests, sequence analysis and preparation of the draft. JL participated in the metal resistance test and phylogenetic analysis. GZ and LL did metal reduction and metal removing tests. HW and BX conducted strain isolation. GW and SL participated in research design and helped to draft the manuscript. All authors read and approved the final manuscript.

Competing interests
The abilities to reduce Cr 6+ and immobilize Pb 2+ and Cd 2+ of strain WH16-1 were described in China Patent, 2015; CN 104,928,213 A [14]. Due to these abilities, strain WH16-1 has a great potential for application in bioremediation of heavy metal. All the authors of this paper and the inventors of the patent [14] declare that they have no commercial or non-commercial competing interests.