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Draft genomic sequence of a chromate- and sulfate-reducing Alishewanella strain with the ability to bioremediate Cr and Cd contamination
Standards in Genomic Sciences volume 11, Article number: 48 (2016)
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 (SO4 2−) 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.
The genus Alishewanella was established by Vogel et al., in 2000 with Alishewanella fetalis as the type species. It belongs to the family Alteromonadaceae of the class Gammaproteobacteria . So far, Alishewanella contains six species: A. fetalis , Alishewanella aestuarii , Alishewanella jeotgali , Alishewanella agri and Alishewanella tabrizica and Alishewanella solinquinati [1–6]. The common characteristics of the genus Alishewanella are Gram-negative, rod-shaped and positive for oxidase and catalase [1–6]. Some Alishewanella strains were able to degrade pectin which is applicable in bioremediation of food industrial wastes [7–11]. Three Alishewanella strains ( A. aestuarii B11T , A. jeotgali KCTC 22429T and A. agri BL06T) have been sequenced and the pectin degradation pathway was found in their genomes [8–11]. Some strains of Alishewanella were reported to tolerate arsenic [12, 13], but the ability of Alishewanella strains to resist or transform other heavy metal(loids) have not been reported.
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 (Cr6+) to the much less toxic and less bioavaliable Cr3+. It could also reduce sulfate (SO4 2−) to S2−. When Cd2+ was present, the S2− reacted with Cd2+ 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 . 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.
Classification and features
Phylogenetic analysis was performed by the neighbor-joining method based on 16S rRNA gene sequences. Strain WH16-1 is closely related to A. agri BL06T (99.7 %) and A. fetalis CCUG 30811T (99.1 %) (Fig. 1). A similar result was obtained based on gyrase B gene (gyrB) sequences (Additional file 1: Figure S1). The gyrB sequences has been successfully used to establish phylogenetic relatedness in Alishewanella , Pseudomonas , Acinetobacter , Vibrio , Bacillus  and Shewanella .
Strain WH16-1 is Gram-negative, facultatively anaerobic, motile and rod-shaped (0.3–0.5 × 1.2–2.0) (Fig. 2). Colonies are white, circular and raised on LB agar plate. Growth occurs at 4–40 °C, in 0–8 % (w/v) NaCl and at pH 4–11. Optimal growth occurs at 37 °C, 1 % (w/v) NaCl and at pH 6.0–8.0 (Table 1). It can grow in LB, trypticase soy broth and R2A medium. API 20NE test (bioMérieux) in combination of traditional classification methods were used to analyze the physiological and biochemical characteristics. Strain WH16-1 is positive for oxidase and catalase activities and is able to reduce nitrate to nitrite. It is positive for aesculinase, gelatinase, arginine dihydrolase and urease but is negative for indole and β-galactosidase. It can use D-sucrose and maltose as the sole carbon sources. It cannot assimilate D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, gluconate, capric acid, adipic acid, malic acid, trisodium citrate or phenylacetic acid. Most of these biochemical characteristics are similar to the other Alishewanella strains [1–6]. However, unlike some Alishewanella strains [8–11], strain WH16-1 cannot degrade pectin.
Interestingly, the strain could reduce 1 mmol/L Cr6+ (added as K2CrO4) in 36 h and remove 60 μmol/L Cd2+ (added as CdCl2) in 60 h (by the production of precipitated CdS  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 incubated at 37 °C for 2 days. The MICs for K2CrO4, CdCl2, PbCl2, CuCl2 and Na3AsO3 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 Cr6+ and SO4 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 Table 2. The final genome consists of 133 scaffolds with approximately 345.3 × coverage. The draft genome sequence was annotated by NCBI PGAP. The genome sequence is available in DDBJ/EMBL/GenBank under accession number LCWL00000000.
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  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 . 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 . 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 × .
The draft genome of WH16-1 was annotated through the NCBI PGAP, which combines the gene caller GeneMarkS+  with the similarity-based gene detection approach. Protein function classification was performed by WebMGA  with E-value cutoff of 1-e10. The transmembrane helices were predicted by TMHMM v. 2.0 . Signal peptides in the genome were predicted by SignalP 4.1 . The translations of the predicted CDSs were also used to search against the Pfam protein family database with E-value cutoff of 1-e5  and the KEGG database . Internal gene clustering was performed by OrthoMCL using Match cutoff of 50 % and E-value Exponent cutoff of 1-e5 [30, 31].
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 genes are identified. The distribution of genes into COGs functional categories is presented in Table 4.
Insights from the genome sequence
Strain WH16-1 has the genes for a complete SO4 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 SO4 2− is uptaken by the putative CysPUWA into the cell ; 2) the intracellular SO4 2− is acetylated to adenylylsulphate (APS) by sulfate adenylyltransferases CysN and CysD ; 3) the APS is phosphorylated to phosphoadenylylsulphate (PAPS) by APS kinase CysC and, 4) the PAPS is reduced to sulfite (SO3 2−) by PAPS reductase CysH  and, 5) the SO3 2− is finally reduced to sulfide (S2−) by sulfite reductase CysIJ . Strain WH16-1 was able to remove Cd2+ most probably due to the reaction between S2− and Cd2+ to form the precipitated CdS . For Cr6+ reduction, a putative chromate reductase YieF was found (Additional file 2: Table S1). YieF was reported to responsible for the reduction of Cr6+ in cytoplasm . 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 Cd2+, Pb2+ and Zn2+ are found [37–39]. Cu2+, As3+ and Hg2+ resistance determinants are also present, such as Cu transporter ATPase , Cu2+ resistance system CopABCD , Ars  and Pst  systems for arsenic resistance and MerTPADE system for mercury resistance  (Additional file 2: Table S2).
Strain WH16-1 has a genome size (3.49 Mbp), similar to A. jeotgali KCTC 22429T (3.84 Mbp), A. aestuarii B11T (3.59 Mbp) and A. agri BL06T (3.49 Mbp) [8–10] (Fig. 4). The G + C content of strain WH16-1 (50.4 %) is also consistent with the other Alishewanella strains ( A. jeotgali KCTC 22429T , 50.7 %, A. aestuarii B11T, 51 % and A. agri BL06T, 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 BL06T, A. jeotgali KCTC 22429T and A. aestuarii B11T were all reported to have the ability of degrading pectin and possess pectin degradation genes [8–11]. However, unlike strains BL06T, KCTC 22429T and B11T, 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.
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.
Prokaryotic Genome Annotation Pipeline
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This work was supported by the National Natural Science Foundation of China (31470226).
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.
The abilities to reduce Cr6+ and immobilize Pb2+ and Cd2+ of strain WH16-1 were described in China Patent, 2015; CN 104,928,213 A . 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  declare that they have no commercial or non-commercial competing interests.
Figure S1. Phylogenetic relationships of Alishewanella sp. WH16-1 based on gyrB sequences. The analysis was performed by MEGA 6.0  with NJ algorithm and 1,000 bootstrap repetitions were computed to estimate the reliability of the tree. The gyrB gene of strain WH16-1 is the gene sequence coding for AAY72_13600. Figure S2. The putative sulfate transport and reduction pathway in Alishewanella sp. WH16-1. APS stands for adenylylsulphate, PAPS stands for phosphoadenylylsulphate. The locus tag numbers of the predicted proteins (CysP, CysU, CysW, CysA, CysN, CysD, CysC, CysH CysJ and CysI) are AAY72_14890, AAY72_14885, AAY72_14880, AAY72_14875, AAY72_03865, AAY72_03870, AAY72_07290, AAY72_07265, AAY72_07255 and AAY72_07260, respectively. (DOCX 355 kb)
Table S1. Putative proteins involved in sulfate and chromate reduction. Table S2. Putative proteins involved in heavy metal resistance. (XLSX 14 kb)
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Xia, X., Li, J., Liao, S. et al. Draft genomic sequence of a chromate- and sulfate-reducing Alishewanella strain with the ability to bioremediate Cr and Cd contamination. Stand in Genomic Sci 11, 48 (2016). https://doi.org/10.1186/s40793-016-0169-3
- Chromate-reducing bacterium
- Sulfate-reducing bacterium