Adaptive Evolution of ExtremeAcidophile Sulfobacillusthermosulfidooxidans Potentially Drivenby Horizontal Gene Transfer and GeneLoss

Xian Zhang,a,b Xueduan Liu,a,b Yili Liang,a,b Xue Guo,a Yunhua Xiao,a Liyuan Ma,a

Bo Miao,a,b Hongwei Liu,a,b Deliang Peng,c Wenkun Huang,c Yuguang Zhang,d

Huaqun Yina,b

School of Minerals Processing and Bioengineering, Central South University, Changsha, Chinaa; Key Laboratoryof Biometallurgy of Ministry of Education, Central South University, Changsha, Chinab; State Key Laboratory forBiology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of AgriculturalSciences, Beijing, Chinac; Institute of Forestry Ecology, Environment and Protection, and the Key Laboratory ofForest Ecology and Environment of State Forestry Administration, Chinese Academy of Forestry, Beijing,Chinad

ABSTRACT Recent phylogenomic analysis has suggested that three strains isolatedfrom different copper mine tailings around the world were taxonomically affiliatedwith Sulfobacillus thermosulfidooxidans. Here, we present a detailed investigation oftheir genomic features, particularly with respect to metabolic potentials and stresstolerance mechanisms. Comprehensive analysis of the Sulfobacillus genomes identi-fied a core set of essential genes with specialized biological functions in the survivalof acidophiles in their habitats, despite differences in their metabolic pathways. TheSulfobacillus strains also showed evidence for stress management, thereby enablingthem to efficiently respond to harsh environments. Further analysis of metabolicprofiles provided novel insights into the presence of genomic streamlining, high-lighting the importance of gene loss as a main mechanism that potentially contrib-utes to cellular economization. Another important evolutionary force, especially inlarger genomes, is gene acquisition via horizontal gene transfer (HGT), which mightplay a crucial role in the recruitment of novel functionalities. Also, a successful inte-gration of genes acquired from archaeal donors appears to be an effective way ofenhancing the adaptive capacity to cope with environmental changes. Taken to-gether, the findings of this study significantly expand the spectrum of HGT and ge-nome reduction in shaping the evolutionary history of Sulfobacillus strains.

IMPORTANCE Horizontal gene transfer (HGT) and gene loss are recognized as majordriving forces that contribute to the adaptive evolution of microbial genomes, al-though their relative importance remains elusive. The findings of this study suggestthat highly frequent gene turnovers within microorganisms via HGT were necessaryto incur additional novel functionalities to increase the capacity of acidophiles toadapt to changing environments. Evidence also reveals a fascinating phenomenonof potential cross-kingdom HGT. Furthermore, genome streamlining may be a criticalforce in driving the evolution of microbial genomes. Taken together, this study pro-vides insights into the importance of both HGT and gene loss in the evolution anddiversification of bacterial genomes.

KEYWORDS Sulfobacillus thermosulfidooxidans, adaptive evolution, genomicstreamlining, horizontal gene transfer

Received 11 November 2016 Accepted 13January 2017

Accepted manuscript posted online 23January 2017

Citation Zhang X, Liu X, Liang Y, Guo X, Xiao Y,Ma L, Miao B, Liu H, Peng D, Huang W, Zhang Y,Yin H. 2017. Adaptive evolution of extremeacidophile Sulfobacillus thermosulfidooxidanspotentially driven by horizontal gene transferand gene loss. Appl Environ Microbiol 83:e03098-16. https://doi.org/10.1128/AEM.03098-16.

Editor Harold L. Drake, University of Bayreuth

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Huaqun Yin,yinhuaqun@gmail.com.

EVOLUTIONARY AND GENOMIC MICROBIOLOGY

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For several decades, it has been acknowledged that genomic rearrangements,including gene duplication and horizontal gene transfer (HGT), play critical roles in

driving the adaptive evolution of microbial genomes in response to intense environ-mental perturbations (1–5). Great attention has thus been paid to gene and/or genomeduplication, which represent dominant forces for functional innovation, includingneofunctionalization and subfunctionalization (6, 7). Furthermore, the HGT process isconsidered a prevalent evolutionary mechanism that contributes to genomic diversi-fication, species-level identification, and a trophic lifestyle (8, 9). Recently, large-scalestudies based on increasing genomic data have significantly expanded the spectrum ofgenome reduction into a pervasive source of genetic modifications that potentiallycause adaptive phenotypic diversity (10). Extensive gene loss events were observedacross a wide range of organisms, including prokaryotes (11–15), protists (16), fungi (17,18), plants (19), and even animals (20–23), thereby serving as robust evidence tosupport the pervasiveness of gene loss in all life kingdoms (10). However, the relativecontributions of different evolutionary forces that shape the organization, structure,and diversification of microbial genomes remain elusive.

Extreme environments are generally known as almost insurmountable physical andchemical barriers to most life forms (24). However, microorganisms have been widelyfound in habitats that are characterized by harsh attributes, such as high temperature(25), high salinity (26), low pH (27, 28), or under strictly anaerobic conditions (29). Toadapt to short-term or longstanding environmental stresses, microbes may have un-dergone frequent gene turnover. Sulfobacillus spp., which are Gram-positive spore-forming bacteria, are taxonomically affiliated with the order Clostridiales (30). Despitetheir poorly characterized low abundance compared to the dominant iron-oxidizingLeptospirillum spp. or certain members of the heterotrophic Thermoplasmatales, mem-bers of the genus Sulfobacillus are metal-tolerant, mildly thermophilic, or thermotoler-ant acidophiles that promote sulfide mineral dissolution and ubiquitously occur invarious acidic habitats (30, 31), such as acid mine drainage (32), acid thermal springs(33), hydrothermal vents (34), and industrial bioleaching operations (35). In the pastseveral decades, a number of papers have discussed issues related to key metabolicfeatures within several isolated Sulfobacillus species, namely, S. thermosulfidooxidans(36), S. acidophilus (33), S. thermotolerans (37), S. sibiricus (38), and S. benefaciens (39). Assuch, the Sulfobacillus genus represents an intriguing consortium of microbes withdisparate ecological distribution and physiological preferences.

In the advent of high-throughput sequencing and development of computationallyderived approaches, an increasing number of available genomes of acidophiles havebeen sequenced, thereby providing the first glimpses into the genomic characteristicsof acidophilic life under a range of environmental conditions (40). To date, the genomicfeatures of two S. acidophilus strains (34, 41) and three S. thermosulfidooxidans strains(31, 42) have been investigated. Furthermore, the draft genomes of several Sulfobacillusstrains have been assembled from metagenomic data (30), including a strain of S.benefaciens, a strain of S. thermosulfidooxidans, and three strains with no culturedrepresentatives. Comparative genomics have yielded valuable insights into the physi-ological diversity, ecological roles, and genome evolution of environmental microor-ganisms (9, 43, 44). Genome-guided studies have revealed novel perspectives on thegenetic features within genus Sulfobacillus; however, our understanding of their evo-lutionary adaptation and the underlying mechanisms of species-level identification islimited.

Here, we present a detailed genomic comparison of various Sulfobacillus species,including three genomes that were sequenced in this study and five other genomesthat have been previously submitted to a public database. Our study provides evidencethat these genomes underwent horizontal gene transfer (HGT) and functional recruit-ment. Findings also showed the prevalence of gene loss coupled with genome stream-lining. Taken together, our results suggest that both HGT and gene loss processesrepresent important driving forces that may have contributed to the evolution ofmicrobial genomes.

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RESULTS AND DISCUSSIONPhylogeny and overview of genome features. The constructed dendrogram

based on the 16S rRNA gene sequences grouped the three newly sequenced strainsinto a phyletic cluster that included S. thermosulfidooxidans (�99% 16S rRNA genesimilarity; Fig. 1 and Table S1). Accordingly, five Sulfobacillus genomes were selected forphylogenomic analysis (Table 1). Comparison of average nucleotide identity (ANI)based on BLAST (ANIb) and MUMmer (ANIm), as well as regression of the tetranucle-otide composition (Tetra), was conducted to further infer their phylogenetic relation-ship. The high ANIb, ANIm, and Tetra values between the new strains (DX, ZBY, and ZJ)and recognized S. thermosulfidooxidans strains Cutipay and ST strongly indicated their

FIG 1 Phylogeny showing relationships among three newly sequenced strains and published Sulfobacillus species using their 16S rRNA sequences. In themaximum likelihood phylogenetic tree, nodes with greater than 50% bootstrap support are shown. These three new strains used in this study are shown inbold.

TABLE 1 Genome-based phylogenetic indicators of Sulfobacillus strainsa

Organism

ANIb (%) Tetra ANIm (%)

DX ZBY ZJ DX ZBY ZJ DX ZBY ZJ

Sulfobacillusthermosulfidooxidans

Cutipay 97.10 97.11 97.10 0.994 0.994 0.994 97.30 97.30 97.30ST 96.99 96.99 96.99 0.999 0.999 0.999 97.22 97.21 97.21CBAR-13 87.41 87.41 87.41 0.989 0.989 0.989 88.98 88.98 88.97

Sulfobacillus acidophilusDSM 10332 66.37 66.43 66.45 0.697 0.696 0.696 91.99 91.78 91.78TPY 65.76 65.79 65.76 0.697 0.697 0.697 92.06 92.06 92.04

aThe average nucleotide identity (ANI) based on BLAST (ANIb) and MUMmer (ANIm), as well asoligonucleotide signature frequencies (Tetra), were calculated using the program JSpecies. Values of ANIband ANIm below 96% and TETRA values below 0.99 indicate that strains used in this study belong todifferent species.

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affiliation. Furthermore, the ANIb, ANIm, and Tetra values of strain CBAR-13 supportedthe hypothesis that this strain belongs to another Sulfobacillus species that is distinctfrom S. thermosulfidooxidans (Table 1). Based on these phylogenetic indicators, thethree novel isolated strains were designated S. thermosulfidooxidans strains DX, ZBY,and ZJ, and S. thermosulfidooxidans CBAR-13 was thereafter referred to as Sulfobacillussp. CBAR-13.

Based on their complete genomes, we then excluded Sulfobacillus acidophilusstrains TPY and DSM 10332 from our evaluation of genome completeness. Othergenome assemblies of Sulfobacillus strains were assessed by CheckM, suggesting thatthese were near complete. The details on the Sulfobacillus genomes are shown in Table2. Genome sizes and predicted protein-coding sequence (CDS) counts significantlyvaried among Sulfobacillus strains. Further inspection indicated slight variations in thetotal genome size of S. thermosulfidooxidans strains DX, ZBY, and ZJ (approximate size,3.18 Mb), much smaller than those seen in other Sulfobacillus strains (Table 2). S.thermosulfidooxidans Cutipay had the largest genome size (3.86 Mb) and number ofCDSs (4,250), whereas S. thermosulfidooxidans DX had the smallest genome size (3.18Mb) and number of CDSs (3,211). These findings indicated that numerous accessorygenes in S. thermosulfidooxidans Cutipay were likely acquired by HGT, similar to earliercomparative genomic analyses of Sinorhizobium strains (45, 46).

Comparison of Sulfobacillus genome contents. Mira et al. (47) reported that thevariations in the genome sizes of bacteria were attributable to differences in genenumber, in which bacterial genomes are tightly packed, with most regions consistingof functional protein-coding sequences. Functional assignment based on COG classifi-cation revealed that the newly sequenced strains consisted of a high number of genesthat could be assigned to COG categories C (energy production and conversion), E(amino acid transport and metabolism), and G (carbohydrate transport and metabolism;Table S2). Similar to most microorganisms, these essential genes allow acidophiles toefficiently take up nutrients from the environment, as well as maintain a basic lifestyle.Konstantinidis and Tiedje (48) previously reported that genes in large genomes weredisproportionally enriched in regulatory and secondary metabolisms. Similarly, rela-tively larger genomes were predicted to harbor more CDSs that could be assigned tothe COG category Q (secondary metabolites biosynthesis, transport, and catabolism)than in medium- and small-sized genomes (Table S2).

TABLE 2 General features of bacterial genomes of Sulfobacillus strains

Organism Geographic originGenomesequence status

Genomesize (bp)

Coverage(�)

Completeness(%)a

No. ofcontigs

GC content(%)

Sulfobacillusthermosulfidooxidans

ZBY Copper mine tailings, Chambishi,Zambia

Draft 3,180,484 140 99.00 24 48.47

ZJ Copper mine tailings, Fujian, China Draft 3,180,801 136 99.00 20 48.47DX Copper mine tailings, Jiangxi, China Draft 3,180,071 132 99.00 33 48.47ST Acid hot spring, Yunnan, China Draft 3,325,386 98.00 97 48.35Cutipay Acidic mining environments,

northern ChileDraft 3,858,948 116 99.00 47 49.32

Sulfobacillus sp. CBAR-13 Escondida mine, Antofagasta, Chile Draft 3,828,023 99.00 3 48.91

Sulfobacillus acidophilusTPY Hydrothermal vent, Pacific Ocean Complete 3,551,206 26 1 56.76DSM 10332 Coal spoil heap, United Kingdom Complete 3,557,831 168 2 56.75

aThe completeness of draft genome sequences were evaluated by CheckM.bGene prediction was performed using the platform RAST.cUnpublished data of S. Marin-Eliantonio, A. Moya-Beltran, M. Acosta-Grinok, F. Issotta, P. Galleguillos, J. P. Cardenas, V. Zepeda, L. G. Acuna, D. Cautivo, D. S. Holmes,R. Quatrini, and C. Demergasso.

(Continued on next page)

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To identify the pangenome of Sulfobacillus strains, a total of 9,049 CDSs obtainedfrom the three novel genomes plus five reference genomes were clustered by PanOCTusing a 65% sequence identity cutoff. We acquired a core genome of 671 CDSs, whichwas much smaller than that of a pangenome, indicating a significant difference amongvarious Sulfobacillus species. Apart from the core genome, these flexible genes aredispensable and are probably not essential to basic bacterial lifestyle, but they mayconfer niche adaptation. The results showed that the genomes of S. thermosulfidooxi-dans strains harbored relatively fewer group-specific genes than S. acidophilus strainsand Sulfobacillus sp. CBAR-13 (Fig. 2A). Similar to the findings of previous studies thatconducted pangenome analyses (43, 45, 49), the highest number of core genes wasobserved in the functional category J (translation, ribosomal structure, and biogenesis).Furthermore, functional assignment based on COG classification revealed that most

TABLE 2 (Continued)

Maximum contiglength (bp)

Minimum contiglength (bp)

N50 length(bp)

N90 length(bp)

No. of RNA genes

No. of predictedCDSsb

Referenceor source

5SrRNA

16SrRNA

23SrRNA tRNA

716,967 218 404,938 88,978 3 1 1 51 3,211 This study

688,926 231 452,891 108,775 2 1 1 51 3,213 This study688,914 1,285 171,016 52,886 1 1 1 51 3,227 This study408,384 201 139,115 34,608 0 1 1 48 3,408 31671,675 1,007 383,918 38,694 5 5 8 51 4,250 42

3,195,503 25,061 3,195,503 607,459 3 3 3 52 4,123 Unpublisheddatac

3,551,206 3,551,206 3,551,206 3,551,206 0 5 5 52 3,894 343,472,898 84,933 3,472,898 3,472,898 0 5 5 53 3,891 41

FIG 2 Pangenome analysis of Sulfobacillus strains. The shared and strain-specific genes among Sulfobacillusgenomes were calculated using PanOCT (A) and were then assigned to COG categories (B). (C) Numbers of coregenome and unique genes within S. thermosulfidooxidans strains and Sulfobacillus sp. CBAR-13 are depicted in thesix-way Venn diagram. Also, shared and strain-specific genes were used to be aligned against the COG database.

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genes belonged to COG categories C and E (Fig. 2B). Differences in functions encodedby group-specific genes were also identified. A large number of CDSs (1,978) wereshared by S. thermosulfidooxidans strains and Sulfobacillus sp. CBAR-13, thereby high-lighting the most common traits between these two groups.

We further observed orthologous genes within recognized S. thermosulfidooxidansstrains and Sulfobacillus sp. CBAR-13. Approximately 2,649 genes were shared by allstrains (Fig. 2C). Around 1,174 genes were exclusively identified in Sulfobacillus sp.CBAR-13 and thus described as strain specific, further indicating their genomic differ-ences from other S. thermosulfidooxidans strains. In addition, the number of uniquegenes in S. thermosulfidooxidans genomes varied from 9 to 938. Further analysis basedon COG classification showed that the four most abundant genes shared by S. ther-mosulfidooxidans strains and Sulfobacillus sp. CBAR-13 were assigned to COG categoriesE, C, G (carbohydrate transport and metabolism), and J (Fig. 2C).

Identification of inferred metabolic traits and niche adaptation. Sulfobacillusspp., known as a cohort of mildly thermophilic or thermotolerant acidophiles, arefrequently found in various acidic settings worldwide. They are facultative anaerobesthat utilize energy and electrons derived from aerobic oxidation of iron and a widerange of inorganic sulfur compounds for the assimilation of organic and/or inorganiccarbon, as well as other anabolic metabolisms (30, 37, 38). According to the annotationresults of the KEGG Automatic Annotation Server (50), the metabolic potentials ofSulfobacillus strains were reconstructed and compared with each other to investigateshared metabolic and species- and/or strain-specific pathways (Table S3). Also, in thiscontext, a comparison of predicted stress tolerance mechanisms was performed.

(i) Comparison of central metabolisms. Sulfobacillus spp. are known as mix-otrophic acidophiles that are capable of assimilating organic and inorganic carbon (30,34, 42, 51). Similar to the published Sulfobacillus genomes (30, 31), a full suite of genesinvolved in the Calvin-Benson-Bassham cycle were predicted in all strains (Fig. 3 andTable S4), e.g., form I ribulose 1,5-bisphosphate carboxylase (RuBisCO), composed ofeight large and eight small subunits (27). In chemolithoautotrophic acidophiles, such asAcidithiobacillus and Ferrovum spp., a gene cluster potentially encoding several copies

FIG 3 Schematic representation of the predicted metabolic potentials and adaptive strategies for environmental stresses within Sulfobacillusstrains. The potential metabolic traits were focused on carbon metabolism, nitrogen uptake, sulfur metabolism, iron oxidation, as well as hydrogenutilization. Stress management mechanism was discussed, including cell mobility, acidic stress tolerance, and heavy metal resistance. Thecorresponding genes are listed in Table S4. CBB, Calvin-Benson-Bassham cycle; CoA, coenzyme A; cyt, cytochrome; Hyd Grp., hydrogenase group.

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of carboxysome shell proteins, carboxysome-associated carbonic anhydrase, andRuBisCO were identified (9, 27, 43, 44). These microorganisms utilize carboxysome-associated carbonic anhydrase to elevate the concentration of carbon dioxide near theRuBisCO via conversion of accumulated cytoplasmic bicarbonate into CO2 (52), therebyenabling efficient utilization of the limited available carbon in the environment. How-ever, no evidence supported the existence of a corresponding gene/gene cluster inSulfobacillus strains. Instead, genes encoding putative carbonic anhydrases were ob-served to be dispersed in other genomic regions (Table S4). With the exception ofautotrophic growth, Sulfobacillus isolates have the ability to grow mixotrophically andheterotrophically on various organic carbon substrates, such as glucose, fructose, andglycerol (30). Although S. thermosulfidooxidans strains and Sulfobacillus sp. CBAR-13were predicted to lack 6-phosphofructokinase (Table S4), an enzyme commonly presentin the Embden-Meyerhof pathway for glycolysis, enzyme assays using cell extractsshowed that these cells can transform glucose under mixotrophic conditions (53).Metabolic enzymes associated with the oxidative portion of the pentose phosphate path-way, including glucose-6-phosphate 1-dehydrogenase, 6-phosphogluconolactonase, and6-phosphogluconate dehydrogenase, were identified in all organisms. Pyruvate that accu-mulates in other pathways is catalyzed by pyruvate dehydrogenase to generate acetyl-coenzyme A via decarboxylation, and then it enters the tricarboxylic acid (TCA) cycle andcertain macromolecular biosynthetic pathways (Fig. 3). Additionally, genes related to theTCA cycle were complete in all the genomes. An earlier study revealed that aerobic COoxidation performed by the carbon monoxide dehydrogenase (CODH) complex is ubiqui-tously found in bacteria (54). This complex is composed of a medium subunit, a large/catalytic subunit, and a small subunit. Similarly, several copies of multigene clusters that arepotentially related to CODH were identified in Sulfobacillus genomes (Table S4).

The comparison of metabolic profiles in Sulfobacillus strains was performed to revealvariations in nitrogen metabolism. All genomes harbor the set of genes required for theutilization of urea via urease (Fig. 3). Ammonium generated by the catalysis of urea isused as a nitrogen source in all Sulfobacillus strains, and the released bicarbonate(another metabolite derived from the hydrolysis of urea) is predicted to be catalyzed bycarbonic anhydrases. All strains shared the potential for ammonium uptake via ammo-nium transporters and the assimilation of ammonia into central metabolic pathways viaa series of enzymes, including glutamate dehydrogenase, glutamine synthetase, andglutamate synthase (Table S4). Additionally, further analysis showed that S. thermosul-fidooxidans strains and Sulfobacillus sp. CBAR-13 harbored genes for assimilatory nitratereduction, as indicated by the nasA encoding assimilatory nitrate reductase catalyticsubunit and nirA, which encodes ferredoxin-nitrite reductase. However, the electrontransfer subunit NasB that is required for electron transfer from NADH to nitrate (55)was absent in the nitrate reductases, thereby rendering an unidentified electron donor(30). No other components for nitrogen fixation and dissimilatory nitrate reductionwere found. In contrast, S. acidophilus strains shared a copper-containing NO-formingnitrite reductase and a nitric oxide dioxygenase that potentially oxidizes nitric oxide tonitrate (56).

Oxidation of various inorganic sulfur compounds, including sulfur, tetrathionate,and sulfide, has been well documented in Sulfobacillus strains (35, 37–39). Sulfuroxygenase reductase (SOR) catalyzes the disproportionation reaction of linear polysul-fide in the presence of molecular oxygen to generate hydrogen sulfide, sulfite, andthiosulfate (57, 58). SOR was identified in all Sulfobacillus spp., with S. thermosulfidooxi-dans strains and Sulfobacillus sp. CBAR-13 showing two copies (Table S4). Othermetabolic enzymes potentially involved in sulfur oxidation of Sulfobacillus strainsinclude sulfide:quinone oxidoreductase, thiosulfate:quinone oxidoreductase that isencoded by doxDA, and thiosulfate sulfurtransferase. Sulfobacillus genomes were pre-dicted to possess tetrathionate hydrolase, which directs the disproportionation oftetrathionate to generate sulfate, thiosulfate, and other sulfur compounds (57). Anadditional route for tetrathionate metabolism in S. acidophilus strains was predicted tobe driven by tetrathionate reductase, which is a three-subunit enzyme that catalyzes

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the reduction of tetrathionate to produce thiosulfate (59). Another important enzymecomplex associated with sulfur compound oxidation in Sulfobacillus strains is hetero-disulfide reductase-like protein, which is also frequently observed in other acidophilicsulfur oxidizers, such as Acidithiobacillus (57, 60, 61). All Sulfobacillus strains exhibitedthe predicted sulfate reduction, although only the S. acidophilus strains harbored thesulfite reductase (ferredoxin) as an additional enzyme.

Ferrous ion [Fe(II)] is rapidly oxidized to ferric iron in circumneutral environments,whereas it is stable under acidic conditions even in the presence of molecular oxygen(62). Thus, Fe(II) in acidic environments is available to iron-oxidizing acidophiles livingin these habitats. Several Sulfobacillus isolates have been recognized as iron oxidizers(33, 37–39). In other acidophiles, such as Acidithiobacillus ferrooxidans (27), electronsderived from iron oxidation are ultimately transferred to either terminal electronacceptor NADH dehydrogenase (“uphill”) or to molecular oxygen (“downhill”). Similarly,S. thermosulfidooxidans and Sulfobacillus sp. CBAR-13 strains were predicted to harborhomologous redox proteins that are related to iron oxidation and the electron transferchain. Membrane-associated c-type cytochromes potentially involved in iron oxidationwere examined in all Sulfobacillus species. Electrons from membrane c-type cyto-chromes might be transferred to aa3-type cytochrome oxidase via a “downhill electronpathway” (Fig. 3). In Leptospirillum spp. and A. ferrooxidans, a bc complex has beenimplicated in reverse electron flow (62). However, no homologous genes were identi-fied in Sulfobacillus genomes. Two putative sulfocyanins, the blue copper proteinhaving the conserved protein domain family (cd04230), were predicted to transferelectrons during iron oxidation in the S. thermosulfidooxidans strains and Sulfobacillussp. CBAR-13 (Table S4), while no candidate gene encoding a sulfocyanin-like proteinwas identified in S. acidophilus. More details about iron oxidation in Sulfobacillus needto be further validated.

Under both oxic and anoxic environmental conditions, molecular hydrogen is widelyused as an electron donor in bacteria and archaea to support growth (63). An earlierstudy has extensively described five distinct types of nickel-iron [NiFe]hydrogenaseswithin the Sulfobacillus genomes (30). Accordingly, a conserved gene cluster encodinghydrogenase-like proteins was detected in all Sulfobacillus species, and the catalyticsubunit was clustered with group 1 respiratory-uptake [NiFe]hydrogenase, which mightoxidize H2, transferring protons to the quinone pool via a membrane-integral cyto-chrome b subunit (64). Additionally, Sulfobacillus genomes contained the secondmultisubunit gene cluster related to hydrogenase, but the operon structure andcatalytic subunits differed (30). The catalytic subunits in the S. acidophilus strainsshowed high sequence homology with subgroup A of group 2 hydrogenase, whereasthe hydrogenases of the S. thermosulfidooxidans strains and Sulfobacillus sp. CBAR-13were most similar to group 5 hydrogenases (Table S4). Other homologous proteinswere assigned to group 4 hydrogenases, despite the absence of binding-site motifs intheir catalytic subunits (30). Taken together, the presence of hydrogenase complexes inSulfobacillus species indicated that molecular hydrogen might be a key source oflow-potential electrons, although no experimental evidence for hydrogen utilizationwas observed in these microorganisms (30).

(ii) Management strategies for environmental stresses. Each of the Sulfobacillus

genomes analyzed in this study harbored a large number of genes assigned to COGcategories T (signal transduction mechanisms; Table S2) and N (cell mobility), many ofwhich were predicted to be associated with chemotaxis signal transduction systemsand flagellar formation (Table S4). These special traits, such as flagella and chemotaxis,enable Sulfobacillus strains to actively move across environmental gradients, therebyproviding a competitive advantage in aquatic environments.

A high number of strain-specific genes related to the COG category L (replication,recombination, and repair) were observed compared to that in other COG categories(Fig. 2C). This finding might be explained by a previous notion that protein and DNArepair systems in acidophiles play a critical role in dealing with acid stresses (65), given

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that low pH in these particular habitats could cause DNA and protein injury. For a longtime, the potential mechanisms for pH homeostasis of acidophiles have attractedconsiderable interest of researchers. Baker-Austin and Dopson (65) described thatdistinctive structural and functional characteristics, mainly including highly imperme-able cell membranes, reversed membrane potential, and the predominance of second-ary transporters enable acidophiles to survive and grow under these extreme condi-tions. Similar to various acidophiles, Sulfobacillus spp. might generate a reversedmembrane potential via the Kdp-type potassium transport system (Table S4), therebypartially deflecting the inward flow of protons (9, 65, 66). Additionally, genes encodingthe Kef-type potassium efflux system and voltage-gated potassium channel proteinswere identified in Sulfobacillus genomes. Monovalent cation/proton antiporters, i.e.,Na�(K�)/H� antiporters, may facilitate the active efflux of cytoplasmic sodium and/orpotassium from the extracellular matrix in exchange for hydrogen (67). Accordingly,Sulfobacillus strains observed in this study were predicted to exclude intracellularredundant protons by Na�/H� antiporters.

Sulfobacillus genomes were identified to harbor several genes/gene clusters that arepotentially involved in stress tolerance strategies to cope with high metal loads (TableS4). A gene cluster related to the reduction of arsenate was identified in the S.thermosulfidooxidans genomes and Sulfobacillus sp. CBAR-13, whereas S. acidophilusstrains were predicted to employ the arsenical efflux pump for arsenical resistance.Some transporting ATPases for the resistance to various heavy metal ions, includinglead, cadmium, zinc, mercury, and copper, were identified in all species. We also foundthat Sulfobacillus organisms harbored genes that encode mercuric ion reductase andthe cation diffusion facilitator family member CzcD.

Potential driving forces of genome evolution. A comparison of the predicted

metabolic profiles suggested that Sulfobacillus spp. harbor a core of genes that wereessential to metabolic functions (Table S4). Further inspection also revealed distinguish-ing metabolic traits among these species. Apart from the core genome, bacterialgenomes have been reported to have a number of accessory genes that were probablyacquired by horizontal gene transfer (HGT) and were beneficial under environmentalconditions (68). Linking the differences in the inferred metabolic profiles to their owngenome architectures allows a detailed comparison of potential driving forces thatcontribute to genome evolution and diversification of Sulfobacillus strains.

(i) Detection of mobile genetic elements. As an evolutionary force shaping the

content of microbial genomes, HGT events frequently occur in microbes and areregarded as an effective means of rapid adaptation to changing environmental de-mands (69). A significant part of HGT is facilitated by mobile genetic elements (MGE)and is often characterized by certain signatures, e.g., integration sites usually associatedwith tRNA genes, varied codon usage, or abnormal G�C contents (68, 70, 71). Gainand/or loss of MGE, such as integrative conjugative and mobile elements, genomicislands, and insertion sequences (IS), may play a crucial role in adaptation to environ-mental stresses (71). Transposases and integrases were identified and classified in allSulfobacillus genomes by using the online platform ISfinder (Table S5). Generally, ahigher number of transposable elements was predicted in relatively large genomes(Cutipay, 272 transposable elements; CBAR-13, 160; TPY, 298; and DSM 10332, 299) thansmall genomes (DX, 76 transposable elements; ZBY, 79; ZJ, 79; ST, 90), indicating thatgene turnover in larger genomes was relatively frequent. IS classes IS3, Tn3, and ISL3were abundant in all genomes, whereas several IS classes were only identified in thegenomes of individual strains (Table S5). Further inspection revealed the potentialcorrelation between genome size and the number of insertion sequences among S.thermosulfidooxidans strains. For instance, S. thermosulfidooxidans Cutipay, which has alarger genome (3.86 Mb), harbored more transposable elements (272) than the others(Table S5). We thus proposed that HGT might be more frequent in larger genomes,thereby contributing to intraspecific divergence.

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Other characteristic functions related to genomic islands were also investigatedusing the online platform IslandViewer3 (72). A higher number of putative genomicislands was identified in larger genomes than in smaller genomes (Table S6), furthersuggesting highly frequent genetic exchanges. Several CDSs located in genomic islandswere annotated as hypothetical proteins. Further inspection identified numerous mo-bile element proteins, indicating that various genomic islands might have been ac-quired by HGT. Because HGT was critical to the expansion of the gene repertoires ofprokaryotes (69, 73, 74), we deduced that the genomic islands in these species mightplay a predominant role in functional recruitment, thereby enabling them to adapt tospecific environmental niches.

(ii) Linking differences in metabolic profiles of Sulfobacillus spp. to genomearchitectures. The Sulfobacillus strains were further classified into four types of ge-nomes based on their genome sizes (Fig. 4). Each type of genome was then used asreference for BLASTN-based whole-genome comparisons to unravel the presence/absence of genomic regions and to some extent reflect the relationship of Sulfobacillusstrains based on the sequence identity.

Figure 4 shows that a number of genomic regions related to metabolic functionswere only distributed over each type of Sulfobacillus genome. Apparently, somegenomic regions potentially involved in metabolic pathways were only found in certainlarger genomes, such as those of S. thermosulfidooxidans Cutipay (sections 1 to 7 in Fig.4B) and Sulfobacillus sp. CBAR-13 (sections 8 to 13 in Fig. 4C). Several genes located inthese genomic regions were predicted to encode putative proteins with unknownfunctions (Table S7). Further investigation to identify HGT signatures was conducted toinfer the possible origin of these putative genes. Several transposases, integrases, andphage-associated proteins were predicted to disperse in the corresponding genomiclocus (Table S7), thereby suggesting that these clusters might have been acquired byHGT. Also, several ABC transporter-related proteins were predicted to be acquired byHGT, probably indicating a high exchange rate for substances.

The genomic regions of the three new Sulfobacillus strains harbored genes involvedin the utilization of urea (Table S4 and Fig. 4A and D). The urease gene clusters in thethree Sulfobacillus species were distributed in distinct genomic regions, which werefurther visualized by the presence of colinear blocks (Fig. 5). Although several homol-ogous genes were identified in these two urease gene clusters, these differed in termsof gene content and nucleotide sequence identity, thereby suggesting the divergentevolution of the urease gene cluster in the Sulfobacillus species. Two transposases wereidentified in the genomic neighborhood of the urease gene cluster of the S. acidophilusstrains (Fig. 5B), whereas no signature for a recent HGT was identified in the S.thermosulfidooxidans strains and Sulfobacillus sp. CBAR-13, thereby suggesting that theurease gene cluster in S. acidophilus genomes was introduced via HGT. A geneencoding carbonic anhydrase (CA) was predicted upstream of the urease gene clusterin the S. thermosulfidooxidans strains and Sulfobacillus sp. CBAR-13 (Fig. 5A). Unlikeother recognized acidophiles, such as Acidithiobacillus spp. (44) and “Ferrovum” spp. (9),in which CA gene was located in a carboxysome gene cluster, no carboxysome-associated genes were found in the Sulfobacillus strains. Additionally, two gene clustersinvolved in carbon monoxide dehydrogenase (CODH), which were potentially acquiredby HGT, are discussed here. Form I CODH was identified in certain Sulfobacillus strainswith larger genomes (Table S4 and section 24 in Fig. 4D), and form III CODH was onlyfound in S. acidophilus strains (Table S4 and section 19 in Fig. 4D). One or severaltransposases identified in the genomic neighborhoods suggested that these genomicregions underwent several HGT events. In the S. thermosulfidooxidans strains andSulfobacillus sp. CBAR-13, 15 genes located in a genomic region were predicted toencode transport-associated enzymes, such as sugar transporter and glycosyltrans-ferases that are potentially related to the synthesis of the cell envelope polysaccharides(9), and signatures of a recent HGT event, i.e., phage-associated genes, were found inthe genomic neighborhood (Fig. S1). We also identified some transposases around thehydrogenase group II gene cluster, which was only present in S. acidophilus strains

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(section 25 in Table S7). HGT events contribute to the acquisition of novel genes thatwere originally derived from apparently taxonomically unrelated species (70) and playan important role in functional recruitment as a number of accessory genes encodeadaptive traits that might be beneficial to inhabiting various econiches (68, 71). We thus

FIG 4 BLASTN-based whole-genome comparison of Sulfobacillus strains using Circos. The representative genomes from S. thermosulfidooxidans DX (A),Cutipay (B), Sulfobacillus sp. CBAR-13 (C), and S. acidophilus TPY (D) were used as references. Matches to each reference genome are displayed on rings1 to 8 with different colors. Additionally, insertion sequences, tRNA, and G�C content are shown on rings 9 to 11. Cross-references link to other figuresand tables showing more details of special regions.

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inferred that gene acquisition via HGT might be an efficient way of rapidly adapting tochanges in the environment, thereby providing the advantage for bacterial survival,growth, and reproduction.

Interestingly, three clustered regularly short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems were also identified in these genomic regions (sections3, 5, and 27 in Table S7). CRISPR/Cas modules are adaptive immunity systems thatwidely occur in various bacterial species and almost all archaea (75, 76). Based on acomparison to systems in other bacteria (76), the CRISPR/Cas systems in S. thermosul-fidooxidans Cutipay were classified as subtype I-C (Dvulg or CASS1; section 3 in TableS7) and subtype I-E (Ecoli or CASS2; section 5 in Table S7), and the CRISPR/Cas systemin the S. acidophilus strains was assigned to subtype I-B (Tneap-Hmari or CASS7; section27 in Table S7). The presence of the CRISPR/Cas systems in these species indicated thatphage-host coevolution might be a means for HGT and play a key role in the genomicevolution in Sulfobacillus strains.

Comparative analysis of genome architecture revealed that several genomic regionswere present in S. acidophilus strains and absent in S. thermosulfidooxidans strains andSulfobacillus sp. CBAR-13 (Fig. 4 and Table S7). These regions were predicted to harborgenes involved in amino acid (section 16 in Table S7) and cofactor biosynthesis (section17 in Table S7). However, no evidence for the presence of integrases, transposases, orphage-associated proteins was identified in the genomic neighborhoods, suggesting

FIG 5 Comparison of genomic regions in Sulfobacillus strains involved in urease (A), urea carboxylase, and carbon monoxidedehydrogenase (B). More details about genes distributed in selected regions are shown in Table S4.

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that the absence of these genomic regions in the S. thermosulfidooxidans strains mightbe the result of several gene loss events rather than HGT. We then inferred that theabandonment of conditionally essential genes contributed to the reduction in genomesize, as well as shaped the metabolic profiles of the S. acidophilus genomes. Notably,the fourth gene cluster involved in carbon monoxide dehydrogenase (CODH) in S.acidophilus strains (Fig. 5B and section 28 in Table S7) was likely derived from acommon ancestor, whereas the gene cluster in the S. thermosulfidooxidans strains andSulfobacillus sp. CBAR-13 might have been lost during the evolution process, becauseno HGT signatures were identified. Compared to other CODH gene clusters, this clustersignificantly differed in terms of gene order and orientation (Table S4). We thusproposed that alien genes might be integrated into the fourth CODH gene cluster andbe caused by several rearrangements, thereby yielding different locations of thehomologous genes in the respective genome. Additionally, S. acidophilus genomesharbor an 11-subunit NADH-quinone oxidoreductase, which lacks three genes (nuoEFG)encoding the “N-module” subunits that are associated with NADH binding (30), therebyrendering an unclear function (Table S4 and section 18 in Table S7). Accordingly, theresults could provide a plausible adaptive explanation for the widespread loss ofaccessory genes with low contribution to cellular fitness.

The streamlining hypothesis supporting genome reduction has been proposed inthe context of increased cellular economization (77). Examples of genome streamlininghave been documented in various microorganisms, such as Prochlorococcus marinus, inwhich genome reduction was proposed as an adaptive mechanism for the efficientutilization of limited nutrients (78). A strategy to engineer the Streptomyces avermitilisgenome has been performed, in which nonessential segments are deleted (79, 80) toimprove the expression of heterologous pathways (81). Because acidic habitats havenutrient-deficient substrates (27), genome reduction appears to confer adaptive fitnessadvantages that drive the abandonment of expensive genes, thereby enabling theeffective acquisition of limited nutrients. For long-term evolution, the streamliningprocess has allowed bacteria to maintain essential genes, as well as facilitate in the lossof dispensable ones, thereby resulting in a simpler but still fully functional genome.

(iii) Identification of putative cross-kingdom HGT events. Phylogenetic analysisof sulfur oxygenase reductase (a critical enzyme involved in the sulfur oxidation ofmany acidophiles) unraveled a close relationship between the Sulfobacillus genus andarchaeal Ferroplasma acidarmanus (31). Accordingly, it is plausible that archaeal phylaacted as potential gene donors to Sulfobacillus strains. Therefore, the genes of theSulfobacillus genomes were likely to have been acquired via cross-kingdom HGT events.To identify other genes that potentially underwent adaptive evolution, we analyzed theoccurrence of cross-kingdom HGT events within various Sulfobacillus genomes.

The genomic sequences of the three novel S. thermosulfidooxidans strains wereannotated against the truncated database, in which protein sequences belonging tothe Sulfobacillus strains were excluded. Several proteins with apparent archaeal BLASTPfirst hits were then identified, despite the lower percentage than that in all examinedsequences (1.49%; Fig. 6). About 53 genes in the S. thermosulfidooxidans genomes wereof putative archaeal origin (Table S8), with the majority of these genes phylogeneticallyaffiliated with phylum Euryarchaeota (Fig. S2). Archaea constitute a significant fractionof the microbial biomass on Earth, although details and bases for this taxonomicclassification are limited (82). Most of the examined genes that were potentially derivedfrom archaeal donors were annotated as hypothetical proteins, whereas a fraction ofthe genes were predicted to be involved in central metabolism, e.g., CoB-CoM hetero-disulfide reductase, sulfur oxygenase reductase, and TQO small subunit DoxD in sulfuroxidation and glucose-6-phosphate isomerase in glycolysis. Accordingly, it appears thatthese HGT processes represent an evolutionary mechanism that contributed to en-hancement of metabolic capacities and thereby confer rapid adaptation to changingenvironments. Additionally, our analysis also suggested that the archaeal phyla arepotential donors of transport-related genes of S. thermosulfidooxidans species. These

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results indicate that the acquisition of genes via cross-kingdom HGT events mightrepresent a potential mechanism that drives the adaptive evolution of S. thermosulfi-dooxidans strains.

Concluding remarks. Comparative genomics has improved our current knowledgeof genome evolution and species-level identification of Sulfobacillus strains. HGT maybe a key evolutionary force that drives the genome expansion of Sulfobacillus strains,thereby controlling the response and adaptation of microorganisms to environmentalchanges. Furthermore, gene acquisition via a cross-kingdom HGT process is likely to bean effective way to recruit novel functionalities in microorganisms. These genomesundergoing genome minimization events, although relatively small, have thus inheriteda core of essential genes and biological functionalities that are necessary for survivaland stress tolerance in specific environments. Collectively, bacterial genomes haveundergone a variety of processes, such as HGT and the loss of gene/genome segments,thereby contributing to the diversification and adaptive evolution of microorganisms(68, 69, 83).

New insights were gained into the potential evolutionary mechanism of Sulfo-bacillus strains. The assessment of mobile elements was performed, suggesting thatgenetic exchanges might be highly frequent in relatively large genomes. We furtherfocus on certain genomic regions mainly related to metabolic potentials which werepredicted to potentially undergo HGT and/or gene loss events. However, genomicinformation shows us the possible HGT within Sulfobacillus genomes but does notquantify HGT. Thus, experimental quantification of HGT should be conducted in thefuture.

MATERIALS AND METHODSBacterial cultivation and genomic DNA extraction. The bacterial strains used in this study (ZBY,

DX, and ZJ) were originally isolated from various copper mine tailings from around the world. Detailedinformation on the geochemical conditions of two Chinese copper mines is also described in this report(84, 85). Unfortunately, the environmental attributes of the sampling sites in the Zambian copper mineat that time were not assessed. Strains were cultivated aerobically in 100 ml of liquid 9K basic medium[3.0 g/liter (NH4)2SO4, 0.5 g/liter MgSO4·7H2O, 0.1 g/liter KCl, 0.01 g/liter Ca(NO3)2, and 0.5 g/liter K2HPO4

(initial pH 1.6)] with 4.5% (wt/vol) ferrous sulfate (membrane filtered) and 0.02% yeast, as previouslydescribed (31). The temperature and shaking speed of the bacterial cultures were 45°C and 170 rpm,respectively. At mid-exponential-growth phase, the bacterial cells were harvested by centrifugation at12,000 � g for 10 min at 4°C. Total genomic DNA was extracted previously described (57) and then usedfor genome sequencing.

Genome sequencing and assembly. Whole-genome sequencing of all three novel strains (ZBY, DX,and ZJ) was performed on an Illumina MiSeq sequencer (Illumina, Inc., USA). Shotgun libraries with anaverage 300-bp insert size were constructed and were then used for 2 � 150-bp paired-end sequencing.The generated read sequences were then assembled de novo, as previously described (43).

FIG 6 Proportion of genes with nonself BLASTP first hits in three newly sequenced S. thermosulfi-dooxidans genomes. Protein sequences within three novel strains in this study were aligned againstthe truncated NCBI-nr database, in which sequences belonging to Sulfobacillus genomes wereexcluded.

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Bacterial phylogenetic reconstruction. The 16S rRNA gene sequences in the three genomeassemblies were extracted using RNAmmer (86). Phylogenetic reconstruction of the 16S rRNA genes wasthen performed by using MEGA version 5.05 using the maximum likelihood method. Nodal support wasevaluated using 1,000 bootstrap replications. The resulting phylogenetic tree initially showed that thesethree new strains were affiliated with S. thermosulfidooxidans, thereby prompting us to download thegenome sequences of five available Sulfobacillus species from the public database, the National Centerfor Biotechnology Information (NCBI), for subsequent analysis. Contig fragments from all assembledscaffolds of S. thermosulfidooxidans strains ST and Cutipay were extracted using an in-house Perl script.The completeness of all available genome assemblies was estimated using CheckM (87). JSpecies version1.2.1 (88), with default parameters, was employed to further infer the phylogenetic relationship amongthe Sulfobacillus strains by comparing their average nucleotide identity (ANI) (89) by using BLAST (ANIb)and MUMmer (ANIm) (90), as well as tetranucleotide frequencies (Tetra) (91).

Pangenome analysis. Genome functional annotation was performed using the Rapid Annotationsusing Subsystems Technology (RAST) platform (http://rast.nmpdr.org/) (92). Subsequently, protein se-quences and corresponding annotation were acquired as previously described (43, 93). PanOCT version3.18 (94), with an E value cutoff of 0.00001 and sequence identity cutoff of 65%, was applied for theidentification of orthologs that were shared between species or strains. Protein sequences extracted byusing a Perl script were then annotated against the extended Clusters of Orthologous Groups (COG) (95).For functional comparison, the KEGG Automatic Annotation Server (50) was employed to predict theputative metabolism-related genes in Sulfobacillus genomes.

Identification of putative HGT events. Transposases or insertion sequences (IS) within the Sulfo-bacillus genomes were identified using ISfinder (96). A recently developed Web server, IslandViewer 3(72), which integrates a single comparative genomic island prediction method, IslandPick (97), and twosequence composition genomic island prediction methods, SIGI-HMM (70) and IslandPath-DIMOB (98),were used to identify putative genomic islands that were dispersed across the Sulfobacillus genomes. Toidentify potential cross-kingdom HGT events, protein sequences belonging to Sulfobacillus species thatwere detected by NCBI-nr were excluded. The three novel genomes in this study were then alignedagainst the truncated database to identify genes that were potentially derived from archaeal donors.Genes assigned to the archaea were then visualized using MEGAN5 (available at http://ab.inf.uni-tuebingen.de/software/megan5/).

Accession number(s). These whole-genome shotgun projects for three novel strains have been depos-ited at the DDBJ/ENA/GenBank under the accession numbers MDZD00000000 (DX), MDZE00000000 (ZBY),and MDZF00000000 (ZJ). The versions in these papers are versions MDZD01000000, MDZE01000000, andMDZF01000000, respectively.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.03098-16.

SUPPLEMENTAL FILE 1, PDF file, 14.6 MB.SUPPLEMENTAL FILE 2, XLSX file, 0.01 MB.SUPPLEMENTAL FILE 3, XLSX file, 0.07 MB.SUPPLEMENTAL FILE 4, XLSX file, 0.07 MB.SUPPLEMENTAL FILE 5, XLSX file, 0.09 MB.SUPPLEMENTAL FILE 6, XLSX file, 0.02 MB.

ACKNOWLEDGMENTSWe thank Zhili He at the University of Oklahoma for helpful discussion and Ye Deng

at the Chinese Academy of Sciences for useful suggestions. We are also grateful to thethree anonymous reviewers for their insightful and constructive comments. Addition-ally, we thank the NCBI for providing the genome sequences of Sulfobacillus thermo-sulfidooxidans strains ST and Cutipay, Sulfobacillus sp. CBAR-13, and Sulfobacillus aci-dophilus strains TPY and DSM 10332. Finally, we thank Accdon for linguistic assistanceduring the preparation of the manuscript.

This work was funded by the National Natural Science Foundation of China (grants41573072, 31570113, and 31571986), the National Basic Research Programme of China(grant 2013CB127502), and the Fundamental Research Funds for the Central Universi-ties of Central South University (grant 2016zzts102).

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