Comparative genomic and transcriptomic analysis revealed genetic characteristics related to solvent formation and xylose utilization in Clostridium acetobutylicum EA 2018

  • Shiyuan Hu1, 2,

    Affiliated with

    • Huajun Zheng3,

      Affiliated with

      • Yang Gu1, 2,

        Affiliated with

        • Jingbo Zhao1,

          Affiliated with

          • Weiwen Zhang4,

            Affiliated with

            • Yunliu Yang1, 2,

              Affiliated with

              • Shengyue Wang3,

                Affiliated with

                • Guoping Zhao1, 3,

                  Affiliated with

                  • Sheng Yang1, 2Email author and

                    Affiliated with

                    • Weihong Jiang1, 2Email author

                      Affiliated with

                      BMC Genomics201112:93

                      DOI: 10.1186/1471-2164-12-93

                      Received: 5 August 2010

                      Accepted: 2 February 2011

                      Published: 2 February 2011

                      Abstract

                      Background

                      Clostridium acetobutylicum, a gram-positive and spore-forming anaerobe, is a major strain for the fermentative production of acetone, butanol and ethanol. But a previously isolated hyper-butanol producing strain C. acetobutylicum EA 2018 does not produce spores and has greater capability of solvent production, especially for butanol, than the type strain C. acetobutylicum ATCC 824.

                      Results

                      Complete genome of C. acetobutylicum EA 2018 was sequenced using Roche 454 pyrosequencing. Genomic comparison with ATCC 824 identified many variations which may contribute to the hyper-butanol producing characteristics in the EA 2018 strain, including a total of 46 deletion sites and 26 insertion sites. In addition, transcriptomic profiling of gene expression in EA 2018 relative to that of ATCC824 revealed expression-level changes of several key genes related to solvent formation. For example, spo0A and adhEII have higher expression level, and most of the acid formation related genes have lower expression level in EA 2018. Interestingly, the results also showed that the variation in CEA_G2622 (CAC2613 in ATCC 824), a putative transcriptional regulator involved in xylose utilization, might accelerate utilization of substrate xylose.

                      Conclusions

                      Comparative analysis of C. acetobutylicum hyper-butanol producing strain EA 2018 and type strain ATCC 824 at both genomic and transcriptomic levels, for the first time, provides molecular-level understanding of non-sporulation, higher solvent production and enhanced xylose utilization in the mutant EA 2018. The information could be valuable for further genetic modification of C. acetobutylicum for more effective butanol production.

                      Background

                      High oil prices, growing concerns over national security and climate change are driving investment and innovation in the renewable alternative fuels [1, 2]. Among various potentially alternatives, butanol has been proposed as an excellent substitute or supplement for gasoline, and has been demonstrated to work in some vehicles designed for use with gasoline without any engine modification [1]. In addition to manufacture from petroleum through chemical refinery process, industry production of butanol is typically through a so-called ABE fermentation process employing gram-positive, spore forming and anaerobic organism Clostridium acetobutylicum [2]. C. acetobutylicum is capable of producing a mixture of acetone (A), butanol (B) and ethanol (E) from a variety of carbohydrate substrates such as starch [3]. According to an estimate in 2008, butanol accounted for a 7-8.4 billon US dollar market worldwide and has a projected market expansion of 3% per year in the near future [4].

                      Significant efforts have been spent on physiological and genetic characterization of solvent-producing C. acetobutylicum in the past decades [58], and tools for genetic manipulation of C. acetobutylicum were also developed [911]. In 2001, the whole genome of well studied C. acetobutylicum ATCC 824 was sequenced, revealing a 3.94 Mb chromosome which encodes 3740 open reading frames (ORF), and a 192 Kb megaplasmid which encodes 178 ORFs [12]. Afterwards, a series of studies employing global approaches have been performed [1316], and the genome-scale metabolic model of C. acetobutylicum was also constructed [1719]. These efforts have improved the understanding of regulatory and metabolic networks of this industry significant species.

                      However, most of the C. acetobutylicum strains are not optimized systems for butanol production because their spore-forming life cycle decreases the efficiency of industrial fermentation, and the ABE fermentation process also creates a number of by-products, such as H2, acetic, lactic and propionic acids, acetone, isopropanol and ethanol [20]. As a result, the butanol yield is difficult to control and a significant amount of energy is wasted in these by-products. Moreover, it also increases the cost of downstream butanol purification. To address these issues, various modification approaches, such as mutagenesis by chemical or radiation agents, and genetic engineering, have been performed to improve the butanol production [10, 21]. Our laboratory has previously obtained a high butanol producing strain, C. acetobutylicum EA 2018, through butanol resistance screening of N-methyl-N-nitro-N-nitrosoguanidine (NTG) treated Clostridium strain isolated from soil [22]. Preliminary results in a 100-ton continuous fermenter showed that butanol ratio and starch conversion rates of EA 2018 strain were 10% and 5% higher than those reported in recent literature [23]. To explore the genetic difference between EA 2018 and ATCC 824, in this study, the C. acetobutylicum EA 2018 genome was sequenced using Roche 454 pyrosequencing together with traditional Sanger sequencing. In addition, comparative genomic and transcriptomic analyses of EA 2018 and ATCC 824 were also performed. The study, for the first time, provides a molecular-level understanding of higher solvent production, enhanced xylose utilization and non-sporulation in the mutant EA 2018. The information could be valuable for further genetic modification of C. acetobutylicum for more effective butanol production.

                      Results and Discussion

                      Characterization of isolate EA 2018

                      The original solvent producing strain was isolated by our laboratory previously [22]. After several rounds of mutagenesis using NTG (N-methyl-N'-nitro-N-nitrosoguanidine), we obtained a hyper butanol-producing strain designated as EA 2018. This strain was later identified as Clostridium acetobutylicum by the China Center for Type Culture Collection (CCTCC) and was kept in CCTCC under the preservation No. CCTCC M_94061. In this work, the 16S rDNA of C. acetobutylicum EA 2018 was cloned and sequenced. The 1399 bp 16S rDNA sequence of C. acetobutylicum EA 2018 was 100% identical to that of the type strain C. acetobutylicum ATCC 824 (Accession number NC_003030 for ATCC 824 genome sequence) [12]. Furthermore, the sol operon involved in butanol production was also cloned from C. acetobutylicum EA 2018 and sequenced, the comparative analysis showed that the sol operon of C. acetobutylicum EA 2018 was also 100% identical to that of C. acetobutylicum ATCC 824 (Accession number NC_001988 for ATCC 824 mega-plasmid sequence) [24]. The analysis demonstrated that EA 2018 and ATCC 824 belong to the same species.

                      Fermentation experiments were performed to compare the solvent production patterns of C. acetobutylicum EA 2018 and ATCC 824. EA 2018 exhibited higher solvent formation capacity than ATCC 824 strain in either 6% (w/v) glucose or xylose media (Figure 1A, B). After 48 h fermentation, 8.3 g/L glucose remained in the EA 2018 culture, while 16.5 g/L glucose still remained in the ATCC 824 culture after 72 h fermentation (Figure 1A). After 96 h fermentation, 23.6 g/L xylose was present in the EA 2018 culture, while 35.7 g/L xylose was still remained in the ATCC 824 culture (Figure 1B). In most C. acetobutylicum strains, solvent formation is always coupled with initiation of sporulation [10]. However, after fermentation of 72 h, there was no spore found in EA 2018 cultures, while significant spores were found in ATCC 824. With its higher solvent production and non-spore forming characteristics, EA 2018 strain could be an excellent strain for industrial application.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-93/MediaObjects/12864_2010_3221_Fig1_HTML.jpg
                      Figure 1

                      Solvent and acid production, sugar utilization ofC. acetobutylicumEA 2018 versusC. acetobutylicumATCC 824 in P2 medium contained with 6% glucose (A) and 6% xylose (B).

                      Overview of C. acetobutylicum EA 2018 genome

                      For better understanding of the genetic basis of improved butanol producing characteristics in EA 2018, the whole genome of EA 2018 was sequenced. The genome has a circular chromosome consisting of 3,940,230 bp with an average G+C content of 30.93% and a circular megaplasmid of 191,996 bp with an average G+C content of 30.91%. The genome finishing procedures were listed in Additional file 1, 2, 3. A total of 3923 protein coding sequence (CDS) including 3,746 in chromosome and 176 in megaplasmid were indentified in the EA 2018 genome, representing 86.8% of the genome and 83.8% of the megaplasmid, respectively. The functional classification of all EA 2018 genes was listed in Table 1. There are 11 copies of rDNA operons and a total of 75 tRNA genes scattered over the EA 2018 genome. Genomic comparison with type strain ATCC 824 revealed the highly conserved gene content and gene order between these two strains. The base numbering start point of EA 2018 were chosen as the same site in ATCC 824 (Figure 2).
                      Table 1

                      Function Classification of EA 2018 genes

                      Function

                      Numbers in plasmid

                      Numbers in genome

                      Function

                      Numbers in plasmid

                      Numbers in genome

                      Energy production and conversion

                      12

                      121

                      Cell envelope biogenesis, outer membrane

                      10

                      182

                      Cell division and chromosome partitioning

                      3

                      38

                      Cell motility and secretion

                      1

                      92

                      Amino acid transport and metabolism

                      1

                      212

                      Posttranslational modification, protein turnover, chaperones

                      2

                      78

                      Nucleotide transport and metabolism

                      1

                      73

                      Inorganic ion transport and metabolism

                      6

                      93

                      Carbohydrate transport and metabolism

                      22

                      221

                      Secondary metabolites biosynthesis, transport and catabolism

                      3

                      27

                      Coenzyme metabolism

                      1

                      110

                      General function prediction only

                      16

                      321

                      Lipid metabolism

                      6

                      76

                      Function unknown

                      5

                      258

                      translation,ribosomal structure and biogenesis

                      0

                      159

                      Signal transduction mechanisms

                      4

                      126

                      transcription

                      22

                      243

                      Intracellular trafficking and secretion

                      0

                      14

                      DNA replication, recombination and repair

                      5

                      137

                      Defense mechanisms

                      5

                      106

                         

                      Not in this system

                      55

                      1059

                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-93/MediaObjects/12864_2010_3221_Fig2_HTML.jpg
                      Figure 2

                      Atlas of the chromosome and the megaplasmid ofC. acetobutylicumEA 2018 and its comparison withC. acetobutylicumATCC 824. Moving inside, each concentric circle represents genomic data for C. acetobutylicum EA 2018 and its comparison with C. acetobutylicum ATCC 824. For chromosome atlas, the outer circle illustrates predicted coding sequences on the plus and minus strands, respectively, colored by functional categories according to COG classification. The 2nd circle represents EA 2018 variation genes compared with ATCC 824. The 3rd circle displays IS elements in EA 2018. The 4th circle shows rDNA genes in EA 2018, distinguished by plus strand (gold) and minus strand (red). The 5th circle shows tRNA genes in EA 2018, distinguished by plus strand (pink) and minus strand (blue). The 6th circle represent GC content, red for GC content above average and blue for GC content below average. The 7th circle (innermost) represents GC skew (G-C)/(G+C) calculated using a 100 kb window. For megaplasmid atlas, the outer circle illustrates predicted coding sequences on the plus and minus strands, respectively, colored by functional categories according to COG classification. 2nd circle represents EA 2018 variation genes compared with ATCC 824. 3th circle represent GC content, red for GC content above average and blue for GC content below average. The 4th circle (innermost) represents GC skew (G-C)/(G+C) calculated using a 100 kb window.

                      Comparative genomic analysis of EA 2018 and ATCC 824

                      The size of the EA 2018 chromosome is 650 bp smaller than that of ATCC 824, and the size of the EA 2018 megaplasmid is 4 bp smaller than that of ATCC 824. Compared with ATCC 824, a total of 46 deletion sites and 26 insertion sites were found across the EA 2018 genome, including 1 deletion site in the megaplasmid (Additional file 4). Among them, 55 sites are single nucleotide indel (i.e. insertion or deletion), and only 7 indel sites are larger than 100 bp. The largest insertion is 1768 bp in 1276337-1278091 which is located within a hypothetical protein gene (CEA_G1125, corresponding to CAC1113 in ATCC 824), while the largest deletion is 1812 bp in 1112030-1112031 which is located within a predicted member protein gene (CEA_G0978, corresponding to CAC0966 in ATCC 824). Apart from those indels, 440 single nucleotide variations (SNVs) were identified between the two chromosomes, and 11 SNVs were identified between the megaplasmid of EA 2018 and ATCC 824 (Additional file 5). The 523 variations (including 72 indels and 451 SNVs) affected a total of 229 protein encoding genes (including 10 genes in megaplasmid) and 38 predicted promoters (including 1 in megaplasmid) in the EA 2018 genome. Excluding the synonymous substitution, a total of 189 proteins had amino acids changes, while 47 of those proteins were with similar amino acid variations (Additional file 6). The 38 genes with their putative promoters affected were listed in Additional file 7. In Table 2 and 3, variations within putative promoters and genes related to solvent formation, xylose utilization, and spore formation (i.e. three key aspects related to enhanced butanol production in EA 2018) were listed.
                      Table 2

                      EA 2018 gene variations associated with key phenotypes

                      Gene locus in EA 2018

                      Gene locus in ATCC 824

                      Gene variation sites in EA 2018

                      Gene variation sites in ATCC 824

                      Protein variation sites in EA 2018

                      Protein variation sites in ATCC 824

                      Product name

                      Solvent formation related genes

                      CEA_G1048

                      CAC1036

                      899(T)

                      899(C)

                      300(V)

                      300(A)

                      Pyruvate kinase

                      CEA_G1755

                      CAC1742

                      391(A)

                      391(G)

                      131(N)

                      131(D)

                      phosphotransacetylase

                      CEA_G2463

                      CAC2449

                      256(A)

                      256(G)

                      86(S)

                      86(G)

                      Predicted flavoprotein

                      CEA_G2485

                      CAC2471

                      439(A)

                      438-439(-)

                      147-150(R,L,P,I)

                      147-150 (G,C,L,Stop codon)

                      Transcriptional regulator, TetR/AcrR family

                      CEA_G2556

                      CAC2543

                      241-249 (G,T,A,G, A,T,C,A,T)

                      240-241(---)

                      81-83(V,D,H)

                      80-81(---)

                      Electron-transferring flavoprotein large subunit

                      CEA_G2806

                      CAC2798

                      286(A)

                      286(G)

                      96(M)

                      96(V)

                      NADH:flavin oxidoreductase

                      CEA_P0058

                      CA_P0059

                      754(A)

                      754(C)

                      252(N)

                      252(H)

                      Alcohol dehydrogenase

                      CEA_P0077

                      CA_P0078

                      91(A)

                      91(G)

                      31(T)

                      31(A)

                      acetyl-CoA acetyltransferase

                      CEA_P0140

                      CA_P0141

                      566(T)

                      566(C)

                      189(I)

                      189(T)

                      Periplasmic hydrogenase small subunit, dehydrogenase

                      Substrate utilization related genes

                      CEA_G0239

                      CAC0234

                      1072(T)

                      1072(C)

                      358(Stop codon)

                      358(Q)

                      PTS system, fructoso-specific IIBC component

                      CEA_G1333

                      CAC1319

                      5(T)

                      5(C)

                      2(I)

                      2(T)

                      Glycerol uptake facilitator protein, GLPF

                      CEA_G1472

                      CAC1456

                      974(T)

                      974(A)

                      325(M)

                      325(K)

                      Sugar-binding periplasmic protein

                      CEA_G2622

                      CAC2613

                      270(T)

                      270(G)

                      90(C)

                      90(W)

                      Transcriptional regulators of NagC/XylR family

                      CEA_G2919

                      CAC2912

                      97(A)

                      97(C)

                      33(T)

                      33(P)

                      Sugar-binding periplasmic protein

                      CEA_P0052

                      CA_P0053

                      317(C)

                      317(T)

                      106(P)

                      106(L)

                      Xylanase, glycosyl hydrolase family 10

                      Sporulation related genes

                      CEA_G0080

                      CAC0080

                      1160(A)

                      1159-1160(-)

                      387-392(N,I,Q,D,L,Stop codon)

                      387-391(I,Y,K,I,Y,K)

                      Histidine kinase-like ATPase

                      CEA_G0656

                      CAC0644

                      1226(T)

                      1226(G)

                      409(V)

                      409(G)

                      Spore germination protein gerKA

                      CEA_G0710

                      CAC0699

                      416(C)

                      416(T)

                      139(T)

                      139(I)

                      Spore photoproduct lyase, splB

                      CEA_G2066

                      CAC2052

                      688(A)

                      687-688(-)

                      230-248 (18 amino acid)

                      230-238 (7 amino acid and a Stop Codon)

                      DNA-dependent RNA polymerase sigma subunit

                      CEA_G3736

                      CAC3729

                      374(T)

                      374(C)

                      125(L)

                      125(P)

                      Stage 0 sporulation J, ParB family of DNA-binding proteins

                      CEA_P0016

                      CA_P0017

                      11,245(G,A)

                      11,245(T,C)

                      4,82(E,G)

                      4,82(A,V)

                      Spore germination protein, GRKB

                      CEA_P0019

                      CA_P0020

                      1120(C)

                      1120(T)

                      407(A)

                      407(V)

                      Spore germination protein, GRKA

                      CEA_P0021

                      CA_P0022

                      -151(T)

                      104(C)

                      1(---)

                      1-85(85 amino acids insertion)

                      Spore germination protein, GRKB

                      Numbers in gene or protein variation sites lines indicated the variation sites in genes; the letters in bracket means the corresponding variation bases or amino acid; the symbol "-" means deletion in genes; the symbol "---" means consecutive deletion in genes; "No" means no amino acid variation; "Stop codon" means this site is mutated to stop codon.

                      Table 3

                      EA 2018 putative promoter variations related to key phenotypes

                      Gene locus in EA 2018

                      Gene locus in ATCC 824

                      Gene variation sites in EA 2018

                      Gene variation sites in ATCC 824

                      Product Name

                      Solvent formation related genes

                      CEA_G0028

                      CAC0028

                      -12(T)

                      -12(C)

                      Hydrogen dehydrogenase

                      CEA_P0034

                      CA_P0035

                      -84(T)

                      -84(C)

                      Aldehyde-alcohol dehydrogenase, ADHEII

                      Substrate utilization related genes

                      CEA_G3043

                      CAC3037

                      -80(T)

                      -80(C)

                      Catabolite control protein, LacI family transcriptional regulator

                      CEA_G3455

                      CAC3451

                      -84(T)

                      -84(G)

                      Sugar/Na+(H+) simporter

                      CEA_G1086

                      CAC1075

                      -56(T)

                      -56(G)

                      Beta-glucosidase family protein

                      CEA_G1365

                      CAC1351

                      -97(T)

                      -97(C)

                      Periplasmic sugar-binding protein

                      Sporulation related genes

                      CEA_G1634

                      CAC1620

                      -136(T)

                      -136(G)

                      Small acid-soluble spore protein

                      CEA_G3742

                      CAC3735

                      -(8-7)(-)

                      -7(C)

                      Predicted RNA-binding protein Jag, SpoIIIJ-associated

                      Numbers in gene or protein variation sites lines indicated the variation sites in genes; the letters in bracket means the corresponding variation bases or amino acid; the symbol "-" means deletion in genes.

                      Comparative transcriptomic analysis of EA 2018 and ATCC 824

                      To further explore the molecular mechanism of enhanced butanol production in the EA 2018 strain, DNA microarray of C. acetobutylicum were manufactured and used for a comparative analysis between EA 2018 and ATCC 824. The complete set of DNA array data was available in Additional file 8. Microarray analysis showed that a total of 2215 genes were differentially regulated at transcriptional level in at least 1 cultivation time point. Among them, differentially regulated genes related to some important metabolic pathways were listed in Table 4. And some putative promoter variation genes such as adhEII were found with differential expression level in the EA 2018 strain (Table 5). The detailed comparative analysis of transcriptomic data, along with genomic data (i.e. variation gene sequence) and biochemical phenotypes will be provided below from three key aspects related to enhanced butanol production (i.e. solvent formation, xylose utilization, and spore formation).
                      Table 4

                      List of differentially regulated genes in key functional groups.

                      Gene locus in EA 2018

                      Gene locus in ATCC 824

                      9 h (Log2Ratio 2018/824)

                      13 h (Log2Ratio 2018/824)

                      17 h (Log2Ratio 2018/824)

                      21 h (Log2Ratio 2018/824)

                      24 h (Log2Ratio 2018/824)

                      30 h (Log2Ratio 2018/824)

                      Product Name

                      Carbohydrate transportant and metabolism

                           

                      CEA_G0343

                      CA_C0332

                      -2.97436

                      -3.22501

                      -2.37758

                      -2.08064

                      -2.57556

                      -1.49158

                      Beta-mannanase

                      CEA_G0501

                      CA_C0490

                      2.331505

                      2.410311

                      2.244172

                      2.361906

                      2.703307

                      2.474026

                      sugar kinase, N-terminal region - uncharacterized protein

                      CEA_G0552

                      CA_C0539

                      -3.20909

                      -3.00324

                      -2.93013

                      -2.29917

                      -3.34939

                      -3.01922

                      ChW repeat-containing mannanase ManB

                      CEA_G0553

                      CA_C0540

                      -3.097

                      -3.03398

                      -2.7227

                      -2.28306

                      -3.3665

                      -2.72934

                      ChW repeat-containing mannanase ManB

                      CEA_G1677

                      CA_C1664

                      1.744408

                      2.013214

                      2.53088

                      1.134795

                      1.816259

                      2.062687

                      glycogen phosphorylase

                      CEA_G2012

                      CA_C1997

                      3.052977

                      3.503142

                      3.004392

                      2.8712

                      2.075025

                      1.372718

                      glycosyltransferase

                      CEA_G2022

                      CA_C2007

                      3.179932

                      3.308867

                      2.812136

                      2.509044

                      1.656072

                      1.958591

                      glycosyltransferase

                      CEA_G2528

                      CA_C2514

                      2.588809

                      2.748177

                      3.625871

                      4.329215

                      2.708288

                      1.042021

                      Beta galactosidase

                      CEA_G2815

                      CA_C2807

                      2.40568

                      2.124155

                      2.476058

                      2.653992

                      2.808752

                      1.737289

                      endo-1,3(4)-beta-glucanase family protein 16

                      CEA_G2818

                      CA_C2810

                      1.835243

                      2.287891

                      2.800041

                      3.929157

                      3.739976

                      4.256289

                      glucoamylase family protein

                      CEA_G3051

                      CA_C3045

                      -2.41601

                      -2.05151

                      -2.19775

                      -2.37262

                      -2.3321

                      -2.16444

                      PHP family hydrolase

                      CEA_G3060

                      CA_C3054

                      -3.63818

                      -4.38617

                      -4.64833

                      -5.02922

                      -5.12371

                      -3.50751

                      phosphoheptose isomerase

                      CEA_G3426

                      CA_C3422

                      -1.07396

                      -2.79221

                      -3.22016

                      -2.15086

                      -2.69786

                      -1.37931

                      sugar:proton symporter (xylulose)

                      CEA_P0052

                      CA_P0053

                      2.477697

                      2.868013

                      3.649329

                      3.695172

                      3.721454

                      5.987476

                      xylanase

                      CEA_P0053

                      CA_P0054

                      4.25084

                      2.735168

                      2.447001

                      3.424303

                      3.598697

                      5.185082

                      xylanase/chitin deacetylase family protein

                      CEA_P0065

                      CA_P0066

                      3.062167

                      1.145145

                      2.159875

                      4.902259

                      3.81378

                      2.953822

                      mannose-specific phosphotransferase system component IIAB

                      CEA_P0066

                      CA_P0067

                      2.925021

                      1.579742

                      2.174524

                      4.883877

                      3.683632

                      2.826095

                      mannose/fructose-specific phosphotransferase system component IIC

                      CEA_P0067

                      CA_P0068

                      3.006142

                      1.74298

                      2.611356

                      4.717371

                      3.502957

                      2.456002

                      mannose-specific phosphotransferase system component IID

                      CEA_P0115

                      CA_P0116

                      1.777221

                      2.342324

                      2.902238

                      3.126694

                      3.321994

                      5.378643

                      xylanase

                      Amino acid transport and metabolism

                           

                      CEA_G0180

                      CA_C0176

                      2.56498

                      2.063235

                      2.36635

                      2.08086

                      3.251555

                      3.705152

                      oligopeptide-binding protein, periplasmic component

                      CEA_G0327

                      CA_C0316

                      -4.09229

                      6.429377

                      7.957548

                      6.881528

                      4.185199

                      2.037144

                      ornithine carbomoyltransferase

                      CEA_G0390

                      CA_C0380

                      -4.23921

                      5.06975

                      5.365591

                      5.142555

                      3.394778

                      2.099342

                      periplasmic amino acid-binding protein

                      CEA_G0984

                      CA_C0973

                      -4.67305

                      6.390879

                      8.78089

                      7.141168

                      3.889621

                      2.522783

                      argininosuccinate synthase

                      CEA_G0985

                      CA_C0974

                      -4.03279

                      6.033303

                      8.738086

                      7.66813

                      4.662246

                      3.462605

                      argininosuccinate lyase

                      CEA_G2392

                      CA_C2377

                      -4.81354

                      -4.50847

                      -4.89131

                      -5.25303

                      -5.6598

                      -6.94396

                      oligopeptide ABC-type transporter, periplasmic binding component (frameshift)

                      CEA_G2403

                      CA_C2388

                      -3.653

                      6.58659

                      9.490741

                      7.214547

                      3.839977

                      1.277684

                      acetylornithine aminotransferase

                      CEA_G2405

                      CA_C2390

                      -4.029

                      7.044851

                      9.332151

                      8.593887

                      5.225523

                      1.729156

                      N-acetyl-gamma-glutamyl-phosphate reductase

                      CEA_G2406

                      CA_C2391

                      -3.63924

                      6.659273

                      9.430206

                      8.041522

                      4.889782

                      1.658329

                      bifunctional ornithine acetyltransferase/N-acetylglutamate synthase protein

                      CEA_G2531

                      CA_C2517

                      2.601974

                      2.875402

                      3.244092

                      4.199853

                      5.407919

                      5.659197

                      extracellular neutral metalloprotease, NPRE

                      CEA_G3059

                      CA_C3053

                      -3.80847

                      -4.46351

                      -4.85998

                      -5.20027

                      -5.64176

                      -4.16398

                      histidinol phosphatase related enzyme

                      CEA_G3625

                      CA_C3618

                      -2.33371

                      4.975742

                      4.579403

                      4.356899

                      3.79613

                      1.400104

                      ABC-type polar amino acid transport system, ATPase component

                      CEA_G3626

                      CA_C3619

                      -2.49499

                      4.94909

                      4.797836

                      4.831871

                      4.227636

                      1.349231

                      amino acid ABC transporter permease

                      CEA_G3627

                      CA_C3620

                      -2.79225

                      4.869307

                      2.77014

                      4.039679

                      3.923914

                      1.658129

                      amino acid ABC transporter periplasmic-binding protein

                      CEA_G3629

                      CA_C3622

                      3.744723

                      1.616914

                      -1.60643

                      -2.30924

                      -3.70552

                      -4.20925

                      benzoyl-CoA reductase/2-hydroxyglutaryl-CoA dehydratase

                      CEA_G3648

                      CA_C3641

                      3.877563

                      5.273677

                      6.532841

                      7.05381

                      6.150331

                      2.645978

                      oligopeptide ABC transporter, ATPase component

                      CEA_G3649

                      CA_C3642

                      3.862767

                      5.064748

                      6.213521

                      7.100308

                      6.123778

                      2.328286

                      oligopeptide ABC transporter, ATPase component

                      CEA_G3651

                      CA_C3644

                      3.540737

                      4.731217

                      4.796332

                      6.751584

                      6.151055

                      3.301733

                      oligopeptide ABC transporter, permease component

                      Lipid transport and metabolism

                            

                      CEA_P0077

                      CA_P0078

                      -1.03663

                      -2.39938

                      -2.90422

                      -5.61457

                      -6.37659

                      -6.669

                      acetyl-CoA acetyltransferase

                      CEA_G0500

                      CA_C0489

                      2.843362

                      2.727643

                      3.104514

                      2.258672

                      2.789674

                      2.994569

                      4'-phosphopantetheinyl transferase

                      CEA_G2024

                      CA_C2009

                      3.02793

                      3.412819

                      2.753976

                      2.880761

                      2.465438

                      1.317947

                      3-hydroxyacyl-CoA dehydrogenase

                      CEA_G2027

                      CA_C2012

                      3.139119

                      3.519089

                      2.760385

                      2.466674

                      1.747722

                      1.295802

                      enoyl-CoA hydratase

                      CEA_G2023

                      CA_C2008

                      3.21547

                      3.393795

                      2.65468

                      2.624962

                      1.849978

                      1.696553

                      3-oxoacyl-(acyl-carrier-protein) synthase

                      CEA_G0825

                      CA_C0814

                      3.447389

                      2.690101

                      3.341654

                      3.492821

                      2.738719

                      2.617536

                      3-oxoacyl-

                      CEA_G3630

                      CA_C3623

                      3.78405

                      1.698615

                      -1.62094

                      -2.13197

                      -3.66189

                      -4.47732

                      2-hydroxyglutaryl-CoA dehydratase activator

                      Coenzyme transport and metabolism

                            

                      CEA_G2539

                      CA_C2526

                      -5.80423

                      -4.29934

                      -2.18936

                      -3.20732

                      -2.04208

                      -2.09088

                      6-pyruvoyl-tetrahydropterin synthase related protein

                      CEA_G0110

                      CA_C0109

                      -1.93293

                      -1.10289

                      -4.51818

                      -4.22606

                      -3.40598

                      -5.95312

                      sulfate adenylyltransferase subunit 2

                      CEA_G2240

                      CA_C2226

                      1.175604

                      1.630568

                      1.933437

                      3.44313

                      4.132037

                      3.011011

                      branched-chain amino acid aminotransferase

                      CEA_G2817

                      CA_C2809

                      1.909506

                      2.362089

                      1.59545

                      3.297164

                      2.183049

                      1.029092

                      HD superfamily hydrolase

                      CEA_G2037

                      CA_C2022

                      2.45013

                      1.945668

                      2.521958

                      2.427533

                      2.187431

                      3.238565

                      molybdopterin biosynthesis protein MoaB

                      CEA_G3633

                      CA_C3626

                      2.627562

                      1.339719

                      -1.38437

                      -2.32524

                      -3.57634

                      -5.06491

                      GTP cyclohydrolase I

                      CEA_G2036

                      CA_C2021

                      2.849463

                      2.144804

                      2.666979

                      4.297463

                      3.319713

                      3.863173

                      molybdopterin biosynthesis protein MoeA

                      CEA_G2009

                      CA_C1994

                      3.136184

                      3.75827

                      3.408469

                      2.84809

                      1.881193

                      1.268359

                      molybdopterin biosynthesis protein MoaB

                      CEA_G2035

                      CA_C2020

                      3.146626

                      3.515592

                      5.182665

                      4.677354

                      4.692789

                      4.012931

                      molybdopterin biosynthesis protein MoeA

                      CEA_G3631

                      CA_C3624

                      3.416604

                      1.490697

                      -1.38827

                      -2.23319

                      -3.83641

                      -6.15768

                      6-pyruvoyl-tetrahydropterin synthase

                      Signal transduction

                             

                      CEA_G0078

                      CA_C0078

                      -4.99228

                      -8.71284

                      -9.43594

                      -9.21854

                      -9.14927

                      -7.97564

                      putative accessory gene regulator protein

                      CEA_G3328

                      CA_C3325

                      -1.00804

                      -2.85267

                      -4.3502

                      -5.19446

                      -4.39209

                      -5.05262

                      periplasmic amino acid binding protein

                      CEA_G0921

                      CA_C0909

                      -2.84748

                      -1.88833

                      -1.93256

                      -1.50557

                      -2.64087

                      -2.89144

                      methyl-accepting chemotaxis protein

                      CEA_G2085

                      CA_C2071

                      2.455498

                      2.263411

                      2.413649

                      1.803983

                      2.20351

                      2.550339

                      Spo0A protein

                      CEA_G2422

                      CA_C2407

                      3.784844

                      1.475039

                      1.927948

                      1.823945

                      2.526403

                      2.502499

                      CheY-like domain-containing protein

                      CEA_G0448

                      CA_C0437

                      2.192653

                      1.378372

                      2.665966

                      1.827148

                      2.252286

                      2.342264

                      sensory transduction histidine kinase

                      CEA_G3025

                      CA_C3019

                      -2.65448

                      3.491201

                      3.449102

                      2.735229

                      1.336433

                      1.266833

                      sensory transduction protein

                      CEA_G0296

                      CA_C0289

                      1.792323

                      1.978035

                      2.438903

                      2.751395

                      3.435751

                      3.052853

                      response regulator

                      CEA_G2782

                      CA_C2774

                      1.625932

                      1.895382

                      3.060796

                      2.846554

                      2.092881

                      1.726376

                      methyl-accepting chemotaxis protein

                      CEA_G0334

                      CA_C0323

                      3.362504

                      2.662176

                      2.652177

                      3.222052

                      4.952742

                      2.037345

                      sensory transduction histidine kinase

                      CEA_G3627

                      CA_C3620

                      -2.79225

                      4.869307

                      2.77014

                      4.039679

                      3.923914

                      1.658129

                      amino acid ABC transporter periplasmic-binding protein

                      CEA_G0390

                      CA_C0380

                      -4.23921

                      5.06975

                      5.365591

                      5.142555

                      3.394778

                      2.099342

                      periplasmic amino acid-binding protein

                      Energy production and convertion

                            

                      CEA_G1083

                      CA_C1072

                      3.271648

                      3.230747

                      4.254617

                      2.59071

                      2.723209

                      3.766376

                      Fe-S oxidoreductase

                      CEA_G2012

                      CA_C1997

                      3.052977

                      3.503142

                      3.004392

                      2.8712

                      2.075025

                      1.372718

                      glycosyltransferase

                      CEA_G2015

                      CA_C2000

                      2.963078

                      3.638083

                      2.745804

                      2.983214

                      2.587284

                      2.204476

                      indolepyruvate oxidoreductase subunit beta

                      CEA_G2016

                      CA_C2001

                      2.832561

                      3.688549

                      3.007107

                      3.355002

                      2.718563

                      2.206461

                      indolepyruvate ferredoxin oxidoreductase, subunit

                      CEA_G2022

                      CA_C2007

                      3.179932

                      3.308867

                      2.812136

                      2.509044

                      1.656072

                      1.958591

                      glycosyltransferase

                      CEA_G2025

                      CA_C2010

                      3.282342

                      3.533526

                      2.84142

                      3.003615

                      2.50221

                      2.005231

                      Fe-S oxidoreductase

                      CEA_G2555

                      CA_C2542

                      8.22595

                      5.876659

                      5.423124

                      5.135223

                      4.224852

                      4.280766

                      FAD/FMN-containing dehydrogenase

                      CEA_G2556

                      CA_C2543

                      8.451735

                      6.100494

                      5.401602

                      5.24343

                      4.426238

                      3.987954

                      electron-transferring flavoprotein large subunit

                      CEA_G2557

                      CA_C2544

                      7.904188

                      6.040402

                      4.848558

                      4.578106

                      4.214698

                      3.969443

                      electron-transferring flavoprotein small subunit

                      CEA_G3411

                      CA_C3408

                      -2.71993

                      -3.64684

                      -4.53359

                      -4.7967

                      -4.89809

                      -4.45863

                      NADH oxidase

                      Cell mobility

                              

                      CEA_G0921

                      CA_C0909

                      -2.84748

                      -1.88833

                      -1.93256

                      -1.50557

                      -2.64087

                      -2.89144

                      methyl-accepting chemotaxis protein

                      CEA_G3572

                      CA_C3565

                      1.783589

                      1.966743

                      1.626123

                      1.303128

                      1.522492

                      1.56009

                      cell adhesion domain-containing protein

                      CEA_G3091

                      CA_C3085

                      1.669031

                      1.879947

                      1.641572

                      3.329325

                      2.811493

                      2.558158

                      TPR repeat-containing cell adhesion protein

                      CEA_G3092

                      CA_C3086

                      1.689695

                      1.7646

                      1.823344

                      3.167841

                      2.629106

                      2.423856

                      cell adhesion domain-containing protein

                      CEA_G2782

                      CA_C2774

                      1.625932

                      1.895382

                      3.060796

                      2.846554

                      2.092881

                      1.726376

                      methyl-accepting chemotaxis protein

                      CEA_P0159

                      CA_P0160

                      2.878477

                      3.739495

                      3.515178

                      3.50976

                      3.19802

                      3.547315

                      cell-adhesion domain-containing protein

                      Table 5

                      DNA and transcriptional variations of putative promoter variations between EA 2018 and ATCC 824

                      Gene locus in EA 2018

                      Gene locus in ATCC 824

                      Gene varition site in 2018

                      Gene varition site in 824

                      9 h (Log2Ratio 2018/824)

                      13 h (Log2Ratio 2018/824)

                      17 h (Log2Ratio 2018/824)

                      21 h (Log2Ratio 2018/824)

                      24 h (Log2Ratio 2018/824)

                      30 h (Log2Ratio 2018/824)

                      Product Name

                      CEA_G0334

                      CA_C0323

                      -96(A)

                      -(97-96)(-)

                      3.362504

                      2.662176

                      2.652177

                      3.222052

                      4.952742

                      2.037345

                      Sensory transduction histidine kinase

                      CEA_G0390

                      CA_C0380

                      -135,-131(A,A)

                      -135,-131(T,T)

                      -4.23921

                      5.06975

                      5.365591

                      5.142555

                      3.394778

                      2.099342

                      Periplasmic amino acid-binding protein

                      CEA_G2504

                      CA_C2490

                      -(13-12),-53(-,T)

                      -12,-(54-53)(C,-)

                      -2.39555

                      -3.35613

                      -3.80832

                      -2.2032

                      -2.7562

                      -1.10185

                      Xre family DNA-binding domain and TPR repeats containing protein

                      CEA_G3701

                      CA_C3694

                      -43(T)

                      -43(C)

                      4.045439

                      3.430925

                      3.859781

                      3.69948

                      4.131629

                      3.75554

                      TPR-repeat-containing protein

                      CEA_P0034

                      CA_P0035

                      -84(T)

                      -84(C)

                      3.331902

                      5.740285

                      6.007483

                      8.056546

                      6.576379

                      7.664691

                      Aldehyde-alcohol dehydrogenase, AdhEII

                      Changes in expression of adhEII, spo0A and hydrogenase gene may contribute to enhanced solvent formation in EA 2018

                      Comparative genomic analysis identified a set of solvent-relevant genes with variations within their coding sequences (Table 2). And most of which were SNV variations, such as genes encoding phosphotransacetylase and acetyl-CoA acetyltransferase. In addition, comparative genomics analysis also identified some variations up-stream of solvent-relevant genes (Table 3), which could potentially affect expression level of these genes. For example, a SNV site was found 84 bases upstream of the start codon of the AdhEII encoded gene CEA_P0034 (CA_P0035 in ATCC 824), under the s 1 transcription start point [25]. Consistent with the variations at genomic level, we also found that transcription level of solvents formation genes, such as adhEII were highly expressed in EA 2018 relative to ATCC 824(Table 5 and Figure 3).
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-93/MediaObjects/12864_2010_3221_Fig3_HTML.jpg
                      Figure 3

                      Expression profiles of variation genes involved in sporulation related two-component system and solvent formation pathway ofC. acetobutylicumEA 2018 versus ATCC 824.

                      Spo0A is a central regulator of sporulation and solvent formation in C. acetobutylicum. Previous studies showed that C. acetobutylicum spo0A inactivation mutant stops producing spores and solvent, while over-expression of spo0A gene can enhance solvent production since 0A binding boxes have been identified in the promoter regions of solvent formation genes [26]. Therefore, it is speculative that the higher expression of spo0A could be one of the reasons for the higher butanol formation in EA 2018. Comparative transcriptomic analysis confirmed our speculation that higher transcriptional level of spo0A was found in EA 2018 (Table 4 and Figure 3). There were three ways for Spo0A to be phosphorylated (i.e. by a sensory kinase; through a novel phosphorylation system; by butyryl-P or acetyl-P) [27]. Using trans-membrane domain along with transcriptional analysis, four orphan kinases (CAC0437, CAC0323, CAC0903, and CAC2730) were identified as plausible kinases that might phosphorylate Spo0A in B. subtilis [27]. In our study, we also found that the transcriptional level of an orphan kinase CEA_G0344 (corresponding to CAC0323 in ATCC 824) was higher in EA 2018 (Figure 3), and the result is consistent well with the spo0A gene expression data.

                      During solvent fermentation process in C. acetobutylicum, a considerable amount of NADH was consumed by hydrogenase via reduced Fd (FeH2) to form hydrogen [28]. Previous reports showed that butanol production by C. acetobutylicum can be elevated by inhibiting hydrogen formation through adding viologen dyes or increasing hydrogen partial pressure [28], and knockdown of hupCBA cluster which encoded hydrogen uptake genes in C. saccharoperbutylacetonicum strain N1-4 decreased butanol formation (to 75.6% compared to the control strain) successfully [29]. Biochemical analysis showed that hydrogen formation in EA 2018 was nearly 29% lower than in ATCC 824 (Table 6). Interestingly, comparative genomic analysis also revealed SNVs in NiFe-hydrogenase coded gene CEA_P0140 (CA_P0141 in ATCC 824) and in the promoter of Fe-only hydrogenase coded gene CEA_G0028 (CAC 0028 in ATCC 824) (Table 2, 3). The variation site of CEA_G 0028 was located on the 12th base upstream of the start codon of hydA and altered the ribosome binding site (RBS) of this important gene (GGGAGG in ATCC 824 versus AGGAGG in EA 2018). In addition, the higher expression level of hydrogen uptake genes mbhs and mbhl were also revealed in EA 2018 (Figure 3). The result showed that hydrogen uptake could be an important factor for butanol formation, and increased expression level of hydrogen uptake gene mbhs and mbhl was closely correlated to the lower hydrogen formation in EA 2018, which can eventually help balance the NAD(P)H needed for higher production of butanol.
                      Table 6

                      Hydrogen production of C. acetobutylicum EA 2018 and C. acetobutylicum ATCC 824 in 6% glucose contained P2 medium

                       

                      Fermentation

                      Time (h)

                      Glucose

                      consumed (mM)

                      Hydrogen

                      production (mM)

                      Hydrogen formation

                      ratio (mM/100

                      mM glucose)

                      EA 2018

                      72

                      260.6 ± 3.7

                      244.1 ± 1.3

                      93.9 ± 1.6

                      ATCC 824

                      72

                      239.4 ± 5.0

                      315.4 ± 0.5

                      131.7 ± 3.1

                      Using non-replicating plasmid pO1X, putative solvent formation repressor solR gene was inactivated in ATTC 824, and its fermentation experiment revealed that more solvent were produced in the solR inactivation mutant [5]. Although there are different speculations on the function of SolR [25], it has been confirmed that low expression of solR will enhance solvent formation [26]. Transcriptomic analysis revealed a lower expression level of solR, especially in the solventogenic phase in EA 2018, which might be related to hyper-butanol formation (Figure 3).

                      It has been suggested that the onset of solvent production is closely related to the accumulation of acid end products [30], and the addition of acetate and butyrate might result in a rapid induction of solventogenesis [31]. For example, it was reported that the concentration of undissociated butyric acid might play an important role in the induction of solventogenesis [32]. Transcriptomic analysis showed that expression of ack, pta, buk and ptb were all lower in EA 2018 than in ATCC 824 (Figure 3), consistent with the biochemical analysis (Figure 1). In addition, the results also suggested that the transition to the solventogenesis took place at a lower acetate and butyrate acid concentration in EA 2018 compared to ATCC 824.

                      Analysis of substrate utilization genes and inactivation of CAC2613 revealed genetic bases of better xylose utilization in EA 2018

                      Solvent production from agriculturally based lignocellulosic substrates (i.e. cellulose or hemicellulose) was studied previously and results showed that a large part of the lignocellulosic substrates were hydrolyzed into glucose and xylose [33]. Therefore, utilization of these substrates, especially xylose, can be important in determining the efficiency of solvent production. Comparative genomic analysis identified several mutations in the putative promoters and within the coding region of genes which might be involved in substrates utilization (Table 2, 3). Among them, three out of seven mutated genes encode sugar-binding periplasmic proteins. One interesting gene was CEA_G2622 (CAC2613 in ATCC 824), which encodes a transcriptional regulator of NagC/XylR family and the sequence variation could cause a putative W90C substitution. The gene is located on the upstream of xylB (xylulose kinase) gene (Figure 4A). Since most of the known xylR genes in other AT-rich gram-positive species such as B. subtillus and C. difficile were located upstream of xyl operon [34, 35], we speculated that this gene (CEA_G2622) may function similarly as xylR in EA 2018 (Figure 4B). Transcriptomic analysis showed that even in the glucose-based medium, the expression level of xylB was higher in EA 2018 (Figure 3). However, evidence is still needed to confirm the direct regulatory function of CEA_G2622 on xyl operon. To do so, we disrupted CAC2613 gene in C. acetobutylicum ATCC 824 (corresponding to CEA_G2622 in EA 2018) using Targetron system (Figure 4C). Batch fermentation showed that xylose utilization in CAC2613 disrupted mutant was faster than ATCC 824 (constant pH 5.0). In addition, the time of butanol formation and acids reassimilation in the mutant were 24 h earlier than ATCC 824 strain, although the final concentration of end products and xylose were nearly the same (Figure 4D). The similarities, in terms of the time of butanol formation and acid reassimilation, between EA 2018 and the CAC2613 disrupted ATCC 824 derived mutant, suggested that better xylose utilization in EA 2018 could be related to the mutation in CEA_G2622 (CAC2613 in ATCC 824).
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-93/MediaObjects/12864_2010_3221_Fig4_HTML.jpg
                      Figure 4

                      Inactivation of putativexylRCAC2613 in ATCC 824 leaded to faster xylose utilization inC. acetobutylicum. A: location of CAC2613 in C. acetobutylicum and xylR in different organisms; B: Alignment of CAC2613 and xylR in different organisms; C: CAC2613 inactivation in ATCC 824 using Targetron system; D: Accurate pH-controlled fermentation profile of mutant strain versus wild type in 6% xylose contained P2 medium.

                      Previous study showed that ATCC 824 harbors extracellular and cell bound xylanase activities when grown under xylose or glucose-based media, and most of the putative xylanase encoded genes were located on the megaplasmid [36]. Two endoxylanase genes, thermostable xylanase 10A gene (CA_P0053 in ATCC 824 and CEA_P0052 in EA 2018) and xylanase 10B gene (CA_P0116 in ATCC 824 and CEA_P0115 in EA 2018) located on mega-plasmid were identified in EA 2018 [37]. In addition, transcriptomic analysis showed higher expression level of those two genes in EA 2018 (Figure 5). Xylan is the major component of hemicelluloses. The higher expression level of xylanase in EA 2018 could make it suitable for hemicellulosic fermentation, and could offer potential economic benefits in the future [1].
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-93/MediaObjects/12864_2010_3221_Fig5_HTML.jpg
                      Figure 5

                      Expression profiles of substrate utilization related genes. Gene functions were shown below the expression profile. Red and green indicated higher or lower expression, respectively.

                      Among all putative promoter variations, there were 4 sites which may affect substrate utilization in EA 2018 (Table 3). Among them, CEA_G3043 (CAC3037 in ATCC 824) gene that encodes a catabolite control protein (CcpA) has a variation site located on 80 bp upstream of the start codon. CcpA play an important role in catabolite repression and inactivation of this gene will release catabolic repression in many gram-positive organisms [8, 38, 39]. However, no significant regulation was observed for the expression level of CcpA gene in EA 2018, and the potential effect of the variation in ccpA promoter region still needs further investigation.

                      agrC and sigma factor variations may involve in spore formation in EA 2018

                      Comparative genomic analysis identified several genes which may be accounted for difference in terms of spore formation (Table 2). Among these muted genes, CEA_G2066 (CAC2052 in ATCC 824) encodes a putative sigma factor. It has been known that the transcription of this gene was closely related to spore-formation in ATCC 824 [40]. CEA_G2066 has a single nucleotide (A) insertion site in 687-688th bases, which altered the C-terminal protein sequence. Transcriptomic analysis showed that the transcription level of CEA_G2066 and other 4 putative sporulation related sigma factors [40] was lower in EA 2018 at 21 h, 24 h and 30 h (Figure 6), which might contribute to non-sporulation property in EA 2018.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-93/MediaObjects/12864_2010_3221_Fig6_HTML.jpg
                      Figure 6

                      Expression profiles of sporulation related genes. Gene functions were shown below the expression profile. Red and green indicated higher or lower expression, respectively.

                      Quarum sensing is related to some important characteristics of bacteria, such as sporulation, virulence, and biofilm formation [41]. In C. perfringens, the virulence gene was regulated by agr system, and agrBD knockout mutant did not express theta-toxin gene, and transcription of the alpha- and kappa-toxin genes was also significantly decreased in the mutant strain [42]. A two-component system gene, CEA_G0080 (CAC0080 in ATCC 824), encoding a histidine kinase-like ATPase (AgrC), has a single nucleotide (A) insertion site in 1159-1160th bases when compared with agrC gene in ATCC 824, which may truncate the protein encoding sequence. In addition, the expression level of agrB (CEA_G0078) was constantly lower in EA 2018 (Figure 3). Early studies suggested that deletion of any agr system genes could result in no spore formation in ATCC 824 [43]. Therefore, mutation in agrC gene and lower expression of agrB gene in EA 2018 might be responsible for to the non-sporulation property in EA 2018.

                      Conclusion

                      A hyper-butanol, non spore-forming C. acetobutylicum EA 2018 strain we isolated previously can produce 10% more butanol than the type strain C. acetobutylicum ATCC 824 [23]. To seek molecular basis of these characteristics in EA 2018 strain, we completed the genome sequencing of this strain using 454 GS FLX pyrosequceing and performed a detailed genomic comparison with a C. acetobutylicum type strain ATCC 824. Although EA 2018 was found more than 99.8% identical to ATCC 824, 72 indels (i.e. insertions and deletions) and 451 SNVs were identified, some of which may be related to the enhanced butanol production in EA 2018. In addition, we performed a comparative transcriptomic analysis of C. acetobutylicum EA 2018 and ATCC 824 using oligonucleotide microarrays. The results showed that increased expression of several key genes related to solvent formation, and decreased expression of the acid formation related genes may be related to the enhanced butanol production in EA 2018. Furthermore, the results also showed that the variation in CEA_G2622 (CAC2613 in ATCC 824), a putative transcriptional regulator involved in xylose utilization, may be able to accelerate utilization of substarte xylose. The comparisons of hyper-butanol EA 2018 and type strain ATCC 824 at both genomic and transcriptomic levels not only improved our understanding of the hyper butanol-producing, xylose utilization as well as non-spore formation properties in EA 2018 strain, but also provided some useful clues for the future genetic modification of C. acetobutylicum to produce solvents, especially butanol more effectively.

                      Methods

                      Bacteria strain and genome sequencing

                      C. acetobutylicum EA 2018 (CCTCC M 94061) used for this study [22] is deposited in China Center for Type Culture Collection (http://​www.​cctcc.​org/​). C. acetobutylicum ATCC 824 was the wild type strain we purchased from American Type Culture Collection (ATCC) [23]. The C. acetobutylicum EA 2018 was grown anaerobically (Thermo Electron Crop., San Jose, USA). Colonies picked from Clostridia growth medium (CGM) plate were inoculated into 5 ml liquid CGM and cultured at 37°C overnight [44], and then the cells were transferred into 100 ml CGM and incubated at 37°C for 16-20 h until they reach late-exponential phase. Cells collected were used for chromosomal DNA isolation as described previously [45]. Roche 454 GS FLX pyrosequencing was used to sequence the DNA. A total of 60.3 Megabases was generated, with an average read length of 200 bp. The GS FLX reads were assembled into a total of 198 contigs using a GS de novo assembler, among them, 157 contigs are larger than 500 bp. The gaps were closed by PCR procedure using the ATCC 824 genome sequence as reference. The large PCR products were sequenced via primer walking. The whole sequence was assembled by using the software phredPhrap (http://​www.​phrap.​org) and was visualized by Consed [46]. The low-quality sequences were verified by PCR resequencing using ABI 3730 (Applied Biosystem Inc.). The sequence accuracy of the final genome was 99.9919%. All the variation between EA 2018 and ATCC 824 were verified by PCR resequencing using ABI 3730.

                      Genome annotation and bioinformatic analysis

                      CDSs were identified by combining the results of ZCURVE 1.0 [47] and Glimmer 3.2 (http://​www.​cbcb.​umd.​edu/​software/​glimmer). Transfer RNA genes were predicted by tRNAscan-SE [48]. Functional annotation of CDSs was performed through comparison with NCBI non-redundant protein database using BLASTP, followed by manual curation. Comparative genomic analysis was performed by using the Artemis Comparison Tool (ACT; http://​www.​sanger.​ac.​uk/​resources/​software/​act/​). The atlas of the genome is drawn by using GenomeViz1.1 [49].

                      Nucleotide sequence accession number

                      The annotated genome sequence has been deposited into GenBank under accession no. CP002118 (Chromosome) and no. CP002119 (Plasmid).

                      Oligonucleotide microarray experiments

                      Cells for RNA isolation were grown on P2 medium and collected at 9 h, 13 h, 17 h, 21 h, 24 h and 30 h by centrifugation at 4°C and 4500 × g for 10 min. Total RNA was extracted and purified by using Trizol (Invitrogen, Carlsbad, CA, USA) and RNeasy cleanup kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. The total RNA yield was quantified by spectrophotometric analysis (NanoDrop Technology, Cambridge, UK) and the quality was verified by gel electrophoresis. Agilent oligonucleotide microarrays technology was used for monochromic analysis, in which probes (size: 60 bp; three replicates for each ORF) from the two groups were labeled by incorporation of cyanine 3 (Cy3) (Agilent Technologies, Palo Alto, CA, USA). The experiment procedures and data normalization were performed using the methods described previously [50]. Average linkage hierarchical clustering was performed using Cluster 3.0, and gene clusters were visualized in Treeview [51].

                      Oligonucleotide microarray accession number

                      The Oligonucleotide microarray data has been deposited into GEO under accession no. GSE23071.

                      Gene disruption

                      Gene disruption in C. acetobutylicum ATCC 824 was performed as described previously [11]. The disruption procedures were shown in Additional file 9. The selected site for CAC2613 disruption was 532/533a, where the group II intron will insert into gene CAC2613 coding region between amino acid 532 and 533 sites in the antisense direction. The primers (CAC2613-532-533-IBS, CAC2613-532-533-EBS1d and CAC2613-532-533-EBS2), for retargeting the RNA portion of the intron for C. acetobutylicum CAC2613 gene disruption, are listed in Additional file 10. The 350 bp targetron fragment was obtained by PCR based on the plasmid pACD4K-C and protocol provided by the TargeTron™ Gene Knockout System Kit (Sigma-Aldrich, St Louis, MO, USA). The 350 bp PCR fragment was digested with XhoI and BsrGI, and then inserted into pSY6 [11] digested with the same restriction enzymes, to generate the plasmid pSY6-2613. The plasmid pSY6-2613 was methylated in E. coli ER2275 (pANS1) first [52], and then electroporated into C. acetobutylicum ATCC 824. Cells were plated on CGM agar containing 50 μg/mL erythromycin and incubated at 37°C for about 2-3 days. The positive transformants containing the inserted intron were identified by colony PCR, using primers CAC2613-ID-fw and CAC2613-ID-rev (Additional file 10).

                      Fermentation conditions

                      Solvent production and sugar utilization of C. acetobutylicum ATCC 824 and C. acetobutylicum EA 2018 were determined when the cultures were grown on P2 medium [53]. 6% Glucose or xylose contained P2 solution I (840 ml) and KH2PO4 (0.5 g/L), K2HPO4 (0.5 g/L), CH3COONH4 (2.2 g/L) contained P2 solution II (100 ml) were boiled for 20 min, then cooled by flushing O2-free N2 gas, and autoclaved at 121°C for 15 min separately. After autoclaving, 100 ml P2 solution II, 10 ml filter-sterilized P2 medium stock solution III (MgSO4·7H2O, 20 g/L; MnSO4·H2O, 1 g/L; FeSO4·7H2O, 1 g/L; NaCl, 1 g/L) and 1 ml solution IV (Para-amino-benzoicacid, 0.1 g/L; thiamin, 0.1 g/L; biotin, 0.001 g/L) were added into P2 solution I. The stock solutions were filter sterilized through a 0.2 μm pore-size filter. An inoculum of 5% from a CGM grown culture was typically used. Batch fermentation was carried out in 250 ml sealed bottle with 100 ml medium. 1 ml samples were taken every 12 h and analyzed for solvent and sugar. Accurate pH-controlled fermentations were carried out in BioFlo 110 bioreactors with 1.5 L working volume (New Brunswick Scientific, Edison, NJ). The pH control was achieved by using 9% (w/v) aqueous ammonia. Anaerobic conditions of fermentors were maintained through aeration of filtered nitrogen.

                      Analytical methods

                      The surface morphology of C. acetobutylicum EA 2018 was studied using a JSM-6360 scanning electron microscope (JEOL Co. Ltd. Japan). Spore formation analysis was performed by growing the cells in P2 medium. After 48 h fermentation, cells were collected by centrifuging at 10,000 × g for 5 min, and stained with crystal violet for imaging analysis using a U-CTR30-2 microscope (Olympus Optical Co. Ltd. Japan). Butanol, acetone, ethanol, acetic acid and butyric acid were determined using a gas chromatograph (7890A, Agilent, Wilmington, DE, USA) equipped with a capillary column (Alltech EC™-WAX) and a flame ionization detector. The analysis was carried out under the following conditions: oven temperature, programmed from 80 to 140°C at a rate of 25°C/min; injector temperature, 200°C; detector temperature, 200°C; nitrogen (carrier gas) flow rate, 13 ml/min; hydrogen flow rate, 20 ml/min; air flow rate, 140 ml/min. Total solvent was defined as the sum of ABE. Isobutylalcohol and isobutyric acid were used as the internal standards for ABE and acid determination, respectively. Glucose and xylose were determined using a HPLC system (Model 1200, Agilent) equipped with a sugar-pak I column (Waters) and a refractive index detector. The analysis was carried out with water as mobile phase at a rate of 0.6 ml/min, and the column temperature was set up at 70°C. The composition of the gas produced (mainly H2 and CO2) was measured using the method described previously [54].

                      Abbreviations

                      ABE: 

                      Acetone-Butanol-Ethanol

                      ATCC: 

                      American Type Culture Collection

                      CCTCC: 

                      China Center for Type Culture Collection

                      ORF: 

                      Open Reading Frames

                      NTG: 

                      N-methyl-N-nitro-N-nitrosoguanidine

                      SNVs: 

                      Single Nucleotide Variations

                      RBS: 

                      Ribosome Binding Site

                      CcpA: 

                      Catabolite Control Protein A

                      CGM: 

                      Clostridia Growth Medium

                      Declarations

                      Acknowledgements

                      This work was supported by National Basic Research Program of China (2007CB707803, 2011CBA00806), Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-G-1, KSCX2-EW-J-12), National Natural Science Foundation of China (31070075) and Key Program of Science and Technology Commission of Shanghai Municipality (08DZ1207100).

                      We thank Prof. Peter Duerre for providing the manipulation methods on Clostridium acetobutylicum. We thank Jun Chen, Huiqi He, Lijun Shao, Cong Ren, Yongqiang Zhu, Qiuping Hu, Yiwei Yang and Qing Tang for their kindly help on this project.

                      During the review process we realized there is a concern from one of the reviewers regarding the origin of EA 2018 used in this research due to its high genome sequence similarity with ATCC 824. Although all the historical records we know about indicated that this is an independently isolated strain [23], at this moment we would like to keep the discussion regarding its origin ongoing until further evidences emerge.

                      Authors’ Affiliations

                      (1)
                      Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
                      (2)
                      Research Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
                      (3)
                      Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai
                      (4)
                      Center for Ecogenomics, Biodesign Institute, Arizona State University

                      References

                      1. Durre P: Biobutanol: an attractive biofuel. J Biotechnol 2007, 2:1525–1534.View Article
                      2. Durre P: New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation. Appl Microbiol Biotechnol 1998, 49:639–648.View Article
                      3. Demain AL, Newcomb M, Wu JHD: Cellulase, Clostridia, and Ethanol. Microbiol Mol Biol Rev 2005, 69:124–154.PubMedView Article
                      4. Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS: Fermentative butanol production by Clostridia. Biotechnol Bioeng 2008, 101:209–228.PubMedView Article
                      5. Nair RV, Green EM, Watson DE, Bennett GN, Papoutsakis ET: Regulation of the sol locus genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 by a putative transcriptional repressor. J Bacteriol 1999, 181:319–330.PubMed
                      6. Gorwa MF, Croux C, Soucaille P: Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J Bacteriol 1996, 178:2668–2675.PubMed
                      7. Boynton ZL, Bennet GN, Rudolph FB: Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824. J Bacteriol 1996, 178:3015–3024.PubMed
                      8. Hueck CJ, Kraus A, Schmiedel D, Hillen W: Cloning, expression and functional analyses of the catabolite control protein CcpA from Bacillus megaterium . Mol Microbiol 1995, 16:855–864.PubMedView Article
                      9. Sillers R, Al-Hinai MA, Papoutsakis ET: Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations. Biotechnol Bioeng 2009, 102:38–49.PubMedView Article
                      10. Papoutsakis ET: Engineering solventogenic clostridia. Curr Opin Biotechnol 2008, 19:420–429.PubMedView Article
                      11. Shao L, Hu S, Yang Y, Gu Y, Chen J, Yang Y, Jiang W, Yang S: Targeted gene disruption by use of a group II intron (targetron) vector in Clostridium acetobutylicum . Cell Res 2007, 17:963–965.PubMedView Article
                      12. Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J: Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 2001, 183:4823–4838.PubMedView Article
                      13. Mao SM, Luo YAM, Zhang TR, Li JS, Bao GAH, Zhu Y, Chen ZG, Zhang YP, Li Y, Ma YH: Proteome Reference Map and Comparative Proteomic Analysis between a Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant with Enhanced Butanol Tolerance and Butanol Yield. J Proteome Res 2010, 9:3046–3061.PubMedView Article
                      14. Shi Z, Blaschek HP: Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 and the hyper-butanol-producing mutant BA101 during the shift from acidogenesis to solventogenesis. Appl Environ Microbiol 2008, 74:7709–7714.PubMedView Article
                      15. Paredes CJ, Senger RS, Spath WS, Borden JR, Sillers R, Papoutsakis ET: A general framework for designing and validating oligomer-based DNA microarrays and its a application to Clostridium acetobutylicum . Appl Environ Microbiol 2007, 73:4631–4638.PubMedView Article
                      16. Sullivan L, Bennett GN: Proteome analysis and comparison of Clostridium acetobutylicum ATCC 824 and Spo0A strain variants. J Ind Microbiol Biotechnol 2006, 33:298–308.PubMedView Article
                      17. Lee J, Yun H, Feist AM, Palsson BO, Lee SY: Genome-scale reconstruction and in silico analysis of the Clostridium acetobutylicum ATCC 824 metabolic network. Appl Microbiol Biotechnol 2008, 80:849–862.PubMedView Article
                      18. Ryan S, Senger ETP: Genome-Scale Model for Clostridium acetobutylicum . Part 2: Development of Specific Proton Flux States and Numerically-Determined Sub-Systems. Biotechnol Bioeng 2008, 101:1053–1071.View Article
                      19. Ryan S, Senger ETP: Genome-scale model for Clostridium acetobutylicum . Part 1: Metabolic network resolution and analysis. Biotechnol Bioeng 2008, 101:1036–1052.View Article
                      20. Durre P, Fischer RJ, Kuhn A, Lorenz K, Schreiber W, Sturzenhofecker B, Ullmann S, Winzer K, Sauer U: Solventogenic enzymes of Clostridium acetobutylicum : catalytic properties, genetic organization, and transcriptional regulation. FEMS Microbiol Rev 1995, 17:251–262.PubMedView Article
                      21. Annous BA, Blaschek HP: Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity. Appl Environ Microbiol 1991, 57:2544–2548.PubMed
                      22. Zhang Y, Chen J, Yang Y, Jiao R: Breeding high-ration butanol strains of Clostridium acetobutylicum and application to industrial production. Industrial Microbiology (in Chinese) 1996, 26:1–6.
                      23. Chiao JS, Sun ZH: History of the Acetone-Butanol-Ethanol Fermentation Industry in China: Development of Continuous Production Technology. J Mol Microbiol Biotechnol 2007, 13:12–14.PubMedView Article
                      24. Fischer RJ, Helms J, Durre P: Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum , a chromosomal locus involved in solventogenesis. J Bacteriol 1993, 175:6959–6969.PubMed
                      25. Fontaine L, Meynial-Salles I, Girbal L, Yang XH, Croux C, Soucaille P: Molecular characterization and transcriptional analysis of adhE2, the gene encoding the NADH-dependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of Clostridium acetobutylicum ATCC 824. J Bacteriol 2002, 184:821–830.PubMedView Article
                      26. Harris LM, Welker NE, Papoutsakis ET: Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824. J Bacteriol 2002, 184:3586–3597.PubMedView Article
                      27. Paredes CJ, Alsaker KV, Papoutsakis ET: A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 2005, 3:969–978.PubMedView Article
                      28. Rao G, Mutharasan R: Altered Electron Flow in Continuous Cultures of Clostridium acetobutylicum Induced by Viologen Dyes. Appl Environ Microbiol 1987, 53:1232–1235.PubMed
                      29. Nakayama Si, Kosaka T, Hirakawa H, Matsuura K, Yoshino S, Furukawa K: Metabolic engineering for solvent productivity by downregulation of the hydrogenase gene cluster hupCBA in Clostridium saccharoperbutylacetonicum strain N1–4. Appl Microbiol Biotechnol 2008, 78:483–493.PubMedView Article
                      30. Haggstrom L: Acetone-butanol fermentation and its variants. Biotechnol Adv 1985, 3:13–28.PubMedView Article
                      31. Grupe H, Gottschalk G: Physiological Events in Clostridium acetobutylicum during the Shift from Acidogenesis to Solventogenesis in Continuous Culture and Presentation of a Model for Shift Induction. Appl Environ Microbiol 1992, 58:3896–3902.PubMed
                      32. Monot F, Engasser JM, Petitdemange H: Influence of pH and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum . Appl Microbiol Biotechnol 1984, 19:422–426.View Article
                      33. Fischer CR, Klein-Marcuschamer D, Stephanopoulos G: Selection and optimization of microbial hosts for biofuels production. Metab Eng 2008, 10:295–304.PubMedView Article
                      34. Kreuzer P, Gartner D, Allmansberger R, Hillen W: Identification and Sequence-Analysis of the Bacillus-Subtilis W23 Xylr Gene and Xyl Operator. J Bacteriol 1989, 171:3840–3845.PubMed
                      35. Rodionov DA, Mironov AA, Gelfand MS: Transcriptional regulation of pentose utilisation systems in the Bacillus/Clostridium group of bacteria. FEMS Microbiol Lett 2001, 205:305–314.PubMedView Article
                      36. Lee SF, Forsberg CW, Gibbins LN: Xylanolytic Activity of Clostridium acetobutylicum . Appl Environ Microbiol 1985, 50:1068–1076.PubMed
                      37. Ali MK, Rudolph FB, Bennett GN: Characterization of thermostable Xyn10A enzyme from mesophilic Clostridium acetobutylicum ATCC 824. J Ind Microbiol Biotechnol 2005, 32:12–18.PubMedView Article
                      38. Kraus A, Hillen W: Analysis of CcpA mutations defective in carbon catabolite repression in Bacillus megaterium . FEMS Microbiol Lett 1997, 153:221–226.PubMedView Article
                      39. Chauvaux S: CcpA and HPr(ser-P): mediators of catabolite repression in Bacillus subtilis . Res Microbiol 1996, 147:518–522.PubMedView Article
                      40. Jones SW, Paredes CJ, Tracy B, Cheng N, Sillers R, Senger RS, Papoutsakis ET: The transcriptional program underlying the physiology of clostridial sporulation. Genome Biol 2008, 9:R114.PubMedView Article
                      41. Ni NT, Li MY, Wang JF, Wang BH: Inhibitors and Antagonists of Bacterial Quorum Sensing. Med Res Rev 2009, 29:65–124.PubMedView Article
                      42. Ohtani K, Yuan Y, Hassan S, Wang R, Wang Y, Shimizu T: Virulence Gene Regulation by the agr System in Clostridium perfringens . J Bacteriol 2009, 191:3919–3927.PubMedView Article
                      43. Winzer K: Quorum sensing in solventogenic clostridia. In BBSRC China partnering award workshop on improving biobutanol production by solventogenic clostridia. Shanghai China; 2009.
                      44. Wiesenborn DP, Rudolph FB, Papoutsakis ET: Thiolase from Clostridium acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents. Appl Environ Microbiol 1988, 54:2717–2722.PubMed
                      45. Zhang Y, Garcia MJ, Lathigra R, Allen B, Moreno C, Vanembden JDA, Young D: Alterations in the Superoxide-Dismutase Gene of an Isoniazid-Resistant Strain of Mycobacterium tuberculosis . Infect Immun 1992, 60:2160–2165.PubMed
                      46. Gordon D, Abajian C, Green P: Consed: A graphical tool for sequence finishing. Genome Res 1998, 8:195–202.PubMed
                      47. Guo FB, Zhang CT: ZCURVE_V: a new self-training system for recognizing protein-coding genes in viral and phage genomes. BMC Bioinformatics 2006, 7:9.PubMedView Article
                      48. Lowe TM, Eddy SR: tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997, 25:955–964.PubMedView Article
                      49. Ghai R, Hain T, Chakraborty T: GenomeViz: visualizing microbial genomes. BMC Bioinformatics 2004, 5:198.PubMedView Article
                      50. Wang SY, Shen XY, Wu CY, Pan F, Shen YY, Sheng HH, Chen XM, Gao HJ: Analysis of whole genomic expression profiles of Helicobacter pylori related chronic atrophic gastritis with IL-1B-31CC/-511TT genotypes. Digest Dis 2009, 10:99–106.View Article
                      51. Eisen MB, Spellman PT, Brown PO, Botstein D: Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 1998, 95:14863–14868.PubMedView Article
                      52. Mermelstein LD, Papoutsakis ET: In vivo methylation in Escherichia coli by the Bacillus subtilis phage Φ3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 1993, 59:1077–1081.PubMed
                      53. Annous BA, Blaschek HP: Regulation and localization of amylolytic enzymes in Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 1990, 56:2559–2561.PubMed
                      54. Tao YZ, He YL, Wu YQ, Liu FH, Li XF, Zong WM, Zhou ZH: Characteristics of a new photosynthetic bacterial strain for hydrogen production and its application in wastewater treatment. Int J Hydrogen Energy 2008, 33:963–973.View Article

                      Copyright

                      © Hu et al; licensee BioMed Central Ltd. 2011

                      This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                      Advertisement