Journal of Industrial Microbiology & Biotechnology

, Volume 40, Issue 12, pp 1443–1448 | Cite as

Isolation and bioinformatic analysis of a novel transposable element, ISCbe4, from the hyperthermophilic bacterium, Caldicellulosiruptor bescii

  • Minseok Cha
  • Hao Wang
  • Daehwan Chung
  • Jeffrey L. Bennetzen
  • Janet Westpheling
Genetics and Molecular Biology of Industrial Organisms


Caldicellulosiruptor bescii is an anaerobic thermophilic bacterium of special interest for use in the consolidated bioprocessing of plant biomass to biofuels. In the course of experiments to engineer pyruvate metabolism in C. bescii, we isolated a mutant of C. bescii that contained an insertion in the l-lactate dehydrogenase gene (ldh). PCR amplification and sequencing of the ldh gene from this mutant revealed a 1,609-bp insertion that contained a single open reading frame of 479 amino acids (1,440 bp) annotated as a hypothetical protein with unknown function. The ORF is flanked by an 8-base direct repeat sequence. Bioinformatic analysis indicated that this ORF is part of a novel transposable element, ISCbe4, which is only intact in the genus Caldicellulosiruptor, but has ancient relatives that are present in degraded (and previously unrecognized) forms across many bacterial and archaeal clades.


Transposable elements ISCbe4 Caldicellulosiruptor bescii Lactate dehydrogenase Bioinformatics 



We thank Jennifer Copeland for outstanding technical assistance, and Robert Kelly and Sara Blumer-Schuette for providing the wild-type Caldicellulosiruptor species. The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This study was supported in part by resources and technical expertise from the Georgia Advanced Computing Resource Center, a partnership between the University of Georgia’s Office of the Vice President for Research and Office of the Vice President for Information Technology.

Supplementary material

10295_2013_1345_MOESM1_ESM.docx (49 kb)
Supplementary material 1 (DOCX 48 kb)


  1. 1.
    Blount ZD, Grogan DW (2005) New insertion sequences of Sulfolobus: functional properties and implications for genome evolution in hyperthermophilic archaea. Mol Microbiol 55(1):312–325. doi: 10.1111/j.1365-2958.2004.04391.x PubMedCrossRefGoogle Scholar
  2. 2.
    Blumer-Schuette SE, Giannone RJ, Zurawski JV, Ozdemir I, Ma Q, Yin Y, Xu Y, Kataeva I, Poole FL 2nd, Adams MW, Hamilton-Brehm SD, Elkins JG, Larimer FW, Land ML, Hauser LJ, Cottingham RW, Hettich RL, Kelly RM (2012) Caldicellulosiruptor core and pangenomes reveal determinants for noncellulosomal thermophilic deconstruction of plant biomass. J Bacteriol 194(15):4015–4028. doi: 10.1128/JB.00266-12 PubMedCrossRefGoogle Scholar
  3. 3.
    Blumer-Schuette SE, Lewis DL, Kelly RM (2010) Phylogenetic, microbiological, and glycoside hydrolase diversities within the extremely thermophilic, plant biomass-degrading genus Caldicellulosiruptor. Appl Environ Microbiol 76(24):8084–8092. doi: 10.1128/AEM.01400-10 PubMedCrossRefGoogle Scholar
  4. 4.
    Brynestad S, Synstad B, Granum PE (1997) The Clostridium perfringens enterotoxin gene is on a transposable element in type A human food poisoning strains. Microbiology 143(Pt 7):2109–2115PubMedCrossRefGoogle Scholar
  5. 5.
    Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T (2009) trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25(15):1972–1973. doi: 10.1093/bioinformatics/btp348 PubMedCrossRefGoogle Scholar
  6. 6.
    Chung D, Farkas J, Westpheling J (2013) Detection of a novel active transposable element in Caldicellulosiruptor hydrothermalis and a new search for elements in this genus. J Ind Microbiol Biotechnol 40(5):517–521. doi: 10.1007/s10295-013-1244-z PubMedCrossRefGoogle Scholar
  7. 7.
    Chung D, Farkas J, Westpheling J (2013) Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement. Biotechnol Biofuels 6(1):82. doi: 10.1186/1754-6834-6-82 PubMedCrossRefGoogle Scholar
  8. 8.
    Chung D, Cha M, Farkas J, Westpheling J (2013) Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus. PLoS ONE 8(5):e62881PubMedCrossRefGoogle Scholar
  9. 9.
    Collins CM, Gutman DM (1992) Insertional inactivation of an Escherichia coli urease gene by IS3411. J Bacteriol 174(3):883–888PubMedGoogle Scholar
  10. 10.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797. doi: 10.1093/nar/gkh340 PubMedCrossRefGoogle Scholar
  11. 11.
    Farkas J, Chung D, Cha M, Copeland J, Grayeski P, Westpheling J (2013) Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii. J Ind Microbiol Biotechnol 40(1):41–49. doi: 10.1007/s10295-012-1202-1 PubMedCrossRefGoogle Scholar
  12. 12.
    Fetherston JD, Perry RD (1994) The pigmentation locus of Yersinia pestis KIM6 + is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and HMWP2. Mol Microbiol 13(4):697–708PubMedCrossRefGoogle Scholar
  13. 13.
    Kataeva IA, Yang SJ, Dam P, Poole FL 2nd, Yin Y, Zhou F, Chou WC, Xu Y, Goodwin L, Sims DR, Detter JC, Hauser LJ, Westpheling J, Adams MW (2009) Genome sequence of the anaerobic, thermophilic, and cellulolytic bacterium “Anaerocellum thermophilum” DSM 6725. J Bacteriol 191(11):3760–3761. doi: 10.1128/JB.00256-09 PubMedCrossRefGoogle Scholar
  14. 14.
    Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16(2):111–120PubMedCrossRefGoogle Scholar
  15. 15.
    Kivi M, Liu X, Raychaudhuri S, Altman RB, Small PM (2002) Determining the genomic locations of repetitive DNA sequences with a whole-genome microarray: IS6110 in Mycobacterium tuberculosis. J Clin Microbiol 40(6):2192–2198PubMedCrossRefGoogle Scholar
  16. 16.
    Liyanage H, Holcroft P, Evans VJ, Keis S, Wilkinson SR, Kashket ER, Young M (2000) A new insertion sequence, ISCb1, from Clostridium beijerinckii NCIMB 8052. J Mol Microbiol Biotechnol 2(1):107–113PubMedGoogle Scholar
  17. 17.
    Maamar H, de Philip P, Belaich JP, Tardif C (2003) ISCce1 and ISCce2, two novel insertion sequences in Clostridium cellulolyticum. J Bacteriol 185(3):714–725PubMedCrossRefGoogle Scholar
  18. 18.
    Mahillon J, Chandler M (1998) Insertion sequences. Microbiol Mol Biol Rev 62(3):725–774PubMedGoogle Scholar
  19. 19.
    Needleman SB, Wunsch CD (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48(3):443–453PubMedCrossRefGoogle Scholar
  20. 20.
    Remm M, Storm CE, Sonnhammer EL (2001) Automatic clustering of orthologs and in-paralogs from pairwise species comparisons. J Mol Biol 314(5):1041–1052. doi: 10.1006/jmbi.2000.5197 PubMedCrossRefGoogle Scholar
  21. 21.
    Rice P, Longden I, Bleasby A (2000) EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16(6):276–277PubMedCrossRefGoogle Scholar
  22. 22.
    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425PubMedGoogle Scholar
  23. 23.
    Siguier P, Filee J, Chandler M (2006) Insertion sequences in prokaryotic genomes. Curr Opin Microbiol 9(5):526–531. doi: 10.1016/j.mib.2006.08.005 PubMedCrossRefGoogle Scholar
  24. 24.
    Stanley J, Baquar N, Threlfall EJ (1993) Genotypes and phylogenetic relationships of Salmonella typhimurium are defined by molecular fingerprinting of IS200 and 16S rrn loci. J Gen Microbiol 139(Pt 6):1133–1140PubMedGoogle Scholar
  25. 25.
    Stroeher UH, Jedani KE, Dredge BK, Morona R, Brown MH, Karageorgos LE, Albert MJ, Manning PA (1995) Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139. Proc Natl Acad Sci USA 92(22):10374–10378PubMedCrossRefGoogle Scholar
  26. 26.
    Svetlichnyi VA, Svetlichnaya TP, Chernykh NA, Zavarzin GA (1990) Anaerocellum-Thermophilum Gen-Nov Sp-Nov—an Extremely Thermophilic Cellulolytic Eubacterium Isolated from Hot-Springs in the Valley of Geysers. Microbiol 59(5):598–604Google Scholar
  27. 27.
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739. doi: 10.1093/molbev/msr121 PubMedCrossRefGoogle Scholar
  28. 28.
    Toleman MA, Bennett PM, Walsh TR (2006) ISCR elements: novel gene-capturing systems of the 21st century? Microbiol Mol Biol Rev 70(2):296–316. doi: 10.1128/MMBR.00048-05 PubMedCrossRefGoogle Scholar
  29. 29.
    Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O, Paux E, SanMiguel P, Schulman AH (2007) A unified classification system for eukaryotic transposable elements. Nat Rev Genet 8(12):973–982. doi: 10.1038/nrg2165 PubMedCrossRefGoogle Scholar
  30. 30.
    Xu Z, Hao B (2009) CV Tree update: a newly designed phylogenetic study platform using composition vectors and whole genomes. Nucleic Acids Res 37 (Web Server issue):W174–178. doi: 10.1093/nar/gkp278
  31. 31.
    Zverlov VV, Klupp M, Krauss J, Schwarz WH (2008) Mutations in the scaffoldin gene, cipA, of Clostridium thermocellum with impaired cellulosome formation and cellulose hydrolysis: insertions of a new transposable element, IS1447, and implications for cellulase synergism on crystalline cellulose. J Bacteriol 190(12):4321–4327. doi: 10.1128/JB.00097-08 PubMedCrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2013

Authors and Affiliations

  • Minseok Cha
    • 1
    • 2
  • Hao Wang
    • 1
    • 2
  • Daehwan Chung
    • 1
    • 2
  • Jeffrey L. Bennetzen
    • 1
    • 2
  • Janet Westpheling
    • 1
    • 2
  1. 1.Department of GeneticsUniversity of GeorgiaAthensUSA
  2. 2.The BioEnergy Science CenterOak Ridge National LaboratoryOak RidgeUSA

Personalised recommendations