Advertisement

Applied Microbiology and Biotechnology

, Volume 97, Issue 17, pp 7805–7819 | Cite as

Generation of biologically contained, readily transformable, and genetically manageable mutants of the biotechnologically important Bacillus pumilus

  • Stephanie Wemhoff
  • Friedhelm MeinhardtEmail author
Applied genetics and molecular biotechnology

Abstract

Bacillus pumilus mutants were generated by targeted deletion of a set of genes eventually facilitating genetic handling and assuring biological containment. The well-defined and stable mutants do not form functional endospores due to the deletion of yqfD, an essential sporulation gene; they are affected in DNA repair, as ΔuvrBA rendered them UV hypersensitive and, thus, biologically contained; they are deficient for the uracil phosphoribosyl-transferase (Δupp), allowing for 5-fluorouracil-based counterselection facilitating rapid allelic exchanges; and they are readily transformable due to the deletion of the restrictase encoding locus (ΔhsdR) of a type I restriction modification system. Vegetative growth as well as extracellular enzyme production and secretion are in no case affected. The combination of such gene deletions allows for development of B. pumilus strains suited for industrial use and further improvements.

Keywords

Bacillus pumilus Extracellular enzymes Safety strain Strain improvement Transformation 

Notes

Acknowledgments

We acknowledge the skilful technical assistance of Julia Tietz. This work was supported by the Federal Ministry of Education and Research (BMBF, Bonn-Bad Godesberg, Germany), grant no. 0315594C.

Supplementary material

253_2013_4935_MOESM1_ESM.pdf (114 kb)
MOESM 1 (PDF 114 kb)

References

  1. Andersen PS, Smith JM, Mygind B (1992) Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12. Eur J Biochem 204(1):51–56PubMedCrossRefGoogle Scholar
  2. Aquino de Muro M, Priest FG (2000) Construction of chromosomal integrants of Bacillus sphaericus 2362 by conjugation with Escherichia coli. Res Microbiol 151(7):547–555PubMedCrossRefGoogle Scholar
  3. Berge M, Mortier-Barriere I, Martin B, Claverys JP (2003) Transformation of Streptococcus pneumoniae relies on DprA- and RecA-dependent protection of incoming DNA single strands. Mol Microbiol 50(2):527–536PubMedCrossRefGoogle Scholar
  4. Bickle TA, Krüger DH (1993) Biology of DNA restriction. Microbiol Rev 57(2):434–450PubMedGoogle Scholar
  5. Biedendieck R, Borgmeier C, Bunk B, Stammen S, Scherling C, Meinhardt F, Wittmann C, Jahn D (2011) Systems biology of recombinant protein production using Bacillus megaterium. Methods Enzymol 500:165–195PubMedCrossRefGoogle Scholar
  6. Borgmeier C, Bongaerts J, Meinhardt F (2012) Genetic analysis of the Bacillus licheniformis degSU operon and the impact of regulatory mutations on protease production. J Biotechnol 159(1–2):12–20PubMedCrossRefGoogle Scholar
  7. Brown B, Carlton B (1980) Plasmid-mediated transformation in Bacillus megaterium. J Bacteriol 142(2):508–512PubMedGoogle Scholar
  8. Chandrasegaran S, Smith HO (1988) Amino acid sequence homologies among twenty-five restriction endonucleases and methylases. In: Sarma MH, Sarma RH (eds) Structure and expression from proteins to ribosomes, vol 1. Adenine Press, New York, pp 149–156Google Scholar
  9. Cole RS (1973) Repair of DNA containing interstrand crosslinks in Escherichia coli: sequential excision and recombination. Proc Natl Acad Sci U S A 70(4):1064–1068PubMedCrossRefGoogle Scholar
  10. EFSA (2007) Introduction of a qualified presumption of safety (QPS) approach for assessment of selected microorganisms referred to EFSA. EFSA J 587:1–16Google Scholar
  11. Eichenberger P, Jensen ST, Conlon EM, van Ooij C, Silvaggi J, Gonzalez-Pastor JE, Fujita M, Ben-Yehuda S, Stragier P, Liu JS, Losick R (2003) The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis. J Mol Biol 327(5):945–972PubMedCrossRefGoogle Scholar
  12. Fabret C, Ehrlich SD, Noirot P (2002) A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 46(1):25–36PubMedCrossRefGoogle Scholar
  13. Feucht A, Evans L, Errington J (2003) Identification of sporulation genes by genome-wide analysis of the sigmaE regulon of Bacillus subtilis. Microbiology 149(Pt 10):3023–3034PubMedCrossRefGoogle Scholar
  14. Fritze D (2002) Bacillus identification — traditional approaches. In: Berkeley R, Heyndrickx M, Logan N, De Vos P (eds) Applications and systematics of Bacillus and relatives. Blackwell Science Ltd, Oxford, pp 100–122CrossRefGoogle Scholar
  15. From C, Hormazabal V, Granum PE (2007) Food poisoning associated with pumilacidin-producing Bacillus pumilus in rice. Int J Food Microbiol 115(3):319–324PubMedCrossRefGoogle Scholar
  16. Fu LL, Xu ZR, Li W, Shuai JB, Lu P, Hu CX (2007) Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnol Adv 25:1–12Google Scholar
  17. Gioia J, Yerrapragada S, Qin X, Jiang H, Igboeli OC, Muzny D, Dugan-Rocha S, Ding Y, Hawes A, Liu W, Perez L, Kovar C, Dinh H, Lee S, Nazareth L, Blyth P, Holder M, Buhay C, Tirumalai MR, Liu Y, Dasgupta I, Bokhetache L, Fujita M, Karouia F, Eswara Moorthy P, Siefert J, Uzman A, Buzumbo P, Verma A, Zwiya H, McWilliams BD, Olowu A, Clinkenbeard KD, Newcombe D, Golebiewski L, Petrosino JF, Nicholson WL, Fox GE, Venkateswaran K, Highlander SK, Weinstock GM (2007) Paradoxical DNA repair and peroxide resistance gene conservation in Bacillus pumilus SAFR-032. PLoS One 2(9):e928PubMedCrossRefGoogle Scholar
  18. Harwood CR, Cutting SM (1990) Molecular biological methods for Bacillus. John Wiley & Sons, ChichesterGoogle Scholar
  19. Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2(4):924–932PubMedCrossRefGoogle Scholar
  20. Hitchins AD, Kahn AJ, Slepecky RA (1968) Interference contrast and phase contrast microscopy of sporulation and germination of Bacillus megaterium. J Bacteriol 96(5):1811–1817PubMedGoogle Scholar
  21. Hoffmann K, Wollherr A, Larsen M, Rachinger M, Liesegang H, Ehrenreich A, Meinhardt F (2010) Facilitation of direct conditional knockout of essential genes in Bacillus licheniformis DSM13 by comparative genetic analysis and manipulation of genetic competence. Appl Environ Microbiol 76(15):5046–5057PubMedCrossRefGoogle Scholar
  22. Hunger W, Claus D (1981) Taxonomic studies on Bacillus megaterium and on agarolytic Bacillus strains. In: Berkeley RCW, Goodfellow M (eds) The aerobic endospore-forming bacteria: classification and identification. Academic Press, London, pp 217–239Google Scholar
  23. Kidane D, Carrasco B, Manfredi C, Rothmaier K, Ayora S, Tadesse S, Alonso JC, Graumann PL (2009) Evidence for different pathways during horizontal gene transfer in competent Bacillus subtilis cells. PLoS Genet 5(9):e1000630PubMedCrossRefGoogle Scholar
  24. Kramer JM, Gilbert RJ (1989) Bacillus cereus and other Bacillus species. In: Doyle MP (ed) Food-borne bacterial pathogens. Marcel Dekker, New York, pp 21–70Google Scholar
  25. Lee YJ, Park SJ, Ciccone SL, Kim CR, Lee SH (2006) An in vivo analysis of MMC-induced DNA damage and its repair. Carcinogenesis 27(3):446–453PubMedCrossRefGoogle Scholar
  26. Loenen WA, Daniel AS, Braymer HD, Murray NE (1987) Organization and sequence of the hsd genes of Escherichia coli K-12. J Mol Biol 198(2):159–170PubMedCrossRefGoogle Scholar
  27. Marceau AH (2012) Functions of single-strand DNA-binding proteins in DNA replication, recombination, and repair. Methods Mol Biol 922:1–21PubMedGoogle Scholar
  28. Meinhardt F, Stahl U, Ebeling W (1989) Highly efficient expression of homologous and heterologous genes in Bacillus megaterium. Appl Microbiol Biotechnol 30:343–350CrossRefGoogle Scholar
  29. Meinhardt F, Busskamp M, Wittchen KD (1994) Cloning and sequencing of the leuC and nprM genes and a putative spoIV gene from Bacillus megaterium DSM319. Appl Microbiol Biotechnol 41(3):344–351PubMedCrossRefGoogle Scholar
  30. Murray NE (2000) Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle). Microbiol Mol Biol Rev 64(2):412–434PubMedCrossRefGoogle Scholar
  31. Naclerio G, Baccigalupi L, Zilhao R, De Felice M, Ricca E (1996) Bacillus subtilis spore coat assembly requires cotH gene expression. J Bacteriol 178(15):4375–4380PubMedGoogle Scholar
  32. Nahrstedt H, Meinhardt F (2004) Structural and functional characterization of the Bacillus megaterium uvrBA locus and generation of UV-sensitive mutants. Appl Microbiol Biotechnol 65(2):193–199PubMedCrossRefGoogle Scholar
  33. Nahrstedt H, Wittchen K, Rachman MA, Meinhardt F (2004) Identification and functional characterization of a type I signal peptidase gene of Bacillus megaterium DSM319. Appl Microbiol Biotechnol 64(2):243–249PubMedCrossRefGoogle Scholar
  34. Nahrstedt H, Waldeck J, Gröne M, Eichstädt R, Feesche J, Meinhardt F (2005) Strain development in Bacillus licheniformis: construction of biologically contained mutants deficient in sporulation and DNA repair. J Biotechnol 119(3):245–254PubMedCrossRefGoogle Scholar
  35. Neuhard J (1983) Utilization of preformed pyrimidine bases and nucleosides. In: Munch-Petersen A (ed) Metabolism of nucleotides, nucleosides and nucleobases in microorganisms. Acadmic Press, New York, pp 95–148Google Scholar
  36. Orren DK, Sancar A (1989) The (A)BC excinuclease of Escherichia coli has only the UvrB and UvrC subunits in the incision complex. Proc Natl Acad Sci U S A 86(14):5237–5241PubMedCrossRefGoogle Scholar
  37. Pan J, Huang Q, Zhang Y (2004) Gene cloning and expression of an alkaline serine protease with dehairing function from Bacillus pumilus. Curr Microbiol 49(3):165–169PubMedCrossRefGoogle Scholar
  38. Papamichael D (1999) The use of thymidylate synthase inhibitors in the treatment of advanced colorectal cancer: current status. Oncologist 4:478–487PubMedGoogle Scholar
  39. Pohl S, Harwood CR (2010) Heterologous protein secretion by Bacillus species from the cradle to the grave. Adv Appl Microbiol 73:1–25PubMedCrossRefGoogle Scholar
  40. Rey MW, Ramaiya P, Nelson BA, Brody-Karpin SD, Zaretsky EJ, Tang M, Lopez de Leon A, Xiang H, Gusti V, Clausen IG, Olsen PB, Rasmussen MD, Andersen JT, Jorgensen PL, Larsen TS, Sorokin A, Bolotin A, Lapidus A, Galleron N, Ehrlich SD, Berka RM (2004) Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biol 5(10):R77PubMedCrossRefGoogle Scholar
  41. Richhardt J, Larsen M, Meinhardt F (2010) An improved transconjugation protocol for Bacillus megaterium facilitating a direct genetic knockout. Appl Microbiol Biotechnol 86(6):1959–1965PubMedCrossRefGoogle Scholar
  42. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, vol 3. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  43. Sancar A (1994) Mechanisms of DNA excision repair. Science 266(5193):1954–1956PubMedCrossRefGoogle Scholar
  44. Santi DV, McHenry CS (1973) 5-Fluoro-2′-Deoxyuridylate: covalent complex with thymidylate synthetase. Proc Natl Acad Sci U S A 69:1855–1857CrossRefGoogle Scholar
  45. Schaeffer P, Millet J, Aubert JP (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A 54(3):704–711PubMedCrossRefGoogle Scholar
  46. Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50(1):1–17PubMedCrossRefGoogle Scholar
  47. Schumann W (2007) Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol 62:137–189PubMedCrossRefGoogle Scholar
  48. Simon R, Priefer U, Pühler A (1983) A broad range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1:784–791CrossRefGoogle Scholar
  49. Sladek FM, Munn MM, Rupp WD, Howard-Flanders P (1989) In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5′-exonuclease of DNA polymerase I. J Biol Chem 264(12):6755–6765PubMedGoogle Scholar
  50. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517PubMedCrossRefGoogle Scholar
  51. Stahl U, Esser K (1983) Plasmid heterogeneity in various strains of Bacillus megaterium. Eur J Appl Biotechnol 17:248–251CrossRefGoogle Scholar
  52. Su F, Yu B, Sun J, Ou HY, Zhao B, Wang L, Qin J, Tang H, Tao F, Jarek M, Scharfe M, Ma C, Ma Y, Xu P (2011) Genome sequence of the thermophilic strain Bacillus coagulans 26, an efficient producer of high-optical-purity l-lactic acid. J Bacteriol 193(17):4563–4564PubMedCrossRefGoogle Scholar
  53. Suominen I, Andersson MA, Andersson MC, Hallaksela AM, Kampfer P, Rainey FA, Salkinoja-Salonen M (2001) Toxic Bacillus pumilus from indoor air, recycled paper pulp, Norway spruce, food poisoning outbreaks and clinical samples. Syst Appl Microbiol 24(2):267–276PubMedCrossRefGoogle Scholar
  54. Takamatsu H, Kodama T, Nakayama T, Watabe K (1999) Characterization of the yrbA gene of Bacillus subtilis, involved in resistance and germination of spores. J Bacteriol 181(16):4986–4994PubMedGoogle Scholar
  55. Truglio JJ, Croteau DL, Van Houten B, Kisker C (2006) Prokaryotic nucleotide excision repair: the UvrABC system. Chem Rev 106(2):233–252PubMedCrossRefGoogle Scholar
  56. Van Houten B (1990) Nucleotide excision repair in Escherichia coli. Microbiol Rev 54(1):18–51PubMedGoogle Scholar
  57. Van Houten B, Gamper H, Hearst JE, Sancar A (1986a) Construction of DNA substrates modified with psoralen at a unique site and study of the action mechanism of ABC excinuclease on these uniformly modified substrates. J Biol Chem 261:14135–14141PubMedGoogle Scholar
  58. Van Houten B, Gamper H, Holbrook SR, Hearst JE, Sancar A (1986b) Action mechanism of ABC excision nuclease on a DNA substrate containing a psoralen crosslink at a defined position. Proc Natl Acad Sci U S A 83:8077–8081PubMedCrossRefGoogle Scholar
  59. Veith B, Herzberg C, Steckel S, Feesche J, Maurer KH, Ehrenreich P, Bäumer S, Henne A, Liesegang H, Merkl R, Ehrenreich A, Gottschalk G (2004) The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. J Mol Microbiol Biotechnol 7(4):204–211PubMedCrossRefGoogle Scholar
  60. Vorob'eva IP, Khmel IA, Alföldi L (1980) Polyethylene glycol induction of Bacillus megaterium protoplast transformation by plasmid DNA. Dokl Akad Nauk SSSR 251(4):977–980PubMedGoogle Scholar
  61. Waldeck J, Daum G, Bisping B, Meinhardt F (2006) Isolation and molecular characterization of chitinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin. Appl Environ Microbiol 72(12):7879–7885PubMedCrossRefGoogle Scholar
  62. Waldeck J, Meyer-Rammes H, Nahrstedt H, Eichstädt R, Wieland S, Meinhardt F (2007) Targeted deletion of the uvrBA operon and biological containment in the industrially important Bacillus licheniformis. Appl Microbiol Biotechnol 73(6):1340–1347PubMedCrossRefGoogle Scholar
  63. Wan MY, Wang HY, Zhang YZ, Feng H (2009) Substrate specificity and thermostability of the dehairing alkaline protease from Bacillus pumilus. Appl Biochem Biotechnol 159(2):394–403PubMedCrossRefGoogle Scholar
  64. Waschkau B, Waldeck J, Wieland S, Eichstädt R, Meinhardt F (2008) Generation of readily transformable Bacillus licheniformis mutants. Appl Microbiol Biotechnol 78(1):181–188PubMedCrossRefGoogle Scholar
  65. Westers H, Darmon E, Zanen G, Veening JW, Kuipers OP, Bron S, Quax WJ, van Dijl JM (2004) The Bacillus secretion stress response is an indicator for alpha-amylase production levels. Lett Appl Microbiol 39(1):65–73PubMedCrossRefGoogle Scholar
  66. Wilson GG, Murray NE (1991) Restriction and modification systems. Annu Rev Genet 25:585–627PubMedCrossRefGoogle Scholar
  67. Wittchen KD, Strey J, Bültmann A, Reichenberg S, Meinhardt F (1998) Molecular characterization of the operon comprising the spoIV gene of Bacillus megaterium DSM319 and generation of a deletion mutant. J Gen Appl Microbiol 44(5):317–326PubMedCrossRefGoogle Scholar
  68. Woodcock DM, Crowther PJ, Doherty J, Jefferson S, DeCruz E, Noyer-Weidner M, Smith SS, Michael MZ, Graham MW (1989) Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17(9):3469–3478PubMedCrossRefGoogle Scholar
  69. Zheng LB, Donovan WP, Fitz-James PC, Losick R (1988) Gene encoding a morphogenic protein required in the assembly of the outer coat of the Bacillus subtilis endospore. Genes Dev 2(8):1047–1054PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Institut für Molekulare Mikrobiologie und BiotechnologieWestfälische Wilhelms-Universität MünsterMünsterGermany

Personalised recommendations