1887

Abstract

To optimize as a production strain for proteins and low molecular substances by genome engineering, we developed a markerless gene deletion system. We took advantage of a general property of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular the mannose PTS. Mannose is phosphorylated during uptake by its specific transporter (ManP) to mannose 6-phosphate, which is further converted to fructose 6-phosphate by the mannose-6-phosphate isomerase (ManA). When ManA is missing, accumulation of the phosphorylated mannose inhibits cell growth. This system was constructed by deletion of and in Δ6, a 168 derivative strain with six large deletions of prophages and antibiotic biosynthesis genes. The gene was inserted into an plasmid together with a spectinomycin resistance gene for selection in . To delete a specific region, its up- and downstream flanking sites (each of approximately 700 bp) were inserted into the vector. After transformation, integration of the plasmid into the chromosome of by single cross-over was selected by spectinomycin. In the second step, excision of the plasmid was selected by growth on mannose. Finally, excision and concomitant deletion of the target region were verified by colony PCR. In this way, all nine prophages, seven antibiotic biosynthesis gene clusters and two sigma factors for sporulation were deleted and the genome was reduced from 4215 to 3640 kb. Despite these extensive deletions, growth rate and cell morphology remained similar to the 168 parental strain.

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2015-10-01
2019-08-21
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References

  1. Allenby N.E., Watts C.A., Homuth G., Prágai Z., Wipat A., Ward A.C., Harwood C.R.. ( 2006;). Phosphate starvation induces the sporulation killing factor of Bacillus subtilis. J Bacteriol 188: 5299–5303 [CrossRef] [PubMed].
    [Google Scholar]
  2. Altenbuchner J., Viell P., Pelletier I.. ( 1992;). Positive selection vectors based on palindromic DNA sequences. Methods Enzymol 216: 457–466 [CrossRef] [PubMed].
    [Google Scholar]
  3. Arnaud M., Chastanet A., Débarbouillé M.. ( 2004;). New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, Gram-positive bacteria. Appl Environ Microbiol 70: 6887–6891 [CrossRef] [PubMed].
    [Google Scholar]
  4. Auchtung J.M., Lee C.A., Monson R.E., Lehman A.P., Grossman A.D.. ( 2005;). Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response. Proc Natl Acad Sci U S A 102: 12554–12559 [CrossRef] [PubMed].
    [Google Scholar]
  5. Bernard P., Gabant P., Bahassi E.M., Couturier M.. ( 1994;). Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71–74 [CrossRef] [PubMed].
    [Google Scholar]
  6. Bertani G.. ( 1951;). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62: 293–300 .
    [Google Scholar]
  7. Bloor A.E., Cranenburgh R.M.. ( 2006;). An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol 72: 2520–2525 [CrossRef] [PubMed].
    [Google Scholar]
  8. Buescher J.M., Liebermeister W., Jules M., Uhr M., Muntel J., Botella E., Hessling B., Kleijn R.J., Le Chat L., other authors. ( 2012;). Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism. Science 335: 1099–1103 [CrossRef] [PubMed].
    [Google Scholar]
  9. Chung C.T., Niemela S.L., Miller R.H.. ( 1989;). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A 86: 2172–2175 [CrossRef] [PubMed].
    [Google Scholar]
  10. Eichenberger P., Jensen S.T., Conlon E.M., van Ooij C., Silvaggi J., González-Pastor J.E., Fujita M., Ben-Yehuda S., Stragier P., other authors. ( 2003;). The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis. J Mol Biol 327: 945–972 [CrossRef] [PubMed].
    [Google Scholar]
  11. Ellermeier C.D., Hobbs E.C., Gonzalez-Pastor J.E., Losick R.. ( 2006;). A three-protein signaling pathway governing immunity to a bacterial cannibalism toxin. Cell 124: 549–559 [CrossRef] [PubMed].
    [Google Scholar]
  12. Engelberg-Kulka H., Amitai S., Kolodkin-Gal I., Hazan R.. ( 2006;). Bacterial programmed cell death and multicellular behavior in bacteria. PLoS Genet 2: e135 [CrossRef] [PubMed].
    [Google Scholar]
  13. Fabret C., Ehrlich S.D., Noirot P.. ( 2002;). A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 46: 25–36 [CrossRef] [PubMed].
    [Google Scholar]
  14. Fujita M., Losick R.. ( 2005;). Evidence that entry into sporulation in Bacillus subtilis is governed by a gradual increase in the level and activity of the master regulator Spo0A. Genes Dev 19: 2236–2244 [CrossRef] [PubMed].
    [Google Scholar]
  15. Goelzer A., Bekkal Brikci F., Martin-Verstraete I., Noirot P., Bessières P., Aymerich S., Fromion V.. ( 2008;). Reconstruction and analysis of the genetic and metabolic regulatory networks of the central metabolism of Bacillus subtilis. BMC Syst Biol 2: 20 [CrossRef] [PubMed].
    [Google Scholar]
  16. Goh Y.J., Azcárate-Peril M.A., O'Flaherty S., Durmaz E., Valence F., Jardin J., Lortal S., Klaenhammer T.R.. ( 2009;). Development and application of a upp-based counterselective gene replacement system for the study of the S-layer protein SlpX of Lactobacillus acidophilus NCFM. Appl Environ Microbiol 75: 3093–3105 [CrossRef] [PubMed].
    [Google Scholar]
  17. González-Pastor J.E.. ( 2011;). Cannibalism: a social behavior in sporulating Bacillus subtilis. FEMS Microbiol Rev 35: 415–424 [CrossRef] [PubMed].
    [Google Scholar]
  18. Graf N., Altenbuchner J.. ( 2011;). Development of a method for markerless gene deletion in Pseudomonas putida. Appl Environ Microbiol 77: 5549–5552 [CrossRef] [PubMed].
    [Google Scholar]
  19. Harwood C.R., Cutting S.M.. ( 1990;). Molecular Biological Methods for Bacillus Chichester: John Wiley & Sons Ltd;.
    [Google Scholar]
  20. Ho S.N., Hunt H.D., Horton R.M., Pullen J.K., Pease L.R.. ( 1989;). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77: 51–59 [CrossRef] [PubMed].
    [Google Scholar]
  21. Hoang T.T., Karkhoff-Schweizer R.R., Kutchma A.J., Schweizer H.P.. ( 1998;). A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212: 77–86 [CrossRef] [PubMed].
    [Google Scholar]
  22. Inaoka T., Takahashi K., Ohnishi-Kameyama M., Yoshida M., Ochi K.. ( 2003;). Guanine nucleotides guanosine 5′-diphosphate 3′-diphosphate and GTP co-operatively regulate the production of an antibiotic bacilysin in Bacillus subtilis. J Biol Chem 278: 2169–2176 [CrossRef] [PubMed].
    [Google Scholar]
  23. Inaoka T., Takahashi K., Yada H., Yoshida M., Ochi K.. ( 2004;). RNA polymerase mutation activates the production of a dormant antibiotic 3,3′-neotrehalosadiamine via an autoinduction mechanism in Bacillus subtilis. J Biol Chem 279: 3885–3892 [CrossRef] [PubMed].
    [Google Scholar]
  24. Jeske M., Altenbuchner J.. ( 2010;). The Escherichia coli rhamnose promoter rhaP(BAD) is in Pseudomonas putida KT2440 independent of Crp-cAMP activation. Appl Microbiol Biotechnol 85: 1923–1933 [CrossRef] [PubMed].
    [Google Scholar]
  25. Keller K.L., Bender K.S., Wall J.D.. ( 2009;). Development of a markerless genetic exchange system for Desulfovibrio vulgaris Hildenborough and its use in generating a strain with increased transformation efficiency. Appl Environ Microbiol 75: 7682–7691 [CrossRef] [PubMed].
    [Google Scholar]
  26. Kobayashi K., Ogura M., Yamaguchi H., Yoshida K., Ogasawara N., Tanaka T., Fujita Y., Comprehensive D.N.A.. ( 2001;). microarray analysis of Bacillus subtilis two-component regulatory systems. J Bacteriol 183: 7365–7370 [CrossRef] [PubMed].
    [Google Scholar]
  27. Krishnappa L., Dreisbach A., Otto A., Goosens V.J., Cranenburgh R.M., Harwood C.R., Becher D., van Dijl J.M.. ( 2013;). Extracytoplasmic proteases determining the cleavage and release of secreted proteins, lipoproteins, and membrane proteins in Bacillus subtilis. J Proteome Res 12: 4101–4110 [CrossRef] [PubMed].
    [Google Scholar]
  28. Kristich C.J., Manias D.A., Dunny G.M.. ( 2005;). Development of a method for markerless genetic exchange in Enterococcus faecalis and its use in construction of a srtA mutant. Appl Environ Microbiol 71: 5837–5849 [CrossRef] [PubMed].
    [Google Scholar]
  29. Kunst F., Ogasawara N., Moszer I., Albertini A.M., Alloni G., Azevedo V., Bertero M.G., Bessières P., Bolotin A., other authors. ( 1997;). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390: 249–256 [CrossRef] [PubMed].
    [Google Scholar]
  30. Leenhouts K., Buist G., Bolhuis A., ten Berge A., Kiel J., Mierau I., Dabrowska M., Venema G., Kok J.. ( 1996;). A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet 253: 217–224 [CrossRef] [PubMed].
    [Google Scholar]
  31. Marahiel M.A., Nakano M.M., Zuber P.. ( 1993;). Regulation of peptide antibiotic production in Bacillus. Mol Microbiol 7: 631–636 [CrossRef] [PubMed].
    [Google Scholar]
  32. Martínez-García E., de Lorenzo V.. ( 2011;). Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol 13: 2702–2716 [CrossRef] [PubMed].
    [Google Scholar]
  33. Marx C.J., Lidstrom M.E.. ( 2002;). Broad-host-range cre-lox system for antibiotic marker recycling in Gram-negative bacteria. Biotechniques 33: 1062–1067 .
    [Google Scholar]
  34. Michel J.F., Millet J.. ( 1970;). Physiological studies on early-blocked sporulation mutants of Bacillus subtilis. J Appl Bacteriol 33: 220–227 [CrossRef] [PubMed].
    [Google Scholar]
  35. Michna R.H., Commichau F.M., Tödter D., Zschiedrich C.P., Stülke J.. ( 2014;). SubtiWiki-a database for the model organism Bacillus subtilis that links pathway, interaction and expression information. Nucleic Acids Res (D1), D692–D698 [CrossRef] [PubMed].
    [Google Scholar]
  36. Molle V., Fujita M., Jensen S.T., Eichenberger P., González-Pastor J.E., Liu J.S., Losick R.. ( 2003;). The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50: 1683–1701 [CrossRef] [PubMed].
    [Google Scholar]
  37. Monteilhet C., Perrin A., Thierry A., Colleaux L., Dujon B.. ( 1990;). Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endonuclease encoded by a group I intron. Nucleic Acids Res 18: 1407–1413 [CrossRef] [PubMed].
    [Google Scholar]
  38. Mootz H.D., Finking R., Marahiel M.A.. ( 2001;). 4′-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis. J Biol Chem 276: 37289–37298 [CrossRef] [PubMed].
    [Google Scholar]
  39. Morimoto T., Kadoya R., Endo K., Tohata M., Sawada K., Liu S., Ozawa T., Kodama T., Kakeshita H., other authors. ( 2008;). Enhanced recombinant protein productivity by genome reduction in Bacillus subtilis. DNA Res 15: 73–81 [CrossRef] [PubMed].
    [Google Scholar]
  40. Nakano M.M., Corbell N., Besson J., Zuber P.. ( 1992;). Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Mol Gen Genet 232: 313–321 .
    [Google Scholar]
  41. Paik S.H., Chakicherla A., Hansen J.N.. ( 1998;). Identification and characterization of the structural and transporter genes for, and the chemical and biological properties of, sublancin 168, a novel lantibiotic produced by Bacillus subtilis 168. J Biol Chem 273: 23134–23142 [CrossRef] [PubMed].
    [Google Scholar]
  42. Petersohn A., Brigulla M., Haas S., Hoheisel J.D., Völker U., Hecker M.. ( 2001;). Global analysis of the general stress response of Bacillus subtilis. J Bacteriol 183: 5617–5631 [CrossRef] [PubMed].
    [Google Scholar]
  43. Price C.W., Fawcett P., Cérémonie H., Su N., Murphy C.K., Youngman P.. ( 2001;). Genome-wide analysis of the general stress response in Bacillus subtilis. Mol Microbiol 41: 757–774 [CrossRef] [PubMed].
    [Google Scholar]
  44. Reyrat J.M., Pelicic V., Gicquel B., Rappuoli R.. ( 1998;). Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect Immun 66: 4011–4017 [PubMed].
    [Google Scholar]
  45. Sambrook J., Fritsch E.F., Maniatis T.. ( 1989;). Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  46. Stein T.. ( 2005;). Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol Microbiol 56: 845–857 [CrossRef] [PubMed].
    [Google Scholar]
  47. Steller S., Vollenbroich D., Leenders F., Stein T., Conrad B., Hofemeister J., Jacques P., Thonart P., Vater J.. ( 1999;). Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3. Chem Biol 6: 31–41 [CrossRef] [PubMed].
    [Google Scholar]
  48. Sun T., Altenbuchner J.. ( 2010;). Characterization of a mannose utilization system in Bacillus subtilis. J Bacteriol 192: 2128–2139 [CrossRef] [PubMed].
    [Google Scholar]
  49. Tamehiro N., Okamoto-Hosoya Y., Okamoto S., Ubukata M., Hamada M., Naganawa H., Ochi K.. ( 2002;). Bacilysocin, a novel phospholipid antibiotic produced by Bacillus subtilis 168. Antimicrob Agents Chemother 46: 315–320 [CrossRef] [PubMed].
    [Google Scholar]
  50. Tanaka K., Henry C.S., Zinner J.F., Jolivet E., Cohoon M.P., Xia F., Bidnenko V., Ehrlich S.D., Stevens R.L., Noirot P.. ( 2013;). Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate large-scale metabolic model. Nucleic Acids Res 41: 687–699 [CrossRef] [PubMed].
    [Google Scholar]
  51. Tsuge K., Ano T., Hirai M., Nakamura Y., Shoda M.. ( 1999;). The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob Agents Chemother 43: 2183–2192 .
    [Google Scholar]
  52. Turgay K., Hahn J., Burghoorn J., Dubnau D.. ( 1998;). Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor. EMBO J 17: 6730–6738 [CrossRef] [PubMed].
    [Google Scholar]
  53. Wang S.T., Setlow B., Conlon E.M., Lyon J.L., Imamura D., Sato T., Setlow P., Losick R., Eichenberger P.. ( 2006;). The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358: 16–37 [CrossRef] [PubMed].
    [Google Scholar]
  54. Warth L., Altenbuchner J.. ( 2013a;). A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites. Appl Microbiol Biotechnol 97: 6845–6856 [CrossRef] [PubMed].
    [Google Scholar]
  55. Warth L., Altenbuchner J.. ( 2013b;). The tyrosine recombinase MrpA and its target sequence: a mutational analysis of the recombination site mrpS resulting in a new left element/right element (LE/RE) deletion system. Arch Microbiol 195: 617–636 [CrossRef] [PubMed].
    [Google Scholar]
  56. Wenzel M., Altenbuchner J.. ( 2013;). The Bacillus subtilis mannose regulator, ManR, a DNA-binding protein regulated by HPr and its cognate PTS transporter ManP. Mol Microbiol 88: 562–576 [CrossRef] [PubMed].
    [Google Scholar]
  57. Wenzel M., Müller A., Siemann-Herzberg M., Altenbuchner J.. ( 2011;). Self-inducible Bacillus subtilis expression system for reliable and inexpensive protein production by high-cell-density fermentation. Appl Environ Microbiol 77: 6419–6425 [CrossRef] [PubMed].
    [Google Scholar]
  58. Westers H., Dorenbos R., van Dijl J.M., Kabel J., Flanagan T., Devine K.M., Jude F., Seror S.J., Beekman A.C., other authors. ( 2003;). Genome engineering reveals large dispensable regions in Bacillus subtilis. Mol Biol Evol 20: 2076–2090 [CrossRef] [PubMed].
    [Google Scholar]
  59. Westers L., Westers H., Quax W.J.. ( 2004;). Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694: 299–310 [CrossRef] [PubMed].
    [Google Scholar]
  60. Wood H.E., Dawson M.T., Devine K.M., McConnell D.J.. ( 1990;). Characterization of PBSX, a defective prophage of Bacillus subtilis. J Bacteriol 172: 2667–2674 .
    [Google Scholar]
  61. Yanisch-Perron C., Vieira J., Messing J.. ( 1985;). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–119 [CrossRef] [PubMed].
    [Google Scholar]
  62. Yoshida K., Kobayashi K., Miwa Y., Kang C.M., Matsunaga M., Yamaguchi H., Tojo S., Yamamoto M., Nishi R., other authors. ( 2001;). Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis. Nucleic Acids Res 29: 683–692 [CrossRef] [PubMed].
    [Google Scholar]
  63. Zahler S.A., Korman R.Z., Rosenthal R., Hemphill H.E.. ( 1977;). Bacillus subtilis bacteriophage SPbeta: localization of the prophage attachment site, and specialized transduction. J Bacteriol 129: 556–558 [PubMed].
    [Google Scholar]
  64. Zakataeva N.P., Nikitina O.V., Gronskiy S.V., Romanenkov D.V., Livshits V.A.. ( 2010;). A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains. Appl Microbiol Biotechnol 85: 1201–1209 [CrossRef] [PubMed].
    [Google Scholar]
  65. Zhang X.Z., Yan X., Cui Z.L., Hong Q., Li S.P.. ( 2006;). mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis. Nucleic Acids Res 34: e71 [CrossRef] [PubMed].
    [Google Scholar]
  66. Zheng G., Hehn R., Zuber P.. ( 2000;). Mutational analysis of the sbo-alb locus of Bacillus subtilis: identification of genes required for subtilosin production and immunity. J Bacteriol 182: 3266–3273 [CrossRef] [PubMed].
    [Google Scholar]
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