Skip to main content
SpringerLink
Log in

Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

The Bacillus subtilis strain A1/3 shows exceptionally diverse antibiotic capacities compared to other B. subtilis strains. To analyze this phenomenon, mutants for the putative pantotheinyltransferase gene ( pptS), and for several genes involved in non-ribosomal peptide synthesis and polyketide synthesis were constructed and characterized, using bioassays with blood cells, bacterial and fungal cells, and mass spectrometry. Among at least nine distinct bioactive compounds, five antibiotics and one siderophore activity were identified. The anti-fungal and hemolytic activities of strain A1/3 could be eliminated by mutation of the fen and srf genes essential for the synthesis of fengycins and surfactins. Both pptS - and dhb -type mutants were defective in iron uptake, indicating an inability to produce a 2,3-dihydroxybenzoate-type iron siderophore. Transposon mutants in the malonyl CoA transacylase gene resulted in the loss of hemolytic and anti-fungal activities due to the inhibition of bacillomycin L synthesis, and this led to the discovery of bmyLD-LA-LB* genes. In mutants bearing disruption mutations in polyketide ( pksM - and/or pksR -like) genes, the biosynthesis of bacillaene and difficidins, respectively, was inactivated and was accompanied by the loss of discrete antibacterial activities. The formation of biofilms (pellicles) was shown to require the production of surfactins, but no other lipopeptides, indicating that surfactins serve specific developmental functions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2a–e
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7a, b
Fig. 8
Fig. 9a–i

Similar content being viewed by others

References

  • Battchikova N, Koivulehto M, Denesyuk A, Ptitsyn L, Boretsky Y, Hellman J, Korpela T (1996) Aspartate amino transferase from an alkalophilic Bacillus contains an additional 20-aa extension at its functionally important N-terminus. J Biochem 120:425–432

    CAS  PubMed  Google Scholar 

  • Behnke D, Gilmore M (1981) Location of antibiotic resistance determinants, copy control, and replication functions on the double-selective streptococcal cloning vector pGB301. Mol Gen Genet 184:115–120

    CAS  PubMed  Google Scholar 

  • Bernhard F, Demel G, Soltani K, Dohren HV, Blinov V (1996) Identification of genes encoding for peptide synthetases in the gram-negative bacterium Lysobacter sp. ATCC 53042 and the fungus Cylindrotrichum oligospermum. DNA Seq 6:319–330

    CAS  PubMed  Google Scholar 

  • Besson F, Peypoux F, Michel G, Delcambe L (1978) Identification of antibiotics of iturin group in various strains of Bacillus subtilis. J Antibiot 31:284–288

    CAS  PubMed  Google Scholar 

  • Borchert S, Patil SS, Marahiel MA (1992) Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases. FEMS Microbiol Lett 71:175–180

    Article  CAS  PubMed  Google Scholar 

  • Branda SS, Gonzalez-Pastor JE, Ben-Yehuda S, Losick R, Kolter R (2001) Fruiting body formation by Bacillus subtilis. Proc Natl Acad Sci USA 98:11621–11626

    Article  CAS  PubMed  Google Scholar 

  • Chu HH, Hoang V, Kreutzmann P, Hofemeister B, Melzer M, Hofemeister J (2002) Identification and properties of type I-signal peptidases of Bacillus amyloliquefaciens. Eur J Biochem 269:458–469

    Article  CAS  PubMed  Google Scholar 

  • Conrad B, Bashkirov V, Hofemeister J (1992) Imprecise excision of plasmid pE194 from the chromosomes of Bacillus subtilis pE194 insertion strains. J Bacteriol 174:6997–7002

    CAS  PubMed  Google Scholar 

  • Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H, Saenger W, Bernhard F, Reinhardt R, Schmidt M, Ullrich C, Stein T, Leenders F, Vater J (1999) The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc Natl Acad Sci USA 96:13294–13299

    Article  CAS  PubMed  Google Scholar 

  • Elsner A, Engert H, Saenger W, Hamoen L, Venema G, Bernhard F (1997) Substrate specificity of hybrid modules from peptide synthetases. J Biol Chem 272:4814–4819

    Article  CAS  PubMed  Google Scholar 

  • Eshita SM, Roberto NH, Beale JM, Mamiya BM, Workman RF (1995) Bacillomycin Lc, a new antibiotic of the iturin group: isolations, structures, and antifungal activities of the congeners. J Antibiot 48:1240–1247

    CAS  PubMed  Google Scholar 

  • Griesbach E, Lattauschke E (1991) Übertragung von Clavibacter michiganensis subsp. michiganensis in Tomaten-Hydroponikkulturen und Möglichkeiten zur Bekämpfung des Erregers. Nachrichtenbl Deut Pflanzenschutzd 43:69–73

    Google Scholar 

  • Horionuchi S, Weisblum B (1982) Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J Bacteriol 150:804–814

    PubMed  Google Scholar 

  • Huang CC, Ano T, Shoda M (1993) Nucleotide sequence and characteristics of the gene, lpa-14 , responsible for biosynthesis of the lipopeptide antibiotics iturin A and surfactin from Bacillus subtilis RB14. J Ferment Bioeng 76:445–450

    Article  CAS  Google Scholar 

  • Huber J, Griesbach E, Pippig R, Kegler H, Richter S (1991) Mikroorganismus und dessen Verwendung als mikrobielles Pflanzenstärkungsmittel. DD 295 526

  • Kakinuma A, Hori M, Isono M, Tamura G, Arima K (1969) Determination of amino acid sequence in surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis. Agr Biol Chem 33:971–972

    CAS  Google Scholar 

  • Koumoutsi A, Chen XH, Henne A, Liesegang H, Hitzeroth G, Franke P, Vater J, Borriss R (2004) Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. J Bacteriol 186:1084–1096

    Article  CAS  PubMed  Google Scholar 

  • Kowall M, Vater J, Kluge B, Stein T, Franke P, Ziessow (1998) Separation and characterization of surfactin isoforms produced by Bacillus subtilis OKB 105. J Colloid Interface Sci 203:1–8

    Article  Google Scholar 

  • Kunst F et al. (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249–256

    Article  CAS  PubMed  Google Scholar 

  • Lambalot RH, Gehring AM, Flugel RS, Zuber P, LaCelle M, Marahiel MA, Reid R, Khosla C, Walsh CT (1996) A new enzyme superfamily—the phosphopanthetheinyl transferases. Chem Biol 3:923–936

    CAS  PubMed  Google Scholar 

  • Landy M, Warren GH, Rosenman SB, Colio LG (1948) Bacillomycin, an antibiotic from Bacillus subtilis active against pathogenic fungi. Proc Soc Exp Biol & Med 67:539–541

    Google Scholar 

  • Lee SY, Rhee SK, Kim CH, Suh JW (1998) Rapid and efficient isolation of genes for biosynthesis of peptide antibiotics from Gram-positive bacterial strains. J Microbiol Biotechnol 8:310–317

    Article  CAS  Google Scholar 

  • Leenders F, Vater J, Stein T, Franke P (1998) Characterization of the binding site of the tripeptide intermediate D-phenylalanyl L-prolyl-L-valine in gramicidin S biosynthesis. J Biol Chem 273:18011–18014

    Article  CAS  PubMed  Google Scholar 

  • Leenders F, Stein TH, Kablitz B, Franke P, Vater J (1999) Rapid typing of Bacillus subtilis strains by their secondary metabolites using matrix-assisted laser desorption/ionization mass spectrometry of intact cells. Rapid Commun Mass Spectrom 13:943–949

    Article  CAS  Google Scholar 

  • Lin TP, Chen CL, Chang LK, Tschen JS, Liu ST (1999) Functional and transcriptional analyses of a fengycin synthetase gene, fenC , from Bacillus subtilis. J Bacteriol 181:5060–5067

    CAS  PubMed  Google Scholar 

  • Marahiel MA, Stachelhaus T, Mootz HD (1997) Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem Rev 97:2651–2674

    Article  CAS  PubMed  Google Scholar 

  • May JJ, Wendrich TM, Marahiel MA (2001) The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin. J Biol Chem 276:7209–7217

    Article  CAS  PubMed  Google Scholar 

  • Menkhaus M, Ullrich C, Kluge B, Vater J, Vollenbroich D, Kamp RM (1993) Structural and functional organization of the surfactin synthetase multienzyme system. J Biol Chem 268:7678–7684

    CAS  PubMed  Google Scholar 

  • Mootz HD, Finking R, Marahiel MA (2001) 4′-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis. J Biol Chem 276:37289–37298

    Article  CAS  PubMed  Google Scholar 

  • Nakano MM, Corbell N, Besson J, Zuber P (1992) Isolation and characterisation of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Mol Gen Genet 232:313–321

    CAS  PubMed  Google Scholar 

  • Neilan BA, Dittmann E, Rouhiainen L, Bass RA, Schaub V, Sivonen K, Börner T (1999) Nonribosomal peptide synthesis and toxigenicity of cyanobacteria. J Bacteriol 181:4089–4097

    CAS  PubMed  Google Scholar 

  • Pabel CT, Vater J, Wilde C, Franke P, Hofemeister J, Adler B, Bringmann G, Hacker J, Hentschel U (2003) Antimicrobial activities and matrix-assisted laser desorption/ionization mass spectrometry of Bacillus isolates from the marine sponge Aplysina aerophoba. Mar Biotechnol 5:424–434

    Article  CAS  PubMed  Google Scholar 

  • Patel PS, Huang S, Fisher S, Pirnik D, Aklonis C, Dean L, Meyers E, Fernandes P, Mayerl F (1995) Bacillaene, a novel inhibitor of procaryotic protein synthesis produced by Bacillus subtilis: production, taxonomy, isolation, physico-chemical characterization and biological activity. J Antibiot 48:997–1003

    CAS  PubMed  Google Scholar 

  • Petit MA, Bruand C, Janniere L, Ehrlich SD (1990) Tn10-derived transposons active in Bacillus subtilis. J Bacteriol 172:6736–6740

    CAS  PubMed  Google Scholar 

  • Peypoux F, Pommier MT, Das BC, Besson F, Delcambe L, Michel G (1984) Structures of bacillomycin D and bacillomycin L peptidolipid antibiotics from Bacillus subtilis. J Antibiot 37:1600–1604

    CAS  PubMed  Google Scholar 

  • Schwartz D, Alijah R, Nussbaumer B, Pelzer S, Wohlleben W (1996) The peptide synthetase gene phsA from Streptomyces viridochromogenes is not juxtaposed with other genes involved in nonribosomal biosynthesis of peptides. Appl Environ Microbiol 62:570–577

    Google Scholar 

  • Seow KT, Meurer G, Gerlitz M, Wendt-Pienkowski E, Hutchinson CR, Davies J (1997) A study of interactive type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms. J Bacteriol 179:7360–7368

    CAS  PubMed  Google Scholar 

  • Shoda M (2000) Bacterial control of plant disease. J Biosci Bioeng 89:515–521

    Article  CAS  Google Scholar 

  • Sinclair JB (1989) Bacillus subtilis as a biocontrol agent for plant diseases. In: Agrihotri VP, Singh N, Chaube HS, Sinh US, Dwivedi TS (eds) Perspectives in plant pathology. Today and Tomorrow’s Printers and Publishers, New Delhi, pp 367–374

  • Sokolov SL, Zakharov MV, Esikova TZ, Evdokimova EG, Alakhov YB (2002) Identification and cloning of peptide synthetase genes of thermostable bacilli using the polymerase chain reaction. Russ J Genet 38:1614–1620

    CAS  Google Scholar 

  • Sosio M, Bossi E, Bianchi A, Donadio S (2000) Multiple peptide synthetase gene clusters in Actinomycetes. Mol Gen Genet 264:213–221

    Article  CAS  PubMed  Google Scholar 

  • Stachelhaus T, Mootz HD, Marahiel MA (2002) Nonribosomal assembly of peptide antibiotics on modular protein templates. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D.C., pp 415–435

  • Stein T, Borchert S, Conrad B, Feesche J, Hofemeister B, Hofemeister J, Entian KD (2002) Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3. J Bacteriol 184:1703–1711

    Article  CAS  PubMed  Google Scholar 

  • Steinborn G (1998) Zweifach positiv selektive Klonierungsvektoren. DE 196 54 841

    Google Scholar 

  • Steinborn G, Hofemeister J (1984) Verfahren zur Herstellung von alpha-Amylase. DD B5 233852

  • Steinborn G, Hofemeister J (1998/2000) Genes for the biosynthesis of anticapsin and bacilysin, their isolation and use. PCT/DE99/02179, WO 00/03009

  • Steinmetz M, Richter R (1994) Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome. J Bacteriol 176:1761–1763

    CAS  PubMed  Google Scholar 

  • Steller S, Vollenbroich D, Leenders F, Stein T, Conrad B, Hofemeister J, Jacques P, Thonard 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

    Article  CAS  PubMed  Google Scholar 

  • Tsuge K, Akiyama T, Shoda M (2001) Cloning, sequencing, and characterization of the iturin A operon. J Bacteriol 183:6265–6273

    Article  CAS  PubMed  Google Scholar 

  • Turgay K, Marahiel MA (1994) A general approach for identifying and cloning peptide synthetase genes. Pept Res 7:238–241

    CAS  PubMed  Google Scholar 

  • Uozumi T, Hoshino T, Miwa K, Horinouchi S, Beppu T, Arima K (1977) Restriction and modification in Bacillus species. Genetic transformation of bacteria with DNA from different species, part I. Mol Gen Genet 152:65–69

    CAS  PubMed  Google Scholar 

  • Vanittanakom N, Loeffler W, Koch U, Jung G (1986) Fengycin—a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J Antibiot 39:888–901

    CAS  PubMed  Google Scholar 

  • Vater J, Stein T, Vollenbroich D, Kruft V, Wittmann-Liebold B, Franke P, Liu L, Zuber P (1997) The modular organization of multifunctional peptide synthetases. J Protein Chem 16:557–564

    Article  CAS  PubMed  Google Scholar 

  • Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS (2002) Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 68:6210–6219

    Google Scholar 

  • Vater J, Gao X, Hitzeroth G, Wilde C, Franke P (2003) Whole cell-matrix-assisted laser desorption ionization-time of flight-mass spectrometry, an emerging technique for efficient screening of biocombinatorial libraries of natural compounds—present state of research. CCHTS 6:557–567

    CAS  Google Scholar 

  • Wilson KE, Flor JE, Schwartz RE, Joshua H, Smith JL, Pelak BA, Liesch JM, Hensens OD (1987) Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis. II. Isolation and physico-chemical characterization. J Antibiot 40:1682–1691

    CAS  PubMed  Google Scholar 

  • Yao S, Gao X, Fuchsbauer N, Hillen W, Vater J, Wang J (2003) Cloning, sequencing, and characterization of the genetic region relevant to biosynthesis of the lipopeptides iturin A and surfactin in Bacillus subtilis. Curr Microbiol 47:272–277

    Article  CAS  PubMed  Google Scholar 

  • Zimmerman SB, Schwartz CD, Monaghan RL, Pelak BA, Weissberger B, Gilfillan EC, Mochales S, Hernandez S, Currie SA, Tejera E (1987) Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis. I. Production, taxonomy and antibacterial activity. J Antibiot 40:1677–1681

    CAS  PubMed  Google Scholar 

  • Zuber P, Nakano MM, Marahiel MA (1993) Peptide antibiotics. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and other Gram-positive bacteria: biochemistry, physiology, and molecular genetics. ASM Press, Washington D.C., pp 897–916

  • Zweerink MM, Edison A (1987) Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis. III. Mode of action of difficidin. J Antibiot 40:1692–1697

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Strain B. subtilis A1/3 was isolated and provided by Erika Griesbach of the Bundesanstalt für Züchtungsforschung an Kulturpflanzen, Aschersleben. We thank the following collaborators at the IPK, Gatersleben: Susanne König for DNA sequencing, Hans-Wolfgang Jank for computer affairs, and Silke Gorgulla, Ingelore Dommes and Birgit Fischer for expert technical assistance. We are much obliged to Birgit Krebs (FZB Biotechnik GmbH, Berlin) for helpful comments on antibiotic testing. Thanks also to Bettina Kempf and Erhard Bremer (Philipps-Universität Marburg) for providing the plasmid pIC333. The studies in Gatersleben were supported by Grant No. BEO22/0311137 from the Bundesministerium für Bildung und Forschung (BMBF, Germany)

Author information

Author notes

  1. J. Feesche

    Present address: VTB-Enzymtechnologie, Henkel KGaA, Henkelstrasse 67, 40191, Düsseldorf, Germany

  2. F. Leenders

    Present address: Celares GmbH, Gebäude d79, Robert Rössle Str. 10, 13125, Berlin, Germany

Authors and Affiliations

  1. Department of Molecular Genetics, Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstrasse 3, 06466, Gatersleben, Germany

    J. Hofemeister, B. Conrad, B. Adler, B. Hofemeister, J. Feesche, N. Kucheryava & G. Steinborn

  2. Institut für Biochemie, Freie Universität Berlin, Thielallee 63, 14195, Berlin, Germany

    P. Franke

  3. ActinoDrug Pharmaceutical GmbH, Neuendorfer Str.24a, 16761, Hennigsdorf, Germany

    N. Grammel & A. Zwintscher

  4. Arbeitsgruppe Biochemie und Molekulare Biologie, Institut für Chemie, Technische Universität Berlin, Franklinstrasse 29, 10587, Berlin, Germany

    F. Leenders, G. Hitzeroth & J. Vater

Authors
  1. J. Hofemeister
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. B. Conrad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B. Adler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. B. Hofemeister
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. Feesche
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. N. Kucheryava
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. G. Steinborn
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. P. Franke
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. N. Grammel
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. A. Zwintscher
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. F. Leenders
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. G. Hitzeroth
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. J. Vater
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Hofemeister.

Additional information

Communicated by A. Kondorosi

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hofemeister, J., Conrad, B., Adler, B. et al. Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3. Mol Genet Genomics 272, 363–378 (2004). https://doi.org/10.1007/s00438-004-1056-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-004-1056-y

Keywords

Search

Navigation