Inhibition of biofilm in Bacillus amyloliquefaciens Q-426 by diketopiperazines

  • Jian-Hua WangEmail author
  • Cui-Yun Yang
  • Sheng-Tao Fang
  • Jian Lu
  • Chun-Shan QuanEmail author
Original Paper


Biofilm formation can make significant effects on bacteria habits and biological functions. In this study, diketopiperazines (DKPs) produced by strain of Bacillus amyloliquefaciens Q-426 was found to inhibit biofilm formed in the gas–liquid interface. Four kinds of DKPs were extracted from B. amyloliquefaciens Q-426, and we found that 0.04 mg ml−1 DKPs could obviously inhibit the biofilm formation of the strain. DKPs produced by B. amyloliquefaciens Q-426 made a reduction on extracellular polymeric substance (EPS) components, polysaccharides, proteins, DNAs, etc. Real-time PCR was performed to determine that whether DKPs could make an obvious effect on the expression level for genes related to biofilm formation in the strain. The relative expression level of genes tasA, epsH, epsG and remB which related to proteins, extracellular matrix, and polysaccharides, were downregulated with 0.04 mg ml−1 DKPs, while the expression level of nuclease gene nuc was significantly upregulated. The quantitative results of the mRNA expression level for these genes concerted with the quantitative results on EPS levels. All of the experimental results ultimately indicated that DKPs could inhibit the biofilm formation of the strain B. amyloliquefaciens Q-426.


Biofilm Diketopiperazines (DKPs) Extracellular polymeric substance (EPS) Real-time PCR Bacillus amyloliquefaciens Q-426 





Extracellular polymeric substance


Quorum sensing


Atomic force microscope


Colony-forming unit



This study was financially supported by Foundation of Key Laboratory of Marine Environmental Corrosion and Bio-fouling (MCKF201402), Institute of Oceanology, Chinese Academy of Sciences, One Hundred-Talent Plan of Chinese Academy of Sciences (CAS) and Research Program of CAS Key Laboratory of Coastal Environmental Processes and Ecological Remediation (No. 1189010002).

Supplementary material

11274_2016_2106_MOESM1_ESM.tif (1 mb)
Figure S1 Electrophoresis of total RNA extracted from strain Q-426. M: DL2000; 1, 3: Control samples; 2: Treated with 0.01 mg ml-1 Cyclo (Pro-Phe); 4: Treated with 0.04 mg ml-1 Cyclo (Pro-Phe) (TIFF 1068 kb)
11274_2016_2106_MOESM2_ESM.tif (194 kb)
Figure S2 Specific amplification plots of biofilm genes by real time PCR (TIFF 193 kb)
11274_2016_2106_MOESM3_ESM.doc (28 kb)
Supplementary material 3 (DOC 27 kb)


  1. Aguilera A, Souza-Egipsy V, Martín-Úriz PS, Amils R (2008) Extraction of extracellular polymeric substances from extreme acidic microbial biofilms. Appl Microbiol Biotechnol 78:1079–1088CrossRefGoogle Scholar
  2. Allesen-Holm M, Barken KB, Yang L, Klausen M, Webb JS, Kjelleberg S (2006) A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol Microbiol 59:1114–1128CrossRefGoogle Scholar
  3. Barken KB, Pamp SJ, Yang L, Gjiermansen M, Bertrand JJ, Klausen M (2008) Roles of type IV pili, flagellum-mediated motility and extracellular DNA in the formation of mature multicellular structures in Pseudomonas aeruginosa biofilms. Environ Microbiol 10:2331–2343CrossRefGoogle Scholar
  4. Boles BR, Thoendel M, Singh PK (2005) Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms. Mol Microbiol 57:1210–1223CrossRefGoogle Scholar
  5. Borriss R, Chen X, Rueckert C, Blom J, Becker A, Baumgarth B (2011) Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons. Int J Syst Evol Microbiol 61:1786–1801CrossRefGoogle Scholar
  6. Bowman JP (2007) Bioactive compound synthetic capacity and ecological significance of marine bacterial genus Pseudoalteromonas. Mar Drugs 5:220–241CrossRefGoogle Scholar
  7. Byun HG, Zhang HMM, Adachi KSY, Lee WJ (2003) Novel antifungal diketopiperazine from marine fungus. J Antibiot 56:102–106CrossRefGoogle Scholar
  8. Campbell J, Lin Q, Geske GD, Blackwell HE (2009) New and unexpected insights into the modulation of LuxR-type quorum sensing by cyclic dipeptides. ACS Chem Biol 4:1051–1059CrossRefGoogle Scholar
  9. Cho JH, Kang JY, Hong YK, Baek HH, Shin HW, Kim MS (2012) Isolation and structural determination of the antifouling diketopiperazines from marine-derived Streptomyces praecox 291-11. Biosci Biotechnol Biochem 76:1116–1121CrossRefGoogle Scholar
  10. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM (1995) Microbial biofilms. Annu Rev Microbiol 49:711–745CrossRefGoogle Scholar
  11. Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322CrossRefGoogle Scholar
  12. Davey ME, Caiazza NC, O’Toole GA (2003) Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol 185:1027–1036CrossRefGoogle Scholar
  13. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280:295–298CrossRefGoogle Scholar
  14. De Carvalho MP, Abraham W-R (2012) Antimicrobial and biofilm inhibiting diketopiperazines. Curr Med Chem 19:3564–3577CrossRefGoogle Scholar
  15. De Kievit TR (2009) Quorum sensing in Pseudomonas aeruginosa biofilms. Environ Microbiol 11:279–288CrossRefGoogle Scholar
  16. Degrassi G, Aguilar C, Bosco M, Zahariev S, Pongor S, Ventui V (2002) Plant growth-promoting Pseudomonas putida WCS358 produces and secretes four cyclic dipeptides: cross-talk with quorum sensing bacteria sensors. Curr Microbiol 45:250–254CrossRefGoogle Scholar
  17. Du L, Feng T, Zhao B, Li D (2010) Alkaloids from a deep ocean sediment-derived fungus Penicillium sp. and their antitumor activities. J Antibiot 63:165–170CrossRefGoogle Scholar
  18. Elchinger PH, Delattre C, Faure S, Roy O, Badel S, Bernardi T, Taillefumier C, Michaud P (2014) Immobilization of proteases on chitosan for the development of films with anti-biofilm properties. Int J Biol Macromol 72:1063–1068CrossRefGoogle Scholar
  19. El-Gendy BEDM, Rateb ME (2015) Antibacterial activity of diketopiperazines isolated from a marine fungus using t-butoxycarbonyl group as a simple tool for purification. Bioorg Med Chem Lett 25:3125–3128CrossRefGoogle Scholar
  20. Goncalves MDS, Delattre C, Balestrino D, Charbonnel N, Elboutachfaiti R, Wadouachi A, Badel S, Bernardi T, Michaud P, Forestier C (2014) Anti-biofilm activity: a function of Klebsiella pneumoniae capsular polysaccharide. PLoS ONE 9:e99995CrossRefGoogle Scholar
  21. Gowrishankar S, Poornima B, Pandian SK (2014) Inhibitory efficacy of cyclo(L-leucyl-L-prolyl) from mangrove rhizosphere bacterium-Bacillus amyloliquefaciens (MMS-50) toward cariogenic properties of Streptococcus mutans. Res Microbiol 165:278–289CrossRefGoogle Scholar
  22. Holden MTG, Chhabra SR, Denys R, Stead P, Bainton NJ, Hill JP (1999) Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram-negative bacteria. Mol Microbiol 33:1254–1266CrossRefGoogle Scholar
  23. Huber B, Riedel K, Hentzer M, Heydorn A, Gotschlich A, Givskov M (2001) The cep quorum-sensing system of Burkholderia cepacia H111 controls biofilm formation and swarming motility. Microbiology 147:2517–2528CrossRefGoogle Scholar
  24. Kaplan JB, Velliyagounder K, Ragunath C, Rohde H, Mack D, Knobloch JK (2004) Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms. J Bacteriol 186:8213–8220CrossRefGoogle Scholar
  25. Karlowsky JA, Jones ME, Draghi DC, Thornsberry C, Sahm DF, Volturo GA (2004) Prevalence and antimicrobial susceptibilities of bacteria isolated from blood cultures of hospitalized patients in the United States in 2002. Ann Clin Microbiol Antimicrob 3:7CrossRefGoogle Scholar
  26. Klose KE (2006) Increased chatter: cyclic dipeptides as molecules of chemical communication in Vibrio spp. J Bacteriol 188:2025–2026CrossRefGoogle Scholar
  27. Kuier I, Lagendijk E, Pickford R, Derrick J, Lamers G, Thomas-Oates J (2004) Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms. Mol Microbiol 51:97–113Google Scholar
  28. Kumar SN, Mohandas C, Siji JV, Rajasekharan KN, Nambisan B (2012) Identification of antimicrobial compound, diketopiperazines, from a Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode against major plant pathogenic fungi. J Appl Microbiol 113:914–924CrossRefGoogle Scholar
  29. Lawrence JR, Korber DR, Hoyle BD, Costerton JW, Caldwell DE (1991) Optical sectioning of microbial biofilms. J Bacteriol 173:6558–6567Google Scholar
  30. Li X, Dobretsov S, Xu Y, Xiao X, Hung OS, Qian PY (2006) Antifouling diketopiperazines produced by a deep-sea bacterium, Streptomyces fungicidicus. Biofouling 22:187–194CrossRefGoogle Scholar
  31. Li Z, Peng C, Shen Y, Miao X, Zhang H, Lin H (2008) L, L-diketopiperazines from Alcaligenes faecalis A72 associated with south china sea sponge Stelletta tenuis. Biochem Syst Ecol 36:230–234CrossRefGoogle Scholar
  32. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408CrossRefGoogle Scholar
  33. Musthafa KS, Balamurugan K, Pandian SK, Ravi AV (2012) 2,5-Piperazinedione inhibits quorum sensing-dependent factors production in Pseudomonas aeruginosa PAO1. J Basic Microbiol 52:1–8CrossRefGoogle Scholar
  34. O’Toole G, Kaplan H, Kolter R (2000) Biofilm formation as microbial development. Annu Rev Microbiol 54:49–79CrossRefGoogle Scholar
  35. Ortiz-Castro R, Díaz-Pérez C, Martínez-Trujillo M, del Río RE, Campos-García J, López-Bucio J (2011) Transkingdom signaling based on bacterial cyclodipeptides with auxin activity in plants. Proc Natl Acad Sci USA 108:7253–7258CrossRefGoogle Scholar
  36. Prasad C (1995) Bioactive cyclic dipeptides. Peptides 16:151–164CrossRefGoogle Scholar
  37. Purevdorj B, Costerton JW, Stoodley P (2002) Influence of hydrodynamics and cell signaling on the structure and behaviour of Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 68:4457–4464CrossRefGoogle Scholar
  38. Qi SH, Xu Y, Gao J, Qian PY (2009) Antibacterial and antilarval compounds from marine bacterium Pseudomonas rhizosphaerae. Ann Microbiol 59:229–233CrossRefGoogle Scholar
  39. Reading NC, Sperandio V (2006) Quorum sensing: the many languages of bacteria. FEMS Microbiol Lett 254:1–11CrossRefGoogle Scholar
  40. Romero D, Vlamakis H, Losick R, Kolter R (2011) An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms. Mol Microbiol 80:1155–1168CrossRefGoogle Scholar
  41. Stoodley P, Lewandowski Z, Boyle J, Lappin-Scott H (1999) The formation of migratory ripples in a mixed species bacterial biofilm growing in turbulent flow. Environ Microbiol 1:447–455CrossRefGoogle Scholar
  42. Sutherland IW (2001) The biofilm matrix, an immobilized but dynamic microbial environment. Trends Microbiol 9:222–227CrossRefGoogle Scholar
  43. Thomas VC, Thurlow LR, Boyle D, Hancock LE (2008) Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development. J Bacteriol 190:5690–5698CrossRefGoogle Scholar
  44. Tommonaro G, Abbamondi GR, Iodice C, Tait K, De Rosa S (2012) Diketopiperazines produced by the Halophilic Archaeon, Haloterrigena hispanica, activate AHL bioreporters. Microb Ecol 63:490–495CrossRefGoogle Scholar
  45. Trigos A, Reyna S, Cervantes L (1995) Three diketopiperazines from the cultivated fungus Fusarium oxysporum. Nat Prod Lett 6:241–246CrossRefGoogle Scholar
  46. Wang G, Dai S, Chen M, Wu H (2010a) Two diketopiperazine cyclo (Pro-Phe) isomers from marine bacteria Bacillus subtilis sp. 13-2. Chem Nat Compd 46:583–585CrossRefGoogle Scholar
  47. Wang JH, Quan CS, Qi XH, Li X, Fan SD (2010b) Determination of diketopiperazines of Burkholderia cepacia CF-66 by gas chromatography-mass spectrometry. Anal Bioanal Chem 396:1773–1779CrossRefGoogle Scholar
  48. Wang FQ, Tong QY, Ma HR, Xu HF, Hu S, Ma W, Xue YB, Liu JJ, Wang JP, Song HP, Zhang JW, Zhang G, Zhang YH (2015) Indole diketopiperazines from endophytic Chaetomium sp 88194 induce breast cancer cell apoptotic death. Sci Rep 5:9294CrossRefGoogle Scholar
  49. Watnick P, Kolter R (2000) Biofilm, city of microbes. J Bacteriol 182:2675–2679CrossRefGoogle Scholar
  50. Zhao PC, Quan CS, Jin LM, Wang LN, Wang JH, Fan SD (2013) Effects of critical medium components on the production of antifungal lipopeptides from Bacillus amyloliquefaciens Q-426 exhibiting excellent biosurfactant properties. World J Microbiol Biotechnol 29:401–409CrossRefGoogle Scholar
  51. Zhao PC, Quan CS, Wang YG, Wang JH, Fan SD (2014) Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f.sp. spinaciae. J Basic Microbiol 54:448–456CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Yantai Institute of Coastal Zone ResearchChinese Academy of SciencesYantaiChina
  2. 2.Key Laboratory of Coastal Biology and Biological Resources Utilization, Yantai Institute of Coastal Zone ResearchChinese Academy of SciencesYantaiChina
  3. 3.Key Laboratory of Marine Environmental Corrosion and Bio-fouling, Institute of OceanologyChinese Academy of SciencesQingdaoChina
  4. 4.Life Science CollegeDalian Nationalities UniversityDalianChina

Personalised recommendations