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Detection of Quorum Sensing Molecules and Biofilm Formation in Ralstonia solanacearum

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Abstract

Many bacteria use small diffusible signaling molecules to communicate each other termed as quorum sensing (QS). Most Gram-negative bacteria use acyl homoserine lactone (AHL) as QS signal molecules. Using these signaling molecules, bacteria are able to express specific genes in response to population density. This work aimed to detect the production of QS signal molecules and biofilm formation in Ralstonia solanacearum isolated from various diseased tomato plants with symptoms of bacterial wilt. A total of 30 R. solanacearum strains were investigated for the production of QS signal molecules using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1 (pZLR4) biosensor systems. All 30 bacterial isolates from various bacterial wilt-affected tomato plants produced AHL molecules that induced the biosensor. The microtiter plate assay demonstrated that of the 30 bacterial isolates, 60 % formed biofilm, among which four isolates exhibited a higher degree of biofilm formation. The biofilm-inducing factor was purified from these four culture supernatants. The structure of the responsible molecule was solved using nuclear magnetic resonance and mass spectroscopy and was determined to be 2-hydroxy-4-((methylamino)(phenyl)methyl) cyclopentanone (HMCP), which was confirmed by chemical synthesis and NMR. The Confocal laser scanning microscopic analysis showed well-developed biofilm architecture of bacteria when treated with HMCP. The knowledge we obtained from this study will be useful for further researcher on the role of HMCP molecule in biofilm formation.

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References

  1. Álvarez B, Biosca EG, López MM (2010) On the life of Ralstonia solanacearum, a destructive bacterial plant pathogen. In: Mendez-Vilas A (ed), Current research, technology and education topics in applied microbiology and microbial biotechnology, pp 267–279

  2. Anbazhagan D, Mansor M, Yan GO, Md Yusof MY, Hassan H (2012) Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp. PLoS One 7:e36696

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Annous BA, Fratamico PM, Smith JL (2009) Quorum sensing in biofilms: why bacteria behave the way they do. J Food Sci 74:24–37

    Article  Google Scholar 

  4. Camilli A, Bassler BL (2006) Bacterial small-molecule signalling pathways. Science 311:1113–1116

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Cataldi TR, Bianco G, Abate S (2009) Accurate mass analysis of N-acyl-homoserine-lactones and cognate lactone-opened compounds in bacterial isolates of Pseudomonas aeruginosa PAO1 by LC-ESI-LTQ-FTICR-MS. J Mass Spectrom 44:182–192

    Article  CAS  PubMed  Google Scholar 

  6. Cha C, Gao P, Chen YC, Shaw PD, Farrand SK (1998) Production of acyl-homoserine lactone quorum-sensing signals by Gram-negative plant-associated bacteria. Mol Plant-Microbe Interact 11:1119–1129

    Article  CAS  PubMed  Google Scholar 

  7. Chua SL, Hultqvist LD, Yuan M, Rybtke M, Nielsen TE, Givskov M, Tolker-Nielsen T, Yang L (2015) In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm-dispersed cells via c-di-GMP manipulation. Nat Protoc 10(8):1165–1180

    Article  CAS  PubMed  Google Scholar 

  8. Decho AW, Frey RL, Ferry JL (2011) Chemical challenges to bacterial AHL signaling in the environment. Chem Rev 111:86–99

    Article  CAS  PubMed  Google Scholar 

  9. Domka J, Lee J, Bansal T, Wood TK (2007) Temporal gene expression in E. Coli K-12 biofilms. Environ Microbiol 9:332–346

    Article  CAS  PubMed  Google Scholar 

  10. Farrand SK, Qin Y, Oger P (2002) Quorum-sensing system of Agrobacterium plasmids: analysis and utility. Methods Enzymol 358:452–484

    Article  CAS  PubMed  Google Scholar 

  11. Flavier AB, Clough SJ, Schell MA, Deenny TP (1997) Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum. Mol Microbiol 26(2):251–259

    Article  CAS  PubMed  Google Scholar 

  12. Garcia-Aljaro C, Vargas-Cespedes GJ, Blanch AR (2011) Detection of acylated homoserine lactones produced by Vibrio spp. and related species isolated from water and aquatic organisms. J Appl Microbiol 112:383–389

    Article  PubMed  Google Scholar 

  13. Geisenberger O, Givskov M, Riedel K, Højby N, Tu¨mmler B (2000) Production of N-acyl-l-homoserine lactones by P. aeruginosa isolates isolates from chronic lung infections associated with cystic fibrosis. FEMS Microbiol Lett 184:273–278

    CAS  PubMed  Google Scholar 

  14. Genin S, Denny TP (2012) Pathogenomics of the Ralstonia solanacearum species complex. Annu Rev Phytopathol 50:67–89

    Article  CAS  PubMed  Google Scholar 

  15. Kelman A (1954) The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathol 44:693–695

    Google Scholar 

  16. Lemessa F, Zeller W (2007) Isolation and characterisation of Ralstonia solanacearum strains from Solanaceae crops in Ethiopia. J Basic Microbiol 47:40–49

    Article  CAS  PubMed  Google Scholar 

  17. Loehfelm TW, Luke NR, Campagnari AA (2008) Identification and characterization of an Acinetobacter baumannii biofilm-associated protein. J Bacteriol 190:1036–1044

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. McClean KH, Winson MK, Fish L, Taylor A, Chhabra SR (1997) Quorum sensing and Chromobacterium Õiolaceum: exploitation of violacein production and inhibition for the detection of N-acyl homoserine lactones. Microbiology 143:3703–3711

    Article  CAS  PubMed  Google Scholar 

  19. McLean RJC, Whiteley M, Stickler DJ, Fuqua WC (1997) Evidence of autoinducer activity in naturally occurring biofilms. FEMS Microbiol Lett 154:259–263

    Article  CAS  PubMed  Google Scholar 

  20. Morris CE, Monier JM (2003) The ecological significance of biofilm formation by plant-associated bacteria. Annu Rev Phytopathol 41:429–453

    Article  CAS  PubMed  Google Scholar 

  21. Opina N, Tavner F, Holloway G, Wang JF, Li TH (1997) A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum). Ass Pac J Mol Biol Biotechnol 5:19–33

    Google Scholar 

  22. Pinto UM, Viana ES, Martins ML, Vanetti MCD (2007) Detection of acylated homoserine lactones in gram-negative proteolytic psychrotrophic bacteria isolated from cooled raw milk. Food Control 18:1322–1327

    Article  CAS  Google Scholar 

  23. Plener L, Boistard P, Gonzalez A, Boucher C, Genin S (2012) Metabolic adaptation of Ralstonia solanacearum during plant infection: a methionine biosynthesis case study. PLoS One 5:e36877

    Article  Google Scholar 

  24. Ravn L, Christensen AB, Molin S, Givskov M, Gram L (2001) Methods for detecting acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics. J Microbiol Methods 44:239–251

    Article  CAS  PubMed  Google Scholar 

  25. Shaw PD, Ping G, Daly SL, Cha C, Cronan JE (1997) Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc Natl Acad Sci USA 94:6036–6041

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Valls M, Genin S, Boucher C (2006) Integrated regulation of the type III secretion system and other virulence determinants in Ralstonia solanacearum. PLoS Pathog 2:798–807

    Article  CAS  Google Scholar 

  27. Xu K, Li S, Yang W, Li K, Bai Y, Xu Y, Jin J, Wang Y, Bartlam M (2015) Structural and biochemical analysis of tyrosine phosphatase related to biofilm formation A (TpbA) from the opportunistic pathogen Pseudomonas aeruginosa PAO1. PLoS One 10(4):e0124330

    Article  PubMed Central  PubMed  Google Scholar 

  28. Zhang G, Zhang F, Ding G, Li J, Guo X (2012) Acyl homoserine lactone-based quorum sensing in a methanogenic archaeon. ISME J 6:1336–1344

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors thank The Chairman, Department of Studies in Biotechnology, University of Mysore, Karnataka, India for supporting this work. We acknowledge the recognition of University of Mysore as an Institution of Excellence by the Government of India with financial support from the Ministry of Human Resource Development and University Grants Commission, India. We also thank Dr. Vittorio Venturi from the Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy for the donation of strains C. violaceum CV026 and A. tumefaciens NT1 (pZLR4).

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Authors and Affiliations

  1. Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore, Karnataka, 570006, India

    J. Shiva Kumar & S. Umesha

  2. Research Faculty, Manipal Centre for Natural Sciences, Manipal University, Manipal, Udupi, Karnataka, 576 104, India

    K. Shiva Prasad

  3. Department of Studies in Biochemistry, Kuvempu University, Shankaraghatta, Shimoga, Karnataka, 577451, India

    P. Niranjana

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  1. J. Shiva Kumar
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Correspondence to S. Umesha.

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Kumar, J.S., Umesha, S., Prasad, K.S. et al. Detection of Quorum Sensing Molecules and Biofilm Formation in Ralstonia solanacearum . Curr Microbiol 72, 297–305 (2016). https://doi.org/10.1007/s00284-015-0953-0

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