Genomics & Informatics

Korea Genome Organization (한국유전체학회)
권호 표지
DOI QR코드

DOI QR Code

Functional annotation of uncharacterized proteins from Fusobacterium nucleatum: identification of virulence factors

  • Kanchan Rauthan (Department of Biotechnology, H.N.B. Garhwal University) ;
  • Saranya Joshi (Department of Biotechnology, H.N.B. Garhwal University) ;
  • Lokesh Kumar (Department of Biotechnology, H.N.B. Garhwal University) ;
  • Divya Goel (Department of Biotechnology, H.N.B. Garhwal University) ;
  • Sudhir Kumar (Department of Biotechnology, H.N.B. Garhwal University)
  • Received : 2022.09.29
  • Accepted : 2023.05.23
  • Published : 2023.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Fusobacterium nucleatum is a gram-negative bacteria associated with diverse infections like appendicitis and colorectal cancer. It mainly attacks the epithelial cells in the oral cavity and throat of the infected individual. It has a single circular genome of 2.7 Mb. Many proteins in F. nucleatum genome are listed as "Uncharacterized." Annotation of these proteins is crucial for obtaining new facts about the pathogen and deciphering the gene regulation, functions, and pathways along with discovery of novel target proteins. In the light of new genomic information, an armoury of bioinformatic tools were used for predicting the physicochemical parameters, domain and motif search, pattern search, and localization of the uncharacterized proteins. The programs such as receiver operating characteristics determine the efficacy of the databases that have been employed for prediction of different parameters at 83.6%. Functions were successfully assigned to 46 uncharacterized proteins which included enzymes, transporter proteins, membrane proteins, binding proteins, etc. Apart from the function prediction, the proteins were also subjected to string analysis to reveal the interacting partners. The annotated proteins were also put through homology-based structure prediction and modeling using Swiss PDB and Phyre2 servers. Two probable virulent factors were also identified which could be investigated further for potential drug-related studies. The assigning of functions to uncharacterized proteins has shown that some of these proteins are important for cell survival inside the host and can act as effective drug targets.

Keywords

References

  1. Kumar A, Thotakura PL, Tiwary BK, Krishna R. Target identification in Fusobacterium nucleatum by subtractive genomics approach and enrichment analysis of host-pathogen protein-protein interactions. BMC Microbiol 2016;16:84.
  2. Han W, Li X, Fu X. The macro domain protein family: structure, functions, and their potential therapeutic implications. Mutat Res 2011;727:86-103. https://doi.org/10.1016/j.mrrev.2011.03.001
  3. Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M, et al. Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe 2013;14:207-215. https://doi.org/10.1016/j.chom.2013.07.007
  4. Kapatral V, Anderson I, Ivanova N, Reznik G, Los T, Lykidis A, et al. Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586. J Bacteriol 2002;184:2005-2018. https://doi.org/10.1128/JB.184.7.2005-2018.2002
  5. Nimrod G, Schushan M, Steinberg DM, Ben-Tal N. Detection of functionally important regions in "hypothetical proteins" of known structure. Structure 2008;16:1755-1763. https://doi.org/10.1016/j.str.2008.10.017
  6. Gazi MA, Mahmud S, Fahim SM, Islam MR, Das S, Mahfuz M, et al. Questing functions and structures of hypothetical proteins from Campylobacter jejuni: a computer-aided approach. Biosci Rep 2020;40:BSR20193939.
  7. Kaur H, Singh V, Kalia M, Mohan B, Taneja N. Identification and functional annotation of hypothetical proteins of uropathogenic Escherichia coli strain CFT073 towards designing antimicrobial drug targets. J Biomol Struct Dyn 2022;40:14084-14095. https://doi.org/10.1080/07391102.2021.2000499
  8. Mazumder L, Hasan M, Rus'd AA, Islam MA. In-silico characterization and structure-based functional annotation of a hypothetical protein from Campylobacter jejuni involved in propionate catabolism. Genomics Inform 2021;19:e43.
  9. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, et al. Protein identification and analysis tools on the ExPASy server. In: The Proteomics Protocols Handbook (Walker JM, ed.). Totowa, NJ: Humana Press, 2005. pp. 571-607.
  10. Yu CS, Lin CJ, Hwang JK. Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions. Protein Sci 2004;13:1402-1406. https://doi.org/10.1110/ps.03479604
  11. Almagro Armenteros JJ, Tsirigos KD, Sonderby CK, Petersen TN, Winther O, Brunak S, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol 2019;37:420-423. https://doi.org/10.1038/s41587-019-0036-z
  12. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001;305:567-580. https://doi.org/10.1006/jmbi.2000.4315
  13. Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 2014;30:1236-1240. https://doi.org/10.1093/bioinformatics/btu031
  14. Letunic I, Khedkar S, Bork P. SMART: recent updates, new developments and status in 2020. Nucleic Acids Res 2021;49: D458-D460. https://doi.org/10.1093/nar/gkaa937
  15. Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res 2011;39: W29-W37. https://doi.org/10.1093/nar/gkr367
  16. Geer LY, Domrachev M, Lipman DJ, Bryant SH. CDART: protein homology by domain architecture. Genome Res 2002;12:1619-1623. https://doi.org/10.1101/gr.278202
  17. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
  18. Eng J. ROC analysis: web-based calculator for ROC curves. Baltimore: Johns Hopkins Medicine, 2014. Accessed 2022 Sep 29. Available from: http://www.jrocfit.org.
  19. Szklarczyk D, Gable AL, Nastou KC, Lyon D, Kirsch R, Pyysalo S, et al. The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res 2021;49:D605-D612. https://doi.org/10.1093/nar/gkaa1074
  20. Wishart DS, Feunang YD, Guo AC, Lo EJ, Marcu A, Grant JR, et al. DrugBank 5.0: a major update to the DrugBank database for 2018. Nucleic Acids Res 2018;46:D1074-D1082. https://doi.org/10.1093/nar/gkx1037
  21. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 2018;46:W296-W303. https://doi.org/10.1093/nar/gky427
  22. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015;10:845-858. https://doi.org/10.1038/nprot.2015.053
  23. Laskowski RA, Hutchinson EG, Michie AD, Wallace AC, Jones ML, Thornton JM. PDBsum: a Web-based database of summaries and analyses of all PDB structures. Trends Biochem Sci 1997;22:488-490. https://doi.org/10.1016/S0968-0004(97)01140-7
  24. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 1996;8:477-486. https://doi.org/10.1007/BF00228148
  25. Saha S, Raghava GP. VICMpred: an SVM-based method for the prediction of functional proteins of Gram-negative bacteria using amino acid patterns and composition. Genomics Proteomics Bioinformatics 2006;4:42-47. https://doi.org/10.1016/S1672-0229(06)60015-6
  26. Garg A, Gupta D. VirulentPred: a SVM based prediction method for virulent proteins in bacterial pathogens. BMC Bioinformatics 2008;9:62.
  27. Nielsen H. Predicting Secretory Proteins with SignalP. Methods Mol Biol 2017;1611:59-73. https://doi.org/10.1007/978-1-4939-7015-5_6
  28. Rollauer SE, Sooreshjani MA, Noinaj N, Buchanan SK. Outer membrane protein biogenesis in Gram-negative bacteria. Philos Trans R Soc Lond B Biol Sci 2015;370.
  29. Parveen N, Cornell KA. Methylthioadenosine/S-adenosylhomocysteine nucleosidase, a critical enzyme for bacterial metabolism. Mol Microbiol 2011;79:7-20. https://doi.org/10.1111/j.1365-2958.2010.07455.x
  30. Kim J, Hetzel M, Boiangiu CD, Buckel W. Dehydration of (R)-2-hydroxyacyl-CoA to enoyl-CoA in the fermentation of alpha-amino acids by anaerobic bacteria. FEMS Microbiol Rev 2004;28:455-468. https://doi.org/10.1016/j.femsre.2004.03.001
  31. Tanaka S, Maeda Y, Tashima Y, Kinoshita T. Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p. J Biol Chem 2004;279:14256-14263. https://doi.org/10.1074/jbc.M313755200
  32. Aliashkevich A, Cava F. LD-transpeptidases: the great unknown among the peptidoglycan cross-linkers. FEBS J 2022;289:4718-4730. https://doi.org/10.1111/febs.16066
  33. Metzger LE 4th, Lee JK, Finer-Moore JS, Raetz CR, Stroud RM. LpxI structures reveal how a lipid A precursor is synthesized. Nat Struct Mol Biol 2012;19:1132-1138. https://doi.org/10.1038/nsmb.2393
  34. Metzger LE 4th, Raetz CR. An alternative route for UDP-diacylglucosamine hydrolysis in bacterial lipid A biosynthesis. Biochemistry 2010;49:6715-6726. https://doi.org/10.1021/bi1008744
  35. Santa Maria J, Vallance P, Charles IG, Leiper JM. Identification of microbial dimethylarginine dimethylaminohydrolase enzymes. Mol Microbiol 1999;33:1278-1279. https://doi.org/10.1046/j.1365-2958.1999.01580.x
  36. Xiong L, Teng JL, Botelho MG, Lo RC, Lau SK, Woo PC. Arginine metabolism in bacterial pathogenesis and cancer therapy. Int J Mol Sci 2016;17:363.
  37. Whiteman PA, Abraham EP, Baldwin JE, Fleming MD, Schofield CJ, Sutherland JD, et al. Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans. FEBS Lett 1990;262:342-344. https://doi.org/10.1016/0014-5793(90)80224-7
  38. Pingoud A, Jeltsch A. Structure and function of type II restriction endonucleases. Nucleic Acids Res 2001;29:3705-3727. https://doi.org/10.1093/nar/29.18.3705
  39. Diethmaier C, Newman JA, Kovacs AT, Kaever V, Herzberg C, Rodrigues C, et al. The YmdB phosphodiesterase is a global regulator of late adaptive responses in Bacillus subtilis. J Bacteriol 2014;196:265-275. https://doi.org/10.1128/JB.00826-13
  40. Abeyrathne PD, Daniels C, Poon KK, Matewish MJ, Lam JS. Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide. J Bacteriol 2005;187:3002-3012. https://doi.org/10.1128/JB.187.9.3002-3012.2005
  41. Mielecki D, Grzesiuk E. Ada response: a strategy for repair of alkylated DNA in bacteria. FEMS Microbiol Lett 2014;355:1-11. https://doi.org/10.1111/1574-6968.12462
  42. Steczkiewicz K, Muszewska A, Knizewski L, Rychlewski L, Ginalski K. Sequence, structure and functional diversity of PD-(D/E) XK phosphodiesterase superfamily. Nucleic Acids Res 2012;40:7016-7045. https://doi.org/10.1093/nar/gks382
  43. Xu Q, Rawlings ND, Chiu HJ, Jaroszewski L, Klock HE, Knuth MW, et al. Structural analysis of papain-like NlpC/P60 superfamily enzymes with a circularly permuted topology reveals potential lipid binding sites. PLoS One 2011;6:e22013.
  44. Sancho J. Flavodoxins: sequence, folding, binding, function and beyond. Cell Mol Life Sci 2006;63:855-864. https://doi.org/10.1007/s00018-005-5514-4
  45. Salillas S, Sancho J. Flavodoxins as novel therapeutic targets against Helicobacter pylori and other gastric pathogens. Int J Mol Sci 2020;21:1881.
  46. Zeller T, Klug G. Thioredoxins in bacteria: functions in oxidative stress response and regulation of thioredoxin genes. Naturwissenschaften 2006;93:259-266. https://doi.org/10.1007/s00114-006-0106-1
  47. Wall EA, Johnson AL, Peterson DL, Christie GE. Structural modeling and functional analysis of the essential ribosomal processing protease Prp from Staphylococcus aureus. Mol Microbiol 2017; 104:520-532. https://doi.org/10.1111/mmi.13644
  48. Hama H, Kayahara T, Ogawa W, Tsuda M, Tsuchiya T. Enhancement of serine-sensitivity by a gene encoding rhodanese-like protein in Escherichia coli. J Biochem 1994;115:1135-1140. https://doi.org/10.1093/oxfordjournals.jbchem.a124469
  49. Haft DH, Selengut J, Mongodin EF, Nelson KE. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol 2005;1:e60.
  50. Boggild A, Sofos N, Andersen KR, Feddersen A, Easter AD, Passmore LA, et al. The crystal structure of the intact E. coli RelBE toxin-antitoxin complex provides the structural basis for conditional cooperativity. Structure 2012;20:1641-1648. https://doi.org/10.1016/j.str.2012.08.017
  51. Jiang Y, Pogliano J, Helinski DR, Konieczny I. ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase. Mol Microbiol 2002;44:971-979. https://doi.org/10.1046/j.1365-2958.2002.02921.x
  52. Oberer M, Zangger K, Gruber K, Keller W. The solution structure of ParD, the antidote of the ParDE toxin antitoxin module, provides the structural basis for DNA and toxin binding. Protein Sci 2007;16:1676-1688. https://doi.org/10.1110/ps.062680707
  53. Marie L, Rapisarda C, Morales V, Berge M, Perry T, Soulet AL, et al. Bacterial RadA is a DnaB-type helicase interacting with RecA to promote bidirectional D-loop extension. Nat Commun 2017;8:15638.
  54. Rudolph CJ, Upton AL, Briggs GS, Lloyd RG. Is RecG a general guardian of the bacterial genome? DNA Repair (Amst) 2010;9: 210-223. https://doi.org/10.1016/j.dnarep.2009.12.014
  55. Ryazansky S, Kulbachinskiy A, Aravin AA. The expanded universe of prokaryotic argonaute proteins. mBio 2018;9:e01935-18.
  56. Schwartz CJ, Giel JL, Patschkowski T, Luther C, Ruzicka FJ, Beinert H, et al. IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins. Proc Natl Acad Sci U S A 2001;98:14895-14900. https://doi.org/10.1073/pnas.251550898
  57. Missiakas D, Mayer MP, Lemaire M, Georgopoulos C, Raina S. Modulation of the Escherichia coli sigmaE (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins. Mol Microbiol 1997;24:355-371. https://doi.org/10.1046/j.1365-2958.1997.3601713.x
  58. Villanelo F, Ordenes A, Brunet J, Lagos R, Monasterio O. A model for the Escherichia coli FtsB/FtsL/FtsQ cell division complex. BMC Struct Biol 2011;11:28.
  59. Hudson AM, Cooley L. Phylogenetic, structural and functional relationships between WD- and Kelch-repeat proteins. Subcell Biochem 2008;48:6-19.
  60. Caruthers JM, McKay DB. Helicase structure and mechanism. Curr Opin Struct Biol 2002;12:123-133. https://doi.org/10.1016/S0959-440X(02)00298-1
  61. Zhang X, Carter MS, Vetting MW, San Francisco B, Zhao S, Al-Obaidi NF, et al. Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars. Proc Natl Acad Sci U S A 2016;113:E4161-E4169. https://doi.org/10.1073/pnas.1605546113
  62. Cerveny L, Straskova A, Dankova V, Hartlova A, Ceckova M, Staud F, et al. Tetratricopeptide repeat motifs in the world of bacterial pathogens: role in virulence mechanisms. Infect Immun 2013;81:629-635. https://doi.org/10.1128/IAI.01035-12
  63. Ghafoor A, Hay ID, Rehm BH. Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture. Appl Environ Microbiol 2011;77:5238-5246. https://doi.org/10.1128/AEM.00637-11
  64. Andrade MA, Perez-Iratxeta C, Ponting CP. Protein repeats: structures, functions, and evolution. J Struct Biol 2001;134:117-131. https://doi.org/10.1006/jsbi.2001.4392
  65. Rodionov DA, Hebbeln P, Gelfand MS, Eitinger T. Comparative and functional genomic analysis of prokaryotic nickel and cobalt uptake transporters: evidence for a novel group of ATP-binding cassette transporters. J Bacteriol 2006;188:317-327. https://doi.org/10.1128/JB.188.1.317-327.2006
  66. Kim S, Jeon TJ, Oberai A, Yang D, Schmidt JJ, Bowie JU. Transmembrane glycine zippers: physiological and pathological roles in membrane proteins. Proc Natl Acad Sci U S A 2005;102:14278-14283. https://doi.org/10.1073/pnas.0501234102
  67. Ahn VE, Lo EI, Engel CK, Chen L, Hwang PM, Kay LE, et al. A hydrocarbon ruler measures palmitate in the enzymatic acylation of endotoxin. EMBO J 2004;23:2931-2941. https://doi.org/10.1038/sj.emboj.7600320
  68. Deng YM, Liu CQ, Dunn NW. Genetic organization and functional analysis of a novel phage abortive infection system, AbiL, from Lactococcus lactis. J Biotechnol 1999;67:135-149. https://doi.org/10.1016/S0168-1656(98)00175-8
  69. Labrie SJ, Samson JE, Moineau S. Bacteriophage resistance mechanisms. Nat Rev Microbiol 2010;8:317-327. https://doi.org/10.1038/nrmicro2315
  70. Gutierrez JA, Crowley PJ, Cvitkovitch DG, Brady LJ, Hamilton IR, Hillman JD, et al. Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress. Microbiology (Reading) 1999;145(Pt 2):357-366. https://doi.org/10.1099/13500872-145-2-357
  71. Kremer BH, van der Kraan M, Crowley PJ, Hamilton IR, Brady LJ, Bleiweis AS. Characterization of the sat operon in Streptococcus mutans: evidence for a role of Ffh in acid tolerance. J Bacteriol 2001;183:2543-2552. https://doi.org/10.1128/JB.183.8.2543-2552.2001
  72. Fardini Y, Wang X, Temoin S, Nithianantham S, Lee D, Shoham M, et al. Fusobacterium nucleatum adhesin FadA binds vascular endothelial cadherin and alters endothelial integrity. Mol Microbiol 2011;82:1468-1480. https://doi.org/10.1111/j.1365-2958.2011.07905.x
  73. Gerard F, Pradel N, Wu LF. Bactericidal activity of colicin V is mediated by an inner membrane protein, SdaC, of Escherichia coli. J Bacteriol 2005;187:1945-1950. https://doi.org/10.1128/JB.187.6.1945-1950.2005
  74. McCallum M, Tammam S, Little DJ, Robinson H, Koo J, Shah M, et al. PilN binding modulates the structure and binding partners of the Pseudomonas aeruginosa type IVa pilus protein PilM. J Biol Chem 2016;291:11003-11015. https://doi.org/10.1074/jbc.M116.718353
  75. Jankevicius G, Hassler M, Golia B, Rybin V, Zacharias M, Timinszky G, et al. A family of macrodomain proteins reverses cellular mono-ADP-ribosylation. Nat Struct Mol Biol 2013;20:508-514. https://doi.org/10.1038/nsmb.2523
  76. Zhuang N, Zhang H, Li L, Wu X, Yang C, Zhang Y. Crystal structures and biochemical analyses of the bacterial arginine dihydrolase ArgZ suggests a "bond rotation" catalytic mechanism. J Biol Chem 2020;295:2113-2124. https://doi.org/10.1074/jbc.RA119.011752
  77. Kachalova GS, Rogulin EA, Yunusova AK, Artyukh RI, Perevyazova TA, Matvienko NI, et al. Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit. J Mol Biol 2008;384:489-502. https://doi.org/10.1016/j.jmb.2008.09.033
  78. Hou HF, Gao ZQ, Li LF, Liang YH, Su XD, Dong YH. Crystal structure of SMU.848 from Streptococcus mutans. Protein Data Bank. Accessed 2023 Jan 1. Bethesda: National Cancer Institute, 2006. Available from: https://www.rcsb.org/ structure/2g0j.
  79. Peixeiro N, Keller J, Collinet B, Leulliot N, Campanacci V, Cortez D, et al. Structure and function of AvtR, a novel transcriptional regulator from a hyperthermophilic archaeal lipothrixvirus. J Virol 2013;87:124-136. https://doi.org/10.1128/JVI.01306-12
  80. Zhou J, Du XJ, Liu Y, Gao ZQ, Geng Z, Dong YH, et al. Insights into the neutralization and DNA binding of toxin-antitoxin system ParE(SO)-CopA(SO) by structure-function studies. Microorganisms 2021;9:2506.
  81. Freiberg JA, Le Breton Y, Harro JM, Allison DL, McIver KS, Shirtliff ME. The arginine deiminase pathway impacts antibiotic tolerance during biofilm-mediated Streptococcus pyogenes infections. mBio 2020;11:e00919-20. https://doi.org/10.1128/mBio.00919-20
  82. Casiano-Colon A, Marquis RE. Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance. Appl Environ Microbiol 1988;54:1318-1324. https://doi.org/10.1128/aem.54.6.1318-1324.1988
  83. Karkowska-Kuleta J, Bartnicka D, Zawrotniak M, Zielinska G, Kieronska A, Bochenska O, et al. The activity of bacterial peptidylarginine deiminase is important during formation of dual-species biofilm by periodontal pathogen Porphyromonas gingivalis and opportunistic fungus Candida albicans. Pathog Dis 2018;76:fty033.
  84. Wissenbach U, Six S, Bongaerts J, Ternes D, Steinwachs S, Unden G. A third periplasmic transport system for L-arginine in Escherichia coli: molecular characterization of the artPIQMJ genes, arginine binding and transport. Mol Microbiol 1995;17:675-686. https://doi.org/10.1111/j.1365-2958.1995.mmi_17040675.x
  85. Sansone C, Van Houte J, Joshipura K, Kent R, Margolis HC. The association of mutans streptococci and non-mutans streptococci capable of acidogenesis at a low pH with dental caries on enamel and root surfaces. J Dent Res 1993;72:508-516. https://doi.org/10.1177/00220345930720020701
  86. Li YH, Tian X. Quorum sensing and bacterial social interactions in biofilms. Sensors (Basel) 2012;12:2519-2538. https://doi.org/10.3390/s120302519