How bacteria colonize surfaces and how they distinguish the individuals around them are fundamental biological questions. Type IV pili are a widespread and multipurpose class of cell surface polymers. Here we directly visualize the DNA-uptake pilus of Vibrio cholerae, which is produced specifically during growth on its natural habitat—chitinous surfaces. As predicted, these pili are highly dynamic and retract before DNA uptake during competence for natural transformation. Interestingly, DNA-uptake pili can also self-interact to mediate auto-aggregation. This capability is conserved in disease-causing pandemic strains, which typically encode the same major pilin subunit, PilA. Unexpectedly, however, we discovered that extensive strain-to-strain variability in PilA (present in environmental isolates) creates a set of highly specific interactions, enabling cells producing pili composed of different PilA subunits to distinguish between one another. We go on to show that DNA-uptake pili bind to chitinous surfaces and are required for chitin colonization under flow, and that pili capable of self-interaction connect cells on chitin within dense pili networks. Our results suggest a model whereby DNA-uptake pili function to promote inter-bacterial interactions during surface colonization. Moreover, they provide evidence that type IV pili could offer a simple and potentially widespread mechanism for bacterial kin recognition.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 25 October 2022
Protein nanowires with tunable functionality and programmable self-assembly using sequence-controlled synthesis
Nature Communications Open Access 11 February 2022
Nature Communications Open Access 08 October 2020
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data that support the findings of this study are available from the corresponding authors upon request.
Maier, B. & Wong, G. C. L. How bacteria use type IV pili machinery on surfaces. Trends Microbiol. 23, 775–788 (2015).
Berry, J. L. & Pelicic, V. Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives. FEMS Microbiol. Rev. 39, 134–154 (2015).
Giltner, C. L., Nguyen, Y. & Burrows, L. L. Type IV pilin proteins: versatile molecular modules. Microbiol. Mol. Biol. Rev. 76, 740–772 (2012).
Chang, Y. W. et al. Architecture of the type IVa pilus machine. Science 351, aad2001 (2016).
Craig, L. et al. Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol. Cell 23, 651–662 (2006).
Gold, V. A., Salzer, R., Averhoff, B. & Kuhlbrandt, W. Structure of a type IV pilus machinery in the open and closed state. eLife 4, e07380 (2015).
Kolappan, S. et al. Structure of the Neisseria meningitidis type IV pilus. Nat. Commun. 7, 13015 (2016).
Merz, A. J., So, M. & Sheetz, M. P. Pilus retraction powers bacterial twitching motility. Nature 407, 98–102 (2000).
Skerker, J. M. & Berg, H. C. Direct observation of extension and retraction of type IV pili. Proc. Natl Acad. Sci. USA 98, 6901–6904 (2001).
Jakovljevic, V., Leonardy, S., Hoppert, M. & Sogaard-Andersen, L. PilB and PilT are ATPases acting antagonistically in type IV pilus function in Myxococcus xanthus. J. Bacteriol. 190, 2411–2421 (2008).
McCallum, M., Tammam, S., Khan, A., Burrows, L. L. & Howell, P. L. The molecular mechanism of the type IVa pilus motors. Nat. Commun. 8, 15091 (2017).
Waldor, M. K. & Mekalanos, J. J. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272, 1910–1914 (1996).
Chiang, S. L., Taylor, R. K., Koomey, M. & Mekalanos, J. J. Single amino acid substitutions in the N-terminus of Vibrio cholerae TcpA affect colonization, autoagglutination, and serum resistance. Mol. Microbiol. 17, 1133–1142 (1995).
Taylor, R. K., Miller, V. L., Furlong, D. B. & Mekalanos, J. J. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. Natl Acad. Sci. USA 84, 2833–2837 (1987).
Kirn, T. J., Lafferty, M. J., Sandoe, C. M. & Taylor, R. K. Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae. Mol. Microbiol. 35, 896–910 (2000).
Chiavelli, D. A., Marsh, J. W. & Taylor, R. K. The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton. Appl. Environ. Microbiol. 67, 3220–3225 (2001).
Moorthy, S. & Watnick, P. I. Genetic evidence that the Vibrio cholerae monolayer is a distinct stage in biofilm development. Mol. Microbiol. 52, 573–587 (2004).
Utada, A. S. et al. Vibrio cholerae use pili and flagella synergistically to effect motility switching and conditional surface attachment. Nat. Commun. 5, 4913 (2014).
Watnick, P. I., Fullner, K. J. & Kolter, R. A role for the mannose-sensitive hemagglutinin in biofilm formation by Vibrio cholerae El Tor. J. Bacteriol. 181, 3606–3609 (1999).
Watnick, P. I. & Kolter, R. Steps in the development of a Vibrio cholerae El Tor biofilm. Mol. Microbiol. 34, 586–595 (1999).
Tamplin, M. L., Gauzens, A. L., Huq, A., Sack, D. A. & Colwell, R. R. Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters. Appl. Environ. Microbiol. 56, 1977–1980 (1990).
Blokesch, M. Competence-induced type VI secretion might foster intestinal colonization by Vibrio cholerae. Bioessays 37, 1163–1168 (2015).
Colwell, R. R. et al. Reduction of cholera in Bangladeshi villages by simple filtration. Proc. Natl Acad. Sci. USA 100, 1051–1055 (2003).
Meibom, K. L. et al. The Vibrio cholerae chitin utilization program. Proc. Natl Acad. Sci. USA 101, 2524–2529 (2004).
Meibom, K. L., Blokesch, M., Dolganov, N. A., Wu, C. Y. & Schoolnik, G. K. Chitin induces natural competence in Vibrio cholerae. Science 310, 1824–1827 (2005).
Johnston, C., Martin, B., Fichant, G., Polard, P. & Claverys, J. P. Bacterial transformation: distribution, shared mechanisms and divergent control. Nat. Rev. Microbiol. 12, 181–196 (2014).
Seitz, P. & Blokesch, M. DNA-uptake machinery of naturally competent Vibrio cholerae. Proc. Natl Acad. Sci. USA 110, 17987–17992 (2013).
Seitz, P. et al. ComEA is essential for the transfer of external DNA into the periplasm in naturally transformable Vibrio cholerae cells. PLoS Genet. 10, e1004066 (2014).
Seitz, P. & Blokesch, M. DNA transport across the outer and inner membranes of naturally transformable Vibrio cholerae is spatially but not temporally coupled. mBio 5, e01409–14 (2014).
Wolfgang, M. et al. PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol. Microbiol. 29, 321–330 (1998).
Gangel, H. et al. Concerted spatio-temporal dynamics of imported DNA and ComE DNA uptake protein during gonococcal transformation. PLoS Pathog. 10, e1004043 (2014).
Laurenceau, R. et al. A type IV pilus mediates DNA binding during natural transformation in Streptococcus pneumoniae. PLoS Pathog. 9, e1003473 (2013).
Ellison, C. K. et al. Obstruction of pilus retraction stimulates bacterial surface sensing. Science 358, 535–538 (2017).
Lo Scrudato, M. & Blokesch, M. The regulatory network of natural competence and transformation of Vibrio cholerae. PLoS Genet. 8, e1002778 (2012).
Jones, C. J. et al. C-di-GMP regulates motile to sessile transition by modulating MshA pili biogenesis and near-surface motility behavior in Vibrio cholerae. PLoS Pathog. 11, e1005068 (2015).
Fong, J. C., Syed, K. A., Klose, K. E. & Yildiz, F. H. Role of Vibrio polysaccharide (vps) genes in VPS production, biofilm formation and Vibrio cholerae pathogenesis. Microbiology 156, 2757–2769 (2010).
Biais, N., Ladoux, B., Higashi, D., So, M. & Sheetz, M. Cooperative retraction of bundled type IV pili enables nanonewton force generation. PLoS Biol. 6, e87 (2008).
Joelsson, A., Liu, Z. & Zhu, J. Genetic and phenotypic diversity of quorum-sensing systems in clinical and environmental isolates of Vibrio cholerae. Infect. Immun. 74, 1141–1147 (2006).
Aagesen, A. M. & Häse, C. C. Sequence analyses of type IV pili from Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. Microb. Ecol. 64, 509–524 (2012).
Aldova, E., Laznickova, K., Stepankova, E. & Lietava, J. Isolation of nonagglutinable vibrios from an enteritis outbreak in Czechoslovakia. J. Infect. Dis. 118, 25–31 (1968).
Chun, J. et al. Comparative genomics reveals mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae. Proc. Natl Acad. Sci. USA 106, 15442–15447 (2009).
Li, M., Shimada, T., Morris, J. G. Jr., Sulakvelidze, A. & Sozhamannan, S. Evidence for the emergence of non-O1 and non-O139 Vibrio cholerae strains with pathogenic potential by exchange of O-antigen biosynthesis regions. Infect. Immun. 70, 2441–2453 (2002).
DiRita, V. J., Neely, M., Taylor, R. K. & Bruss, P. M. Differential expression of the ToxR regulon in classical and El Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression. Proc. Natl Acad. Sci. USA 93, 7991–7995 (1996).
Jude, B. A. & Taylor, R. K. The physical basis of type 4 pilus-mediated microcolony formation by Vibrio cholerae O1. J. Struct. Biol. 175, 1–9 (2011).
Lim, M. S. et al. Vibrio cholerae El Tor TcpA crystal structure and mechanism for pilus-mediated microcolony formation. Mol. Microbiol. 77, 755–770 (2010).
Rhine, J. A. & Taylor, R. K. TcpA pilin sequences and colonization requirements for O1 and O139 Vibrio cholerae. Mol. Microbiol. 13, 1013–1020 (1994).
Ellison, C. K. et al. Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae. Nat. Microbiol. 3, 773–780 (2018).
Hélaine, S. et al. PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells. Mol. Microbiol. 55, 65–77 (2005).
Shime-Hattori, A. et al. Two type IV pili of Vibrio parahaemolyticus play different roles in biofilm formation. FEMS Microbiol. Lett. 264, 89–97 (2006).
Krebs, S. J. & Taylor, R. K. Protection and attachment of Vibrio cholerae mediated by the toxin-coregulated pilus in the infant mouse model. J. Bacteriol. 193, 5260–5270 (2011).
Hang, L. et al. Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae. Proc. Natl Acad. Sci. USA 100, 8508–8513 (2003).
Oldewurtel, E. R., Kouzel, N., Dewenter, L., Henseler, K. & Maier, B. Differential interaction forces govern bacterial sorting in early biofilms. eLife 4, e10811 (2015).
Chamot-Rooke, J. et al. Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination. Science 331, 778–782 (2011).
Smukalla, S. et al. FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast. Cell 135, 726–737 (2008).
Hirose, S., Benabentos, R., Ho, H. I., Kuspa, A. & Shaulsky, G. Self-recognition in social amoebae is mediated by allelic pairs of tiger genes. Science 333, 467–470 (2011).
Pathak, D. T., Wei, X., Dey, A. & Wall, D. Molecular recognition by a polymorphic cell surface receptor governs cooperative behaviors in bacteria. PLoS Genet. 9, e1003891 (2013).
Strassmann, J. E., Gilbert, O. M. & Queller, D. C. Kin discrimination and cooperation in microbes. Annu. Rev. Microbiol. 65, 349–367 (2011).
Wall, D. Kin recognition in bacteria. Annu. Rev. Microbiol. 70, 143–160 (2016).
Borgeaud, S., Metzger, L. C., Scrignari, T. & Blokesch, M. The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer. Science 347, 63–67 (2015).
Trunk, T., Khalil, H. S. & Leo, J. C. Bacterial autoaggregation. AIMS Microbiol. 4, 140–164 (2018).
Yildiz, F. H. & Schoolnik, G. K. Role of rpoS in stress survival and virulence of Vibrio cholerae. J. Bacteriol. 180, 773–784 (1998).
Matthey, N., Drebes Dörr, N. C. & Blokesch, M. Long-read-based genome sequences of pandemic and environmental Vibrio cholerae strains. Microbiol. Resour. Announc. 7, e01574-18 (2018).
Blokesch, M. A quorum sensing-mediated switch contributes to natural transformation of Vibrio cholerae. Mob. Genet. Elements 2, 224–227 (2012).
Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989).
De Souza Silva, O. & Blokesch, M. Genetic manipulation of Vibrio cholerae by combining natural transformation with FLP recombination. Plasmid 64, 186–195 (2010).
Marvig, R. L. & Blokesch, M. Natural transformation of Vibrio cholerae as a tool—optimizing the procedure. BMC Microbiol. 10, 155 (2010).
Blokesch, M. TransFLP—a method to genetically modify Vibrio cholerae based on natural transformation and FLP-recombination. J. Vis. Exp. 68, e3761 (2012).
Van der Henst, C. et al. Molecular insights into Vibrio cholerae’s intra-amoebal host-pathogen interactions. Nat. Commun. 9, 3460 (2018).
Gurung, I., Berry, J. L., Hall, A. M. J. & Pelicic, V. Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology. Nucleic Acids Res. 45, e40 (2017).
Bao, Y., Lies, D. P., Fu, H. & Roberts, G. P. An improved Tn7-based system for the single-copy insertion of cloned genes into chromosomes of Gram-negative bacteria. Gene 109, 167–168 (1991).
Yamamoto, S. et al. Regulation of natural competence by the orphan two-component system sensor kinase ChiS involves a non-canonical transmembrane regulator in Vibrio cholerae. Mol. Microbiol. 91, 326–347 (2014).
Dalia, A. B., Lazinski, D. W. & Camilli, A. Identification of a membrane-bound transcriptional regulator that links chitin and natural competence in Vibrio cholerae. mBio 5, e01028–01013 (2014).
Yamamoto, S. et al. Identification of a chitin-induced small RNA that regulates translation of the tfoX gene, encoding a positive regulator of natural competence in Vibrio cholerae. J. Bacteriol. 193, 1953–1965 (2011).
Lo Scrudato, M. & Blokesch, M. A transcriptional regulator linking quorum sensing and chitin induction to render Vibrio cholerae naturally transformable. Nucleic Acids Res. 41, 3644–3658 (2013).
Jaskólska, M., Stutzmann, S., Stoudmann, C. & Blokesch, M. QstR-dependent regulation of natural competence and type VI secretion in Vibrio cholerae. Nucleic Acids Res. 46, 10619–10634 (2018).
Blair, K. M., Turner, L., Winkelman, J. T., Berg, H. C. & Kearns, D. B. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science 320, 1636–1638 (2008).
Kearse, M. et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649 (2012).
Keymer, D. P., Miller, M. C., Schoolnik, G. K. & Boehm, A. B. Genomic and phenotypic diversity of coastal Vibrio cholerae strains is linked to environmental factors. Appl. Environ. Microbiol. 73, 3705–3714 (2007).
Purdy, A., Rohwer, F., Edwards, R., Azam, F. & Bartlett, D. H. A glimpse into the expanded genome content of Vibrio cholerae through identification of genes present in environmental strains. J. Bacteriol. 187, 2992–3001 (2005).
The authors thank members of the Blokesch laboratory for scientific discussions and I. Mateus-Gonzalez for assistance with bioinformatics analyses. The authors also thank A. Boehm, S. Pukatzki, J. Mekalanos, J. Reidl and members of the Institut National de Recherche Biomédicale of the Democratic Republic of the Congo for providing V. cholerae strains and V. Pelicic for advice on pheS-mediated counter-selection. Work on this issue was supported by a Marie Skłodowska-Curie Individual Fellowship (703340, CMDNAUP) to D.W.A. and by EPFL intramural funding and an ERC Starting (309064-VIR4ENV) and Consolidator (724630-CholeraIndex) Grant from the European Research Council to M.B. M.B. is a Howard Hughes Medical Institute (HHMI) International Research Scholar (grant no. 55008726).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Table 1, Supplementary References, Supplementary Figs. 1–21, Supplementary Video legends.
Details of the 647 V. cholerae genomes used for bioinformatic analyses.
DNA sequences of pilA and the deduced amino acid sequences of the proteins they encode from a collection of 22 environmental and clinical V. cholerae strains.
DNA-uptake pili exhibit rapid assembly dynamics.
Additional examples of dynamic DNA-uptake pili.
High time-resolution imaging of pilus extension and retraction.
Retraction of an unusually long pilus.
Pilus retraction followed by DNA-uptake.
Cells lacking pilT produce multiple static pili.
Additional examples of static pili in retraction deficient cells.
About this article
Cite this article
Adams, D.W., Stutzmann, S., Stoudmann, C. et al. DNA-uptake pili of Vibrio cholerae are required for chitin colonization and capable of kin recognition via sequence-specific self-interaction. Nat Microbiol 4, 1545–1557 (2019). https://doi.org/10.1038/s41564-019-0479-5
This article is cited by
Protein nanowires with tunable functionality and programmable self-assembly using sequence-controlled synthesis
Nature Communications (2022)
Nature Communications (2022)
Nature Reviews Microbiology (2020)
Nature Communications (2020)
Nature Reviews Microbiology (2019)