Symbiosis between the bobtail squid (Mollusca: Cephalopoda) and bacteria has been a well-studied model for understanding the molecular mechanisms of colonization and adherence to host cells. For example, pilin expression has been observed to cause subtle variation in colonization for a number of Gram-negative bacteria with eukaryotic hosts. To investigate variation amongst genes of closely related strains of vibrios, we amplified genes , , and to determine orientation and sequence similarity to other symbiotic vibrios. The gene was found to be upstream from all other genes, and not contiguous with the rest of the operon. The , and loci were flanked at the 3′ end by , followed by a conserved hypothetical gene. DNA sequences of each gene were aligned and analysed phylogenetically using parsimony for both individual and combined gene trees. Results demonstrate that certain loci ( and ) are conserved among strains of , but differs in sequence between symbiotic and free-living strains. Phylogenetic analysis of all genes gives better resolution of Indo-west Pacific symbionts compared with analysis of the 16S rRNA gene. Hawaiian and Australian symbiotic strains form one monophyletic tree, supporting the hypothesis that strain specificity is selected by the geographical location of their hosts and is not related to specific squid species.


Article metrics loading...

Loading full text...

Full text loading...



  1. Alm, R. A., Hallinan, J. P., Watson, A. A. & Mattick, J. S.(1996). Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein-secretion systems and pilY1 encodes a gonococcal PilC homologue. Mol Microbiol 22, 161–173.[CrossRef] [Google Scholar]
  2. Darzins, A. & Russell, M. A.(1997). Molecular genetic analysis of type-4 pilus biogenesis and twitching motility using Pseudomonas aeruginosa as a model system – a review. Gene 192, 109–115.[CrossRef] [Google Scholar]
  3. Erni, B. & Zanolari, B.(1985). The mannose-permease of the bacterial phosphotransferase system. Gene cloning and purification of the enzyme IIMan/IIIMan complex of Escherichia coli. J Biol Chem 260, 15495–15503. [Google Scholar]
  4. Farris, J. S., Albert, V. A., Källersjö, M., Lipscomb, D. & Kluge, A. G.(1996). Parsimony jackknifing outperforms neighbor-joining. Cladistics 12, 99–124.[CrossRef] [Google Scholar]
  5. Fullner, K. J. & Mekalanos, J. J.(1999). Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae. Infect Immun 67, 1393–1404. [Google Scholar]
  6. Goloboff, P. A.(1999). Analyzing large data sets in reasonable times: solutions for composite optima. Cladistics 15, 415–428.[CrossRef] [Google Scholar]
  7. Graf, J., Dunlap, P. V. & Ruby, E. G.(1994). Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri. J Bacteriol 176, 6986–6991. [Google Scholar]
  8. Graupner, S., Frey, V., Hashemi, R., Lorenz, M. G., Brandes, G. & Wackernagel, W.(2000). Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species. J Bacteriol 182, 2184–2190.[CrossRef] [Google Scholar]
  9. Guerrero-Ferreira, R. C. & Nishiguchi, M. K.(2007). Biodiversity among luminescent symbionts from squid of the genera Uroteuthis, Loliolus and Euprymna (Mollusca: Cephalopoda). Cladistics 23, 497–506.[CrossRef] [Google Scholar]
  10. Jones, B. & Nishiguchi, M.(2004). Counterillumination in the Hawaiian bobtail squid, Euprymna scolopes Berry (Mollusca: Cephalopoda). Mar Biol 144, 1151–1155.[CrossRef] [Google Scholar]
  11. Jones, B. W., Huttenberg, J., Lopez, J. E. & Nishiguchi, M. K.(2006). Population structure between environmentally transmitted vibrios and bobtail squids using nested clade analysis. Mol Ecol 15, 4317–4329.[CrossRef] [Google Scholar]
  12. McFall-Ngai, M. J.(1994). Animal-bacterial interactions in the early life history of marine invertebrates: the Euprymna scolopes/Vibrio fischeri symbiosis. Am Zool 34, 554–561. [Google Scholar]
  13. McFall-Ngai, M. J. & Ruby, E. G.(1998). Sepiolids and vibrios: when first they meet. Bioscience 48, 257–265.[CrossRef] [Google Scholar]
  14. Millikan, D. S. & Ruby, E. G.(2004).Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization. J Bacteriol 186, 4315–4325.[CrossRef] [Google Scholar]
  15. Nair, V. S.(2006).The evolution of symbiosis in Vibrionaceae and factors influencing host colonization in a squid-Vibrio mutualism. PhD dissertation, New Mexico State University, Las Cruces, NM, USA.
  16. Nishiguchi, M. K.(2000). Temperature affects species distribution in symbiotic populations of Vibrio spp. Appl Environ Microbiol 66, 3550–3555.[CrossRef] [Google Scholar]
  17. Nishiguchi, M. K.(2002). Host-symbiont recognition in the environmentally transmitted sepiolid squid-Vibrio mutualism. Microb Ecol 44, 10–18.[CrossRef] [Google Scholar]
  18. Nishiguchi, M. K. & Nair, V. S.(2003). Evolution of symbiosis in the Vibrionaceae: a combined approach using molecules and physiology. Int J Syst Evol Microbiol 53, 2019–2026.[CrossRef] [Google Scholar]
  19. Nishiguchi, M. K., Ruby, E. G. & McFall-Ngai, M. J.(1998). Competitive dominance among strains of luminous bacteria provides an unusual form of evidence for parallel evolution in sepiolid squid-Vibrio symbioses. Appl Environ Microbiol 64, 3209–3213. [Google Scholar]
  20. Nunn, D.(1999). Bacterial type II protein export and pilus biogenesis: more than just homologies? Trends Cell Biol 9, 402–408.[CrossRef] [Google Scholar]
  21. Nyholm, S. V. & McFall-Ngai, M. J.(2004). The winnowing: establishing the squid-Vibrio symbiosis. Nat Rev Microbiol 2, 632–642.[CrossRef] [Google Scholar]
  22. Pepe, J. C. & Lory, S.(1998). Amino acid substitutions in PilD, a bifunctional enzyme of Pseudomonas aeruginosa. Effect on leader peptidase and N-methyltransferase activities in vitro and in vivo. J Biol Chem 273, 19120–19129.[CrossRef] [Google Scholar]
  23. Ruby, E. G., Urbanowski, M., Campbell, J., Dunn, A., Faini, M., Gunsalus, R., Lostroh, P., Lupp, C., McCann, J. & other authors(2005). Complete genome sequence of Vibrio fischeri: a symbiotic bacterium with pathogenic congeners. Proc Natl Acad Sci U S A 102, 3004–3009.[CrossRef] [Google Scholar]
  24. Rudel, T., Boxberger, H. J. & Meyer, T. F.(1995a). Pilus biogenesis and epithelial cell adherence of Neisseria gonorrhoeaepilC double knock-out mutants. Mol Microbiol 17, 1057–1071.[CrossRef] [Google Scholar]
  25. Rudel, T., Facius, D., Barten, R., Scheuerpflug, I., Nonnenmacher, E. & Meyer, T. F.(1995b). Role of pili and the phase-variable PilC protein in natural competence for transformation of Neisseria gonorrhoeae. Proc Natl Acad Sci U S A 92, 7986–7990.[CrossRef] [Google Scholar]
  26. Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  27. Sandkvist, M.(2001). Biology of type II secretion. Mol Microbiol 40, 271–283.[CrossRef] [Google Scholar]
  28. Soto, G. E. & Hultgren, S. J.(1999). Bacterial adhesins: common themes and variations in architecture and assembly. J Bacteriol 181, 1059–1071. [Google Scholar]
  29. Stabb, E. V. & Ruby, E. G.(2003). Contribution of pilA to competitive colonization of the squid Euprymna scolopes by Vibrio fischeri. Appl Environ Microbiol 69, 820–826.[CrossRef] [Google Scholar]
  30. Swofford, D. L.(2002).paup*: Phylogenetic analysis using parsimony (and other methods), version 4. Sunderland, MA: Sinauer Associates.
  31. Thompson, F. L. & Swings, J.(2006). Taxonomy of the vibrios. In The Biology of Vibrios, pp. 29–43. Edited by F. L. Thompson, B. Austin & J. Swings. Washington, DC: American Society for Microbiology.
  32. Thompson, C. C., Thompson, F. L., Vandemeulebroecke, K., Hoste, B., Dawyndt, P. & Swings, J.(2004a). Use of recA as an alternative phylogenetic marker in the family Vibrionaceae. Int J Syst Evol Microbiol 54, 919–924.[CrossRef] [Google Scholar]
  33. Thompson, F. L., Iida, T. & Swings, J.(2004b). Biodiversity of vibrios. Microbiol Mol Biol Rev 68, 403–431.[CrossRef] [Google Scholar]
  34. Villar, M. T., Hirschberg, R. L. & Schaefer, M. R.(2001). Role of the Eikenella corrodenspilA locus in pilus function and phase variation. J Bacteriol 183, 55–62.[CrossRef] [Google Scholar]
  35. Visick, K. L. & Ruby, E. G.(2006).Vibrio fischeri and its host: it takes two to tango. Curr Opin Microbiol 9, 632–638.[CrossRef] [Google Scholar]
  36. Wheeler, W. C.(1995). Sequence alignment, parameter sensitivity, and the phylogenetic analysis of molecular data. Syst Biol 44, 321–331.[CrossRef] [Google Scholar]
  37. Wheeler, W.(1996). Optimization alignment: the end of multiple sequence alignment in phylogenetics? Cladistics 12, 1–9.[CrossRef] [Google Scholar]
  38. Wheeler, W. C., Gladstein, D. S. & DeLaet, J.(2002).poy: the optimization of alignment characters. New York: American Museum of Natural History.
  39. Winther-Larsen, H. C., Hegge, F. T., Wolfgang, M., Hayes, S. F., van Putten, J. P. & Koomey, M.(2001).Neisseria gonorrhoeae PilV, a type IV pilus-associated protein essential to human epithelial cell adherence. Proc Natl Acad Sci U S A 98, 15276–15281.[CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error