A question we were often asked is “”are there any special crab shells required for natural transformation to occur?”". To circumvent the problem of acquiring crab shells we tested commercially available chitin sources including chitosan, chitin

flakes and chitin powder. Except for chitosan we always got highly efficient natural transformation to occur. Our final goal was to make use of a standard minimal medium instead of the complex defined artificial seawater medium. To boost the transformation efficiency we tested Akt inhibitor M9 minimal medium supplemented with four different salts/components: NaCl, HEPES, MgSO4C and CaCl2. As illustrated in Fig. 5 we saw significant positive effects after addition of Mg2+ and/or Ca2+. Both of these cations were also shown to enhance natural transformation of A. calcoaceticus [19]. Conclusion We established an optimized procedure to genetically manipulate V. cholerae by chitin-induced natural competence (see Additional File 1 for a detailed protocol). The advantages of the new protocol are 1) its rapid feasibility (three days in total for the expedite version); 2) that PCR-derived donor DNA can Daporinad clinical trial be used given homologous flanking regions of at least 500 bp are present; 3) the chitin source is commercially available; 4) M9 minimal medium enriched for MgSO4 and CaCl2 can be utilized. Further

studies will demonstrate whether other Vibrio species are also amenable to this new procedure. Authors’ information RLM is a Master student at the Center for Systems Microbiology/Department of Systems

Biology of the Technical University of Denmark. He performed a summer internship in the Blokesch lab at EPFL, Lausanne, Switzerland. Acknowledgements We like to thank Olga de Souza Silva for excellent technical assistance. This work was supported by fellowships to RLM from the Otto Mønsteds Fond, the Frimodt-Heineke Fonden, the Rudolph Als Fondet and the Oticon Fonden. Electronic Bumetanide supplementary material Additional file 1: This file provides a detailed natural transformation protocol based on the results obtained in this study. (PDF 81 KB) References 1. Colwell RR: Global climate and infectious disease: the cholera paradigm. Science 1996,274(5295):2025–2031.PubMedCrossRef 2. Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Umayam L, Gill SR, Nelson KE, Read TD, Tettelin H, Richardson D, Ermolaeva MD, Vamathevan J, Bass S, Qin H, Dragoi I, Sellers P, McDonald L, Utterback T, Fleishmann RD, Nierman WC, White O: DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae . Nature 2000,406(6795):477–483.PubMedCrossRef 3. Dziejman M, Balon E, Boyd D, Fraser CM, Heidelberg JF, Mekalanos JJ: Comparative genomic analysis of Vibrio cholerae : genes that correlate with cholera endemic and pandemic disease. Proc Natl Acad Sci USA 2002,99(3):1556–1561.PubMedCrossRef 4.