Primer combinations used: (I) KanR-100-flank-up & KanR-100-flank-down; (II) KanR-250-flank-up & KanR-250-flank-down; see more (III) KanR-500-flank-up & KanR-500-flank-down; (IV) NheI-lacZ-start & LacZ-end-SalI; (V) Tfm-II-gDNA-1000 & Tfm-II-gDNA+1000; (VI) Tfm-II-gDNA-2000 & Tfm-II-gDNA+2000. Panel B: Plasmid pBR-lacZ-Kan-lacZ was used as PCR template and is shown in a linearized fashion. The following primer pairs were used: (V) Tfm-II-1000 & Tfm-II+1000; (VI) Tfm-II-2000

& Tfm-II+2000. Sizes of the up- and downstream flanking regions with respect to the Kanamycin resistance cassette are indicated in the middle. Blue shading: region homologous to recipient strain (A1552; wild-type); grey shading: heterologous region. Panel C: V. cholerae wild-type strain A1552 was naturally transformed using the crab-shell transformation protocol and PCR-derived DNA according to Panels A and B. Transformation frequencies

are shown on the Y-axis using either 2 ug gDNA of strain A1552-LacZ-Kan as positive control (lane 1; black) or 200 ng of PCR-derived Selleck OSI-027 DNA with varying length of the homologous (in blue; lane 2 to 7; according to Panel A) or homologous + heterologous (in grey; lane 8 and 9; according to Panel B) flanking region. Length of Kan R -flanking DNA: lane 2: 100 bp, lane 3: 250 bp; lane 4: 500 bp; lanes 5: ~1000 bp; lane 6 and 8: ~2000 bp; lane 7 and 9: ~3000 bp. Average of at least three independent experiments. Changing the source of chitin to simplify the natural transformation protocol To uniform the chitin substrate and make it available to researcher without access to crab shells we tested other forms of chitin or chitin-derivatives as inducer of natural competence (Fig. 4). Whereas chitosan, a deacetylated form of chitin, did not result in any detectable transformants (Fig. 4, lane 2), the other chitin sources (chitin flakes, lane 4; Sitaxentan chitin powder, lane 6) worked very well and resulted in comparable transformation frequencies as in the case of

crab-shells (Fig. 4, lane 8). Figure 4 Induction of natural competence by different chitin sources. Different chitin sources and chitin derivatives were tested for their ability to induce natural competence in V. cholerae A1552. Lanes 1 and 2: chitosan; lanes 3 and 4: chitin flakes; lanes 5 and 6: chitin powder; lanes 7 and 8: crab-shell fragments (approx. 1 cm2). The medium was not changed at the time of donor DNA (2 ug LacZ-Kan gDNA) addition for all odd lanes but for all even lanes. Average of four independent experiments. We also tested another variation from the standard transformation protocol using these different chitin sources (Fig. 4, lanes 1, 3, 5 and 7): after culturing the bacteria for 16 hours the surrounding medium was NOT exchanged; instead donor DNA was directly added (see Methods). This resulted in no Cilengitide solubility dmso difference in the case of chitin flakes and chitin powder as substrate (Fig. 4, lanes 3 and 5) in contrast to a 30-fold drop of transformation frequency using the crab shell protocol (Fig.