2 ug (lane 1) and 200 ug (lane

4), respectively. Figure 1 The amount of donor DNA determines transformation frequencies. V. cholerae strains A1552 (WT; lanes 1-4) and A1552Δdns (5-8), respectively, were naturally transformed on crab shell fragments with increasing amounts of donor genomic DNA (gDNA) of strain A1552-LacZ-Kan. Amounts of donor gDNA provided: 0.2 μg (lanes 1 and 5), 2 μg (lanes 2 and 6), 20 μg (lanes 3 and 7) and 200 μg (lanes 4 and 8). Average of at least three independent experiments. Student’s t test: * statistically significant difference between lowest and highest amount of donor gDNA (p < 0.05); ** statistically PI3K Inhibitor Library significant difference between wild-type and nuclease minus strain (p < 0.01). The fact that higher amounts of donor DNA give rise to higher transformation frequencies can have two not mutually exclusive reasons: 1) The amount of DNA is at sub-saturation level and thus the more DNA is provided the more DNA is taken up and might get homologously recombined into the chromosome; 2) The

donor DNA might be degraded before uptake, e.g. outside of the bacteria. To follow up on the latter hypothesis we repeated the experiment using an extracellular nuclease minus strain Daporinad (A1552Δdns; [13]), which was shown to be hypertransformable [13]. Under these conditions we did not observe any statistically significant change in transformation frequency by adding increasing amounts of donor gDNA (Fig. 1, lanes 5 to 8). Thus, the amount of donor gDNA is saturating for this strain with respect to the transformation process itself. This allow us to conclude that in the case of the wild-type Flucloronide strain (Fig. 1, lanes 1 to 4) part of the donor DNA might be degraded before uptake, e.g. outside of the bacteria, so that excess of DNA helps to protect transforming DNA against degradation. PCR fragments can be used as donor DNA for natural transformation Moving genomic fragments, including GW-572016 concentration selective marker(s), from one

strain to another is certainly doable by this method. Nevertheless, to genetically manipulate new strains with the aid of PCR-derived constructs is more desirable. One possibility to do so is to amplify the flanking genomic regions, contemplated for an antibiotic marker insertion by PCR, as well as the antibiotic resistance cassette itself and combining them in a second round of PCR reaction. This has been done successfully resulting in the integration of a Kanamycin resistance cassette (aph) into the O37 antigen region of strain ATCC25873 by chitin-induced natural transformation [9]. In contrast to this, the study of Gulig et al. reported very low efficiency using PCR-derived donor DNA for V. vulnificus [11]. To follow up on this we PCR-amplified approximately 3700 bp of DNA comprising the Kanamycin resistance gene aminoglycoside 3′-phosphotransferase (aph) using plasmid pBR-lacZ-Kan-lacZ as template.