We show that these elements are responsible for the genomic instability of B. petrii observed during long term growth in vitro. Results

and discussion find more Long term survival of B. petrii in river water and appearance of phenotypic variants B. petrii was the first Bordetella species isolated from the environment, i.e. from a river sediment. The analysis of its survival capacity in river water revealed a high survival rate and nearly no decay in viability during a period of 38 weeks, while under the same experimental conditions viability of a B. bronchiseptica strain declined rapidly and no viable bacteria could be detected in the water samples after about three weeks (data not shown). The short survival time of B. bronchiseptica is somewhat surprising, since in a previous study it was shown to persist for more than 20 GDC-0994 weeks in lake water [18]. A possible explanation for this may be that different B. bronchiseptica strains were used in these studies. However, the direct comparison of B. bronchiseptica and B. petrii demonstrates that B. petrii has a much more pronounced capacity to survive in river water for a prolonged time period which is in agreement with its original isolation from river sediment. Interestingly, after about 20 days of the survival experiment stable phenotypic variants of B. petrii with differing colony morphology regarding colour

and colony size appeared when the bacteria were plated on LB agar plates (data not shown). In this study, three of these variants (named f, g, k) were further characterized. All of these variants showed virtually Adriamycin purchase identical growth characteristics at 37°C in liquid LB medium, while two of them (f, k) showed a markedly impaired growth capacity at 15°C as compared to the wild type strain and to variant g (data not shown). Genome rearrangements involving the genomic islands of B. petrii In a previous study we have reported about the spontaneous loss of a huge part comprising more than 500 Kb of the genome of B. petrii during in vitro

culture correlating with the presence of several genomic islands (GI1–GI3) [14]. To investigate whether the frequent appearance of phenotypic variants of B. petrii is in fact correlated with the various genomic islands, we started to characterize the three variants described above by pulsed ADAM7 field gel electrophoresis. Figure 2 shows that after BcuI digestion each of the three variants lack three large bands as compared to the wild type, but they have an identical restriction pattern among each other. To identify those regions of the variants lacking as compared to the wild type we performed hybridization studies with a B. petrii DNA-whole genome microarray. The results presented in Table 1 show that in all three variants the same genes are missing and that the deleted regions correspond to the clc-like elements GI1, GI3, and GI6 and to the island GI5.