Charakterisierung der DNA-Replikation beider Chromosomen von Vibrio cholerae durch Methoden der Genomik und synthetischen Biologie

Vibrio cholerae is, due to the distribution of its genome on two chromosomes, a commonly used model organism for bacteria with multipartite genomes. The origin of replication of its second chromosome, ori2, provides an ideal basis for the development of a synthetic secondary chromosome in monochromosomal bacteria like E. coli. The regulation of initiation of DNA replication, especially of the secondary chromosome, and the coordination of replication of both chromosomes in V. cholerae is still not fully understood. This work should contribute to this understanding. The coordination of replication of Chr1 and Chr2 in V. cholerae is maintained by the chr2 replication triggering site crtS, as replication of the crtS induces initiation of DNA replication at ori2 (Val et al. 2016). The copy number of ori2-based replicons, like the synthetic secondary chromosome synVicII, is increased in E. coli after integration of the crtS into the main chromosome. This effect could be used to analyze the functionality of crtS of different V. cholerae strains. It was shown that the crtS is conserved among the Vibrionaceae. The crtS and ori2 of different Vibrionaceae are, with few exceptions, compatible in E. coli. All analyzed Vibrionaceae showed the order of replication ori1-crtS-ori2, known from V. cholerae, and synchronous termination of replication of both chromosomes. Additionally, a method for synchronization of DNA replication in V. cholerae using serine hydroxamate (SHX) was established. During the course of these experiments, the used strain V. cholerae A1552 was completely sequenced and annotated. Treatment of V. cholerae with SHX leads to induction of the stringent response (SR) (Haralalka et al. 2003). In contrast to E. coli, V. cholerae shows a defined program of cell division and re initiation of DNA replication during SR. This program is controlled by the SR-induced protein biosynthesis and the resulting cell growth and is therefore dependent on the production of the alarmone (p)ppGpp and its regulation of transcription. Additionally, the program is dependent on the concentration of SHX, which was shown experimentally as well as by a mathematical model.