Replicación inducida por estrés térmico en bacterias y plásmidos

Resumen

Genome duplication before cell division is an essential process in the cell cycle. "Escherichia coli" has a single circular chromosome that is precisely replicated once per cell cycle. The research group where this PhD Thesis has been developed has described and characterized the Heat Induced Replication, HIR, for the E. coli chromosome, with different requirements from those of the cyclic replication. Once HIR has been well defined in "E. coli" chromosome, in this PhD Thesis its study is broadened to other model bacterial species ("Salmonella tyhpimurium", "Bacillus subtilis" and "Vibrio cholera") and to "E. coli" plasmids with different replication control (minichromosomes, F, P1?incA, pSC101, R1, p15A and pBR322). The global aim of this work is to know the spreading of HIR mechanism in the bacterial world and determining its requirements in the different replicons. Another interesting point is the effect of extra-replications on other cell cycle parameters, both in E. coli and in other species. With the obtained results we conclude that heat-induced replication is not exclusive of "E. coli" chromosome and HIR can be considered as a stress mechanism widespread in the prokaryotic world. GFPmut2 measurement (fluorescence or gene dose) is an effective method for the study of HIR in plasmids; we propose spectrofluorometry and qPCR as simpler, quicker and more accurate methods than flow cytometry and Southern blot for plasmid copy number quantification.