Estudio de los mecanismos moleculares responsables de la adaptación del desarrollo de los bacteriófagos [phi]29 y Nf al estado fisiológico de la célula de "Bacillus subtilis" infectada

Abstract

Bacillus subtilis cells that are infected during the initial stages of sporulation with the lytic phage φ29 do not become lysed. Rather, under these conditions, the infected φ29 genome gets trapped into the spore thereby postponing its lytic life cycle upon germination of the spore. Thus, the φ29 infection strategy is adapted to the physiological conditions of the host. The molecular mechanisms underlying this alternative infection strategy have been studied in this thesis. The studies showed that the strategy depends on the presence of binding sites on the φ29 genome for the host-encoded proteins Spo0A (0A boxes) and Spo0J (parS sites). Thus, whereas the partition protein Spo0J is involved in spore entrapment of the infecting phage genome, Spo0A, the main transcriptional regulator for entry into sporulation, suppresses the lytic cycle. Interestingly, Spo0A suppresses the phage lytic cycle not only at the level of transcription, but also at the level of DNA replication. In addition, the role of Spo0A on phage φ29 life cycle has been studied all along B. subtilis cell development. Surprisingly, it was found that φ29 development is suppressed in a subpopulation of vegetative growing cells in a spo0A-dependent way. spo0A was known to be bistably expressed during the early stages of sporulation and our findings reveal that it is also expressed in a bistable manner during vegetative growth. Moreover, evidence was obtained that kinC and to a minor extent kinD are responsible for this heterogeneous expression levels of spo0A during vegetative growth whose maximum levels are below the threshold to activate sporulation, but sufficient for suppression of the lytic cycle of φ29. φ29 belongs to a family of phages that can be divided into three groups. Members of group 1 (e.g. φ29) and group 2 (e.g. Nf) infect B. subtilis. To determine if the alternative infection strategy is conserved in phages belonging to group 2, we studied phage Nf. The results obtained show that the lytic cycle of Nf is suppressed in a spo0A-dependent way but to a lesser extent than that observed for φ29. Sequence determination of the entire genome of Nf revealed that it contains only one perfect 0A box and two parS sites whereas the φ29 genome contains six perfect 0A boxes and five parS sites. By in vitro approaches we have shown that, contrary to φ29, Spo0A does not repress the early Nf C2 promoter and that the Nf A2b promoter is only slightly repressed at high Spo0A concentrations. In addition, contrary to φ29, Spo0A-mediated prevention of initiation of DNA replication is not observed in the Nf in vitro system. Therefore, the different adaptations of the life cycle of φ29 and Nf to the physiological state of the infected host are due to the differences in Spo0A-mediated regulation between these phages. Finally, we have shown that Spo0A also functions as a direct inhibitor of B. subtilis initiation of chromosome replication.