Estudio funcional de las proteínas p1 y p17 del bacteriófago phi 29

Abstract

Bacillus subtilis bacteriophage φ29 is a well-studied virus used as a model organism to expand knowledge in aspects concerning to DNA replication and transcription. However, little is known about the in vivo organization of phage φ29 DNA replication and the proteins involved in this process. Phage φ29 proteins p1 and p17 are known to be required for DNA replication in vivo but their temporal and spatial organizations in infected cells have not yet been determined. Although the role of p17 in viral DNA replication in vivo has been partially elucidated, phage φ29 protein p1 function remained elusive. Protein p1 is a membrane-associated protein that forms large protofilament sheets that resemble eukaryotic tubulin and bacterial FtsZ polymers. In the absence of protein p1, phage φ29 DNA replication is impaired. In this Thesis, it is shown that a functional fusion of protein p1 to YFP localizes at the medial region of B. subtilis cells independently of other phage-encoded proteins. Moreover, protein p1 colocalizes with the B. subtilis cell division protein FtsZ and these proteins are associated in a complex. Importantly, the midcell localization of YFP-p1 was disrupted in a strain that does not express FtsZ, and the fluorescent signal was distributed all over the cell. Depletion of penicillin-binding protein 2B (PBP2B) in B. subtilis cells did not affect the subcellular localization of YFP-p1, indicating that its distribution does not depend on septal wall synthesis. Interestingly, when φ29 protein p1 was expressed, B. subtilis cells were about 1.5-fold longer than wild type cells, and the accumulation of φ29 DNA was higher in mutant B. subtilis cells with increased length. We propose that protein p1 promotes viral DNA replication by increasing the length of B. subtilis cells. By using a bacterial two hybrids system, we have shown that protein p1 associates with protein p17 in vivo. Apart from its role in DNA replication, protein p17 is required for the entrance of viral DNA into the cell during the injection process. In this Thesis, it has been observed that p17 localizes in a helical pattern along the whole B. subtilis cell in the absence of other viral components. Also, this localization does not require the cytoskeletal proteins MreB, Mbl or MreBH. The helix-like localization of p17 and its association with the viral protein p16.7, which also distributes in a helical manner, might reflect another role for protein p17, stabilizing p16.7 to redistribute viral genomes at the final stages of φ29 DNA replication.