Disclosing bacillus virus BAM35 and its host. Identification and characterization of the viral SSB, host genomic characterization and phage-bacteria interactome
Título (trad.)Estudio de Bacillus virus Bam35 y su hospedador. Identificación y caracterización de la SSB viral, caracterización genómica del hospedador e interactoma fago-bacteria
EntidadUAM. Departamento de Biología Molecular; Centro de Biología Molecular Severo Ochoa (CBM)
Fecha de edición2020-12-15
MateriasBacterias gram-positivas; Virología; Bacteriófagos; Biología y Biomedicina / Biología
NotaTesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-12-2020
Esta tesis tiene embargado el acceso al texto completo hasta el 15-06-2022
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Bacillus virus Bam35 is the main model virus of the Betatectivirus genus, which belongs to the Tectiviridae family of tailless, icosahedral, emembrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years, because of its possible evolutionary relationship with various groups of prokaryotic and eukaryotic viruses. Also, betatectiviruses infect Bacillus cereus s. l. members, known by their pathogenicity and use in industry. Here, we address Bam35 biology from a broad perspective, from the particular characterization of its single-stranded DNA-binding protein (SSB) to the characterization of its host and the protein-protein interactions (PPIs) with it. The protein-primed DNA replication mechanism of Bam35 has been recently elucidated although no accessory proteins in this process have been described to date. In this work, we have identified and characterized the SSB of Bam35, B35SSB. We described B35SSB as a member of a novel group of SSBs with a conserved fold, highly divergent from the canonical OB-fold. This group mainly includes SSBs from distantly related phages with protein-primed replication. The biochemical characterization of B35SSB showed a high capacity to bind ssDNA although binding to dsDNA and RNA seems to be negligible. B35SSB is a monomer in solution that multimerizes along the substrate in the presence of ssDNA. Thus, B35SSB binds ssDNA in a non-distributive and sequence-independent manner with very high cooperativity, resulting in a high binding efficiency. We have also identified several aromatic and positively charged conserved residues essential for ssDNA-binding capacity, being aromatic residues essential for protein-DNA interaction. Moreover, B35SSB stimulates in vitro processive DNA synthesis, increasing the reaction yield without specificity for its cognate DNA polymerase. Several studies addressing Bam35 features have been performed. However, the Bacillus strains commonly used to study this phage have not been characterized. Thus, we have obtained the complete genome of B. thuringiensis HER1410, a known host strain for a variety of Bacillus phages and particularly useful for Bam35 studies. We have fully resolved the genome enabling the delineation of three plasmids: pLUSID1, a likely conjugative plasmid involved in virulence; pLUSID2, potentially related to sporulation, and a smaller plasmidial prophage pLUSID3, with a dual lifestyle whose integration within the chromosome causes the disruption of a key flagellar gene. We also detected a unique location of the single cry gene, cry1Ba4, found on a genomic island near the chromosomal replication origin. Phylogenetic analysis placed this strain next to B. thuringiensis sv. entomocidus and a within a clade comprising different B. thuringiensis and with B. cereus s. s. strains, in agreement with the intermingled taxonomy of the B. cereus group. Finally, we present the first wide analysis of protein-protein interactions for the Bam35-B. thuringiensis virushost model using a novel approach in which we combined the traditional yeast two-hybrid (Y2H) system and Illumina high-throughput sequencing technology. We generated and thoroughly analyzed a genomic library of Bam35’s host B. thuringiensis HER1410 and used it to screen interactions with all the viral proteins. A total of 158 screens have resulted in the detection of more than 4,000 putative interactions from which we obtained 183 highly-selective interactions that define a proposed virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched in the interactome, showing differing patterns to the known host-phages PPIs. Besides, we have also detected specific and novel host-viral PPIs which have allowed us to propose functions for several Bam35 unknown function proteins and achieve a better understanding of the Bam35-B. thuringiensis interaction
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Google Scholar:Lechuga Mateo, Ana
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