Unlimited cooperativity ofBetatectivirusSSB, a novel DNA binding protein related to an atypical group of SSBs from protein-primed replicating bacterial viruses

Abstract

Bam35 and related betatectiviruses are tail-less bacteriophages that prey on members of the Bacillus cereus group. These temperate viruses replicate their linear genome by a protein-primed mechanism. In this work, we have identified and characterized the product of the viral ORF2 as a single-stranded DNA binding protein (hereafter B35SSB). B35SSB binds ssDNA with great preference over dsDNA or RNA in a sequence-independent, highly cooperative manner that results in a non-specific stimulation of DNA replication. We have also identified several aromatic and basic residues, involved in base-stacking and electrostatic interactions, respectively, that are required for effective protein-ssDNA interaction. Although SSBs are essential for DNA replication in all domains of life as well as many viruses, they are very diverse proteins. However, most SSBs share a common structural domain, named OB-fold. Protein-primed viruses could constitute an exception, as no OB-fold DNA binding protein has been reported. Based on databases searches as well as phylogenetic and structural analyses, we showed that B35SSB belongs to a novel and independent group of SSBs. This group contains proteins encoded by protein-primed viral genomes from unrelated viruses, spanning betatectiviruses and Φ29 and close podoviruses, and they share a conserved pattern of secondary structure. Sensitive searches and structural predictions indicate that B35SSB contains a conserved domain resembling a divergent OB-fold, which would constitute the first occurrence of an OB-fold-like domain in a protein-primed genome.

Comparative Genomics of Prophages Sato and Sole Expands the Genetic Diversity Found in the Genus Betatectivirus

“…Recently it was shown that Bam35 SSB binds ssDNA in a highly cooperative, yet sequence-independent manner. Additionally, Bam35 SSB and the phage phi29-related SSB proteins, seem to be forming a novel clade of SSBs from protein-primed replicating viruses that share a highly divergent OB-fold-like domain [ 46 ]. Maximum likelihood phylogeny reconstruction among SSB proteins with OB-fold ( Figure S2b ) indicated two well separated clusters: one grouping betatectiviruses Sato and Sole with more classical OB-fold SSBs, including the previously mentioned SSBs from S. aureus, B. subtilis and H. pylori and, the second cluster containing other betatectiviruses (i.e., Bam35, GIL01, GIL16 and AP50) along with protein-primed replicating phages belonging to the subfamily Picovirinae (family Salasmaviridae ).…”

“…Inverted terminal repeats (ITR) are shown as red arrows at both ends of the genomes. The color key at the bottom-right indicates known and postulated gene functions [ 4 , 10 , 22 , 23 , 44 , 45 , 46 ]. Three genetic modules based of functional grouping, together with the highly variable region (HVR), are indicated above the genomes.…”

Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative Yeast two hybrid-High throughput sequencing approach

“…Contrary to structural viral proteins that are very interconnected through host proteins, those involved in ‘Gene regulation & genome replication’ interact independently. This is the case, for example, of the B35SSB (P2) [52] which remains outside of the network, interacting only with an Acetate coA transferase and a primosomal protein. In general, the obtained network shows a highly interconnected system where some viral proteins could act as hubs in the bacteria-virus interactome [53].…”