Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins

Abstract

The family Tectiviridae comprises a group of tailless, icosahedral, membranecontaining bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle.IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast twohybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria.KEYWORDS tectivirus, Bam35, yeast two-hybrid, intraviral interactome, ORFan, protein-protein interactions, PPIs

Role of plasmid plasticity and mobile genetic elements in the entomopathogen Bacillus thuringiensis serovar israelensis

“… 2013 ; Gillis and Mahillon 2014c ; Berjón-Otero et al. 2017 ) are colour-coded: rose, regulatory element; grey, unknown function; blue, replication; purple, DNA packaging and assembly; orange, capsid structural component; yellow, membrane structural component; green, lysis. Inverted terminal repeats (ITR) are highlighted by the red block arrows at both ends of the genome.…”

Bacteriophage GC1, a Novel Tectivirus Infecting Gluconobacter Cerinus, an Acetic Acid Bacterium Associated with Wine-Making

“…By contrast, members of the genus Betatectivirus include 6 closely related phages (67–99% nucleotide identity), represented by bacteriophage Bam35 and infecting gram-positive firmicutes of the genus Bacillus [ 25 , 26 ]. Despite close genetic relationship within each genus, members of one genus generally display no detectable sequence similarity to viruses from the other genus [ 27 , 28 ], suggesting an ancient divergence of the two tectivirus lineages. Furthermore, whereas all known alphatectiviruses are strictly lytic [ 29 ], betatectiviruses are temperate, persisting within their hosts as linear plasmids [ 30 , 31 , 32 ].…”

Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue

“…Of the 8 early genes involved in GIL01 regulation and replication (Figure 1A ), ORF1 and ORF6 are the only genes with predicted regulatory functions, as their products possess obvious DNA binding motifs ( 10 , 16 ). Since ORF1 has been linked to establishing and maintaining the lysogenic cycle ( 11 ), we examined the role of the ORF6 gene product, gp6, in the GIL01 lytic/lysogenic switch.…”

“…In doing this gp7 stabilized LexA binding to the promoter DNA, without binding the DNA itself, and prevented LexA auto-cleavage and premature phage reactivation. The internal lytic promoter P3 controls the expression of the GIL01 capsid and lytic genes ( 11 , 16 ). Like P1, P3 appears to be regulated by host LexA in vivo , with LexA potentially binding to two proposed sites, dinBox 2 and dinBox 3 (Figure 1A ) ( 11 , 12 ).…”