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

Bacillus virus Bam35 is the model Betatectivirus and member of the Tectiviridae family, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions network for a tectivirus-host system by studying the Bam35-Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and Illumina high-throughput sequencing. We generated and thoroughly analyzed a genomic library of Bam35’s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. In total, this screen resulted in the detection of over 4,000 potential interactions, of which 183 high-confidence interactions were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage protein-protein interaction networks (PPIs). Our approach also suggests biological roles for several Bam35 proteins of unknown function, resulting in a better understanding of the Bam35-B. thuringiensis interaction at the molecular level. Author summary Members of the family Tectiviridae, composed of non-tailed icosahedral, membrane-containing bacteriophages, have been increasingly scrutinized in recent years for their possible role in the origin of dsDNA viruses. In particular, the genus Betatectivirus receives increased attention as these phages can infect clinical strains as well as industrially relevant members of the B. cereus group. However, little is known about the interactions between these temperate viruses and their hosts. Here, we present the first high-throughput study of tectivirus-host protein-protein interactions focusing on Bam35, model virus of betatectiviruses, and its host B. thuringiensis, an important entomopathogenic bacterium. We adapted the well-known technique yeast-two-hybrid and integrated high-throughput sequencing and bioinformatics for the downstream analysis of the results which enables large-scale analysis of protein-protein interactions. In total, 182 detected interactions show an enrichment in host metabolic proteins and peptidases, in contrast with the current knowledge on host-phage PPIs. Specific host-viral protein-protein interactions were also detected enabling us to propose functions for uncharacterized proteins.


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results which enables large-scale analysis of protein-protein interactions. The Tectiviridae family is defined as a family of tailless, icosahedral viruses with a lipidic inner 121 last decade has seen the emergence of "omics" approaches as investigative tools for the study 122 of biological pathways involved in pathogen replication, host response, and, eventually, infection 123 progression. Among the methods used in high-throughput interactomics, the yeast two-hybrid 124 system (Y2H) remains one of the most widely used techniques to study PPIs [35]. Some of the 125 first Y2H studies involving viruses addressed the interaction between bacteria and their phages. 136 The recent combination of Y2H with high-throughput sequencing technologies (HTS) 137 overcomes the labor-intensive clone-by-clone analysis and has been shown to speed up the study 138 of PPIs, while increasing the efficiency and sensitivity of the method. Different approaches have 139 been followed such as recombination-based methods [45][46][47] or methods based on the screen 140 of a genomic library against one single protein [48,49]. Although these techniques represent a 141 marked improvement of the method, the data analysis and interpretation remain a challenge.

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Indeed, the large amounts of data generated with these approaches require the development of 143 specific bioinformatics pipelines, and a fine-tuning of thresholds to disclose reliable interactions. Integrating the yeast two-hybrid system with high-throughput sequencing 157 To obtain an extensive protein-protein interactome of Bam35 and B. thuringiensis through the 158 development of a novel and customizable approach, we established an experimental setup that 159 combines traditional Y2H with HTS. We used the previously generated Bam35 ORF collection  Table 1). In this 183 case, the difference in the number of represented genes by each library, lower for pPC, is mainly 184 due to the differential final filtering step, which is more stringent for this combination.  of selection when using the HIS3 reporter gene, the autoactivation of the bait proteins was 215 avoided by adding the optimal concentration of the inhibitor 3-amino-1,2,4-triazole (3AT) for each 216 bait construct. Besides, the reduction of cell background and, therefore, the efficient growth and 217 selection of positive colonies was achieved by two steps of replica plating ( Figure 3A). As can be 218 observed in Figure 3A, the screen led to a wide variety of results in terms of color and number of 219 colonies. The assays that included the pPN-Library and were plated on selective media with low 220 3AT concentration (0-0.1 mM) resulted in a pink lawn ( Figure 3A, lane 4). This could correspond 221 to a false positive background caused by the pPN-Library which prevents the recovery of positive 222 colonies. For these cases, the increase of 3AT concentration up to 3 mM effectively avoided the 223 pink lawn, allowing the growth of hidden interacting partners (lane 5). In addition, based on the 224 MEL1 reporter selection system, 5-Bromo-4-chloro-3-indolyl α-D-galactopyranoside (X-α-Gal) 225 was added to identify and amplify reliable interactions by increasing the 3AT concentration in the 226 plates containing mostly white colonies (weak interaction). However, the plates meeting this 227 requirement already contained the highest concentration of 3AT. To identify the prey interaction 228 partners in each assay, cells from the last replica plate (Y2H positive clones) were pool-harvested 229 and the prey fragments were amplified by PCR for subsequent Illumina sequencing ( Figure 3B).

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The PCR products of each assay showed distinct patterns of discrete bands or a smear which 231 corresponds to different sizes within the library size range. Nine combinations showed few or no 232 colonies and, consequently, no PCR product was generated (Supplementary Table 2).   Table 2). These reads were 251 filtered using the bioinformatics pipeline illustrated in Figure 1. After data treatment, mapping of 252 the Y2H-validated fragments allowed us to retrieve a total of 4,477 possible interactions (  346 Table 4. Putative PPIs between Bam35 (B35) proteins and B. thuringiensis (Bt) functional groups a .

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The use of HTS to identify all the possible prey partners involved in the Y2H screening 454 also reduces the false-negative rate to the minimum that Y2H can attain. We also used several 455 methods aiming to maximally reduce false-positive interactions such as the competitive inhibitor 3AT which prevents bait self-activation, the addition of negative controls including the empty 457 plasmids, and the identification and removal of "sticky" preys. Remarkably, a large percentage 458 (97%) of the prey proteins found in the empty combinations were also tagged as "sticky".  Figure 6). Strikingly, although a higher frequency in protein processing and gene 498 regulation has been associated for phage lambda with its lysogenic state, these categories are 499 not specifically represented for Bam35 and Giles phages, which are also temperate.

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One of the most relevant interactions detected with our approach is the described direct 586 interaction between the viral P7 and the host LexA, key for maintaining lysogeny [19,28]. Here, 587 the LexA fragment comprises the second half of the protein (residues 121 to 206) suggesting that 588 the interaction domain is located in this region and it is sufficient to establish the interaction.

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Importantly, the detection of this previously known (and unique) interaction between Bam35 and 590 B. thuringiensis provides confidence in our method. Only another viral protein interacts with the 591 host LexA, P8. Interestingly, the ORF8 is located in a highly variable region of tectiviruses whose 592 ORFans may alter phage regulatory functions, influencing phage and possibly also host fitness 593 [80]. However, the lack of sequence similarity does not provide any hint about its specific function.

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Since the only detected host partner of P8 is LexA, it is tempting to suggest a role of P8 in viral 595 cycle control. Particularly, as the ORF8 is located at the end of a gene cassette responsible for     Table 3 interacting with a large number of baits were tagged as potential promiscuous or "sticky preys" Table 4). Thus, interactions involving HER1410 protein fragments that interact 780 with more than six different Bam35 proteins, the average of interactors after selection, were 781 excluded from further evaluation. Third, prey interacting partners of bait pBC and pBN empty 782 plasmids were considered as false-positive generating preys. Interactions involving these prey 783 plasmids were also removed from the dataset (Supplementary Table 5 Table 9).

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Pairwise screens of the selected interactions were performed, as detailed in Table 3