Phylogenetic Distribution of WhiB- and Lsr2-Type Regulators in Actinobacteriophage Genomes

ABSTRACT Viruses that infect different actinobacterial host species are known as actinobacteriophages. They are composed of highly divergent and mosaic genomes due to frequent gene exchange between their bacterial hosts and related viral species. This is also reflected by the adaptive incorporation of host transcription factors (TFs) into phage regulatory networks. Previous studies discovered Lsr2-type and WhiB-type regulators encoded by actinobacteriophage genomes. However, limited information is available about their distribution, evolution, and impact on host species. In this study, we computationally screened the distribution of known bacterial and phage TFs inside 2951 complete actinobacteriophage genomes and identified 13 different TF domains. Among those, WhiB, Lsr2, MerR, and Cro/CI-like proteins were widespread and found in more than 10% of the analyzed actinobacteriophage genomes. Neighboring genomic context analysis of the whiB and lsr2 loci showed group-specific conservation of gene synteny and potential involvement of these genes in diverse regulatory functions. Both genes were significantly enriched in temperate phages, and the Lsr2-encoding genomes featured an overall lower GC content. Phylogenetic analysis of WhiB and Lsr2 proteins showed the grouping of phage sequences within bacterial clades, suggesting gene acquisition by phages from their bacterial host species or by multiple, independent acquisition events. Overall, our study reports the global distribution of actinobacteriophage regulatory proteins and sheds light on their origin and evolution. IMPORTANCE Actinobacteriophages are viruses that infect bacterial species of the diverse phylum of Actinobacteria. Phages engage in a close relationship with their bacterial host. This is also reflected by the adoption of genetic material from their host and its incorporation into phage regulatory circuits. In this study, we systematically searched the genomes of actinobacteriophages for the presence of transcription factor domains. We show that proteins belonging to the regulator families of WhiB and Lsr2 belong to the most abundant regulatory proteins encoded by actinobacteriophages. Further phylogenetic analysis shed light on their origin and evolution. Altogether, this study provides an important basis for further experimental investigation of their role in the coordination of the phage life cycle and their interaction with the host regulatory network in this important bacterial phylum.

Line 76. There are far more than 24 distinct clusters. Including sub clusters that number goes well over 100.
Line 77. Are they "Highly divergent phages"? Not sure what is meant by this line. I've always assumed it is a sampling issue and that singletons are just a future cluster. The way it is written suggests that they are somehow different than other phages. I'd suggest that the line be "Phages without close relatives are assigned as singletons".
Line86. "make a decision" is inappropriate language for viruses. We all do this when talking about viruses (including myself). Viruses aren't capable of deciding anything. They also don't own the host cell ("infection of their host cell"). Maybe change it to "temperate phages follow one of two lifecycle paths upon infection of the host cell"? Line 95. Perhaps making it clearer that most tailed bacteriophages studied to date infect the Proteobacteria? I think the work in this paper is made more interesting because it is looking at the phages of a completely different bacterial phylum.
How is Table S1 organized? Looks almost random to me. Is almost by accession number but isn't. I'd suggest ordering them based on cluster name.
I'm not going to ask you do it for this paper but in the future (if you do similar analyses) could you use more than just accession number in table S2. It would have been useful to include phage name and cluster. Would make possible cluster patterns in table S2 more obvious to a reader.
Line 138. "10% of the analyzed phage genomes". I couldn't see anything about cluster bias. A and F clusters are huge and could well account for 10% of phage genomes. Line 468. Did you mean secondary structure? Just the helicies and beta sheets? Or did you mean the overall fold of the proteins, the tertiary structure?

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