Prevalence, Diversity and UV-Light Inducibility Potential of Prophages in Bacillus subtilis and Their Possible Roles in Host Properties

Abstract

Bacillus subtilis is an important bacterial species due to its various industrial, medicinal, and agricultural applications. Prophages are known to play vital roles in host properties. Nevertheless, studies on the prophages and temperate phages of B. subtilis are relatively limited. In the present study, an in silico analysis was carried out in sequenced B. subtilis strains to investigate their prevalence, diversity, insertion sites, and potential roles. In addition, the potential for UV induction and prevalence was investigated. The in silico prophage analysis of 164 genomes of B. subtilis strains revealed that 75.00% of them contained intact prophages that exist as integrated and/or plasmid forms. Comparative genomics revealed the rich diversity of the prophages distributed in 13 main clusters and four distinct singletons. The analysis of the putative prophage proteins indicated the involvement of prophages in encoding the proteins linked to the immunity, bacteriocin production, sporulation, arsenate, and arsenite resistance of the host, enhancing its adaptability to diverse environments. An induction study in 91 B. subtilis collections demonstrated that UV-light treatment was instrumental in producing infective phages in 18.68% of them, showing a wide range of host specificity. The high prevalence and inducibility potential of the prophages observed in this study implies that prophages may play vital roles in the B. subtilis host.

Keywords: Bacillus subtilis; bacteriophages; diversity; in silico prophage analysis; induction; insertion sites; prevalence; prophages.

Figure 1

Distribution of intact prophages (IP), questionable prophages (QP), and incomplete prophages (InP) in 164 sequenced B. subtilis genomes. Host genome size is indicated on the X-axis. The distributions of the types of IPs according to host genome sizes are indicated on the Y-axis. Closed circles represent predicted prophages, and their colors indicate IP abundance, as indicated at the top.

Figure 2

The prevalence of Ips in B. subtilis strains and their distributions as integrated or plasmid forms.

Figure 3

Characterization of intact prophages (IPs) in the genomes of B. subtilis strains. (a) Dotplot illustrating IP abundance and genome proportion per genome of their host and their associations. (b) Density plot showing the distribution and association of IPs in terms of their genome size and GC-content.

Figure 4

Comparison of 172 IP genome sequences predicted in B. subtilis (a,b) Gegenees heatplot generated using BLASTN and TBLASTX, respectively, with 200/100 settings. Heatplot colors indicate similarity ranging from nearly identical (green) to similar (yellow) or dissimilar (red). The corresponding numbers of group members of each cluster are indicated on the y-axis. Labeled boxes at the top represent IP clusters. (c) Dotplot alignment of IP sequences generated by Gepard.

Figure 5

Phylogenomic relationships of B. subtilis IPs. The distances of the IP sequence were calculated using Gegenees. Then, the tree was constructed by SplitsTree4 and visualized by iTOL. Clusters are coded by different colors, and singletons are in red.

Figure 6

Functional annotation analysis of putative proteins encoded by B. subtilis prophages.

Figure 7

The survival rate of B. subtilis to UV-light treatment. UV-light tolerance of the B. subtilis was measured at three distances between the sample and the UV-lamp for seven varying exposure times.

Figure 8

Prevalence of in silico-predicted prophages and UV-light inducible of prophages in B. subtilis.