Pervasive prophage recombination occurs during evolution of spore-forming Bacilli

Abstract

Phages are the main source of within-species bacterial diversity and drivers of horizontal gene transfer, but we know little about the mechanisms that drive genetic diversity of these mobile genetic elements (MGEs). Recently, we showed that a sporulation selection regime promotes evolutionary changes within SPβ prophage of Bacillus subtilis, leading to direct antagonistic interactions within the population. Herein, we reveal that under a sporulation selection regime, SPβ recombines with low copy number phi3Ts phage DNA present within the B. subtilis population. Recombination results in a new prophage occupying a different integration site, as well as the spontaneous release of virulent phage hybrids. Analysis of Bacillus sp. strains suggests that SPβ and phi3T belong to a distinct cluster of unusually large phages inserted into sporulation-related genes that are equipped with a spore-related genetic arsenal. Comparison of Bacillus sp. genomes indicates that similar diversification of SPβ-like phages takes place in nature. Our work is a stepping stone toward empirical studies on phage evolution, and understanding the eco-evolutionary relationships between bacteria and their phages. By capturing the first steps of new phage evolution, we reveal striking relationship between survival strategy of bacteria and evolution of their phages.

Fig. 1. Changes within B. subtilis prophage sequence and integration site observed after prolonged sporulation selection regime.

a Experimental evolution with sporulation selection regime leads to spontaneous release of phage particles by the evolved strains [43]. b Overnight culture of evolved B. subtilis strain B410mB (amyE::mKate, shown in red) was diluted 100× and spotted on the lawn of undiluted B. subtilis ancestor strain (amyE::gfp, shown in green), resulting in a clearance zone, and growth of B410mB in that zone. The same experiment was performed using 100x diluted culture of ancestor strain (amyE::mKate) on a lawn of undiluted ancestor (amyE::gfp), as control. Scale bar = 2.5 mm. c Schematic representation of genome rearrangements in one of the phage-releasing evolved strains (B310mA), compared to the ancestor (Anc). The evolved strains carry a hybrid prophage phi3Ts-SPβ. Fragments of phi3Ts are shown in black, while fragments of SPβ are shown in pink. Below, schematic representation of phage genomes, spontaneously released by B310mA. d Schematic comparison of phi3Ts genome, with genome of Bacillus phage phi3T (KY030782.1). Fragment ‘s’ which is unique for phi3Ts, can be detected within prophage genomes of 6 B. subtilis strains, isolated in different parts of the world, specifically: SRCM103612 (South Korea), MB9_B1 and MB8_B1 (Denmark), JAAA (China), HMNig-2 (Egypt) and SSJ-1 (South Korea).

Fig. 2. Hybrid phages and extrachromosomal fragments of phage DNA, detected in the evolved strains.

a Top: Genome comparison of phi3Ts and SPβ (Query cover = 58%, Percent Identity = 99.73%), where regions of high homology (73.6–100%) are shown in gray, and regions of 99% homology are connected. Segments that are unique for phi3Ts, or SPβ are highlighted in black and pink, respectively. Phage genomes are arranged according to their integration into the host chromosome, which is represented in red. hybrid phi3Ts-SPβ phages spontaneously released by the evolved strains. Below: Each hybrid phage contains DNA segments that are unique to SPβ or phi3Ts. Based on the position of these segments, the simplified schemes of recombinant phages were created (below the hybrid name). Hyb^phi3Ts-SPβ additionally carries fragment of the host chromosome, adjacent to the left att site. Picture of corresponding plaque is placed on the right site of phage maps. Numbers on phage maps indicate unique recombination sites, which are characterized in Supplementary Table 4. b Extrachromosomal phage DNA fragments detected during PacBio sequencing, colored according to their homology to phi3Ts, SPβ, or fragments of host chromosome flanking phage integration sites. Fragments are ordered according to sequencing coverage relative to the chromosomal region, which is represented as bar chart on the left. Numbers on epDNA maps indicate unique recombination sites, which are characterized in Supplementary Table 4.

Fig. 3. Detection of phi3Ts DNA in the ancestor strain B. subtilis 168 through mapping of raw sequencing reads.

Top: Representation of phi3Ts genome according to its homology to SPβ prophage. Fragments of high homology to SPβ (73.6–100%) are shown in gray, while fragments that are unique to phi3Ts are shown in black. Bars 1, 2, and 3 correspond to DNA sequences that are unique for phi3Ts and that were used as targets for raw reads mapping (lower part). Green and red bars represent reads obtained from forward and reverse strands, respectively.

Fig. 4. Effect of phi3T lysogenization on B. subtilis sporulation and germination dynamics.

a Sporulation dynamics. Percentage of spores compared to total cell count, were examined in B. subtilis 3610 and the same strain lysogenized by phi3T phage, in three different time points of growth in minimal medium (MSgg). Data represent an average from four biological replicates, error bars correspond to standard error.

Fig. 5. Overview of prophage elements of natural Bacillus sp. isolates.

a Prophage elements were extracted from fully assembled genomes of Bacillus sp. and plotted according to size and integration position in the chromosome. Cluster of large prophages, integrated in the area of replication terminus could be detected (black dotted line). b Schematic representation of SPβ-like prophage found in B. subtilis SRCM 103612, isolated from traditional Korean food. The prophage genome was colored according to its homology to phi3Ts (black) and SPβ (pink), or both (gray). Extrachromosomal phage DNA found in this strain is matching left and right arms of the chromosomal prophage.

Fig. 6. Natural diversity of SPβ-like phages.

a Phylogenetic tree of B. subtilis strains that carry SPβ-like prophage in spsM or kamA gene, and two control strains that are free from such prophage. The tree was arbitrarily divided into six clades. b Phylogenetic tree of SPβ-like prophages hosted by the strains in (a). Inner circle shows prophage integration site, while outer circle indicates presence/absence of conjugative element ICEBs1, which blocks SPβ lytic cycle (c). Selected prophages of Bacillus sp. colored according to its homology to phi3Ts (black) and SPβ (pink), or both (gray). The upper four sequences integrate either in kamA or spsM and clearly belong to SPβ-like phages. Bottom four sequences come from other Bacillus species, and although they are more distant to phi3T or SPβ, they still carry segments of high homology with these phages. Explanation of ICEBs1 figure legend: intact—intact copy (100% identity to B. subtilis 168 or NCBI 3610) of ICEBs1 conjugative element is present; negative—lack of BLAST hits to ICEBs1 sequence; partial—at least 70% of ICEBs1 sequence is present; residual—<5% of ICEBs1 sequence is present.