Genome-Based Identification of Active Prophage Regions by Next Generation Sequencing in Bacillus licheniformis DSM13

Abstract

Prophages are viruses, which have integrated their genomes into the genome of a bacterial host. The status of the prophage genome can vary from fully intact with the potential to form infective particles to a remnant state where only a few phage genes persist. Prophages have impact on the properties of their host and are therefore of great interest for genomic research and strain design. Here we present a genome- and next generation sequencing (NGS)-based approach for identification and activity evaluation of prophage regions. Seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Analysis of isolated phage particles (induced by mitomycin C) from the wild-type strain and prophage deletion mutant strains revealed activity of the prophage regions BLi_Pp2 (PBSX-like), BLi_Pp3 and BLi_Pp6. In contrast to BLi_Pp2 and BLi_Pp3, neither phage DNA nor phage particles of BLi_Pp6 could be visualized. However, the ability of prophage BLi_Pp6 to generate particles could be confirmed by sequencing of particle-protected DNA mapping to prophage locus BLi_Pp6. The introduced NGS-based approach allows the investigation of prophage regions and their ability to form particles. Our results show that this approach increases the sensitivity of prophage activity analysis and can complement more conventional approaches such as transmission electron microscopy (TEM).

Closely relatedVibrio alginolyticusstrains encode an identical repertoire of prophages and filamentous phages

“…Without a proof-of principle which would again require knock-out versions of the filamentous phages, we can only speculate that the Caudovirales Vibrio phage VALGΦ1 is supressed by the two actively replicating filamentous phages. Indeed, within-host competition between different Caudovirales has been found in other systems, for instance in Bacillus licheniformis (29). Alternatively, Vibrio phage VALGΦ1 might be not induced within the conditions of our experimental set up or could have been wrongly predicted to be complete by the software but is, however not able to actively replicate suggesting prophage domestication, which has also been predicted for Vibrio phage VALGΦ2 and 2b.…”

“…From the sequenced supernatant we could only identify filamentous phages but no head-tail phages, suggesting that in the present strains, filamentous phages are the only active replicating phages. To locate the exact positions of the induced prophages, we performed a PHAGE-seq experiment (29). In control experiments, the complete procedure has been applied without mitomycin C where the reference genomes were sequenced using Illumina technology.…”

“…Again, we hypothesise, that one phage, in this case Vibrio phage VALGΦ8, negatively affects the replication of another phage, here Vibrio phage VALGΦ6. As above, to verify or falsify this hypothesis, knock-out versions of strains containing both filamentous phages would be required, as have been used in (29). Within-host competition is common among different head-tail prophages within the same host leading to strong selection for short lysis time (55).…”

“…The supernatant was sterile filtered using 0.45 µm pore size filter (Sarstedt, Nümbrecht, Germany). We added lysozyme from chicken egg white (10µg/ml, SERVA Heidelberg, Germany) to disrupt the cell walls of potentially remaining host cells, RNAse A (Quiagen, Hilden, Germany) and DNAse I (Roche Diagnostics, Mannheim, Germany) at a final concentration of 10 µg/ml to remove free nucleic acids and remaining host cells as described in (29). After incubation at 25°C for 16 hours phage particles were sedimented by ultracentrifugation using a Sorvall Ultracentrifuge OTD50B with a 60Ti rotor applying 200,000 g for 4 hours.…”

“…Electron microscopy was carried out on a Jeol 1011 electron microscope (Eching, Germany). Negative staining and transmission electron microscopy (TEM) of phage-containing particles was performed as described previously (29, 32). Phosphotungstic acid dissolved in pure water (3%; pH 7) served as staining solution.…”