Page 102 - Read Online

Page 10 of 19 Ambros et al. Microbiome Res Rep 2023;2:34 https://dx.doi.org/10.20517/mrr.2023.18

Figure 2. Phylogenetic tree after the neighbor-joining method (distance measure: Maximum Composite Likelihood method) of
L. curvatus phage integrases. Phages infecting other lactobacilli were included as outgroups (grey lettering). The assignment of groups
corresponding to closely related integrase genes (reflected by different letter colouring) was based on phages infecting L. curvatus (dots
[21]
and bold lettering) and L. sakei. The groups I to VI (including phages infecting L. sakei) were described in a previous study . 1,000
replicates were used in the bootstrap analysis (Jukes-Cantor model). L. curvatus: Latilactobacillus curvatus.

A second gene (terminase large subunit) was analysed to further demonstrate the phylogenetic diversity
between the intact prophages within this study [Supplementary Figure 3]. While no distinct clustering was
observable when comparing the relationships between integrases with the relationship between terminase
(large subunit) genes, the phylogenic relationship of terminase (large subunits) genes follows the
relationship of prophages [Figure 1] more closely.

Prophage inducibility
Screening for prophages in bacterial genomes provides insight into whether a species might be a target for
temperate phages. However, phage functions such as the inducibility of the intact predicted prophages and
correct virion assembly must be checked by performing induction experiments.

Six available lysogenic strains with intact predicted prophages were selected and exposed to one of two
common phage inducers, UV light or mitomycin C. If induction and virion assembly were successful,
host lysis and release of fully assembled virions would be expected. To assess the successful phage induction
after the treatment, we monitored the growth of the lysogenic strains after induction treatment, analysed the
lysates via transmission electron microscopy, and sequenced viral DNA from the potentially released
virions.