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As expected, all induced lysogens lysed after the induction process, although we experienced strain-
dependent differences in lysis intensity and time [Figure 3]. Lysis started approximately 2-5 h after
induction and ranged from a slight halt in growth (L. curvatus TMW 1.1447; UV light induction) to a rapid,
nearly complete lysis of the bacterial culture (L. curvatus TMW 1.591; UV light induction).

The micrographs [Figure 4] showed virions with the siphovirus morphotype in four of the six lysate samples
(A: Lysate of L. curvatus strain TMW 1.591, B: Lysate of L. curvatus strain TMW 1.706, C: Lysate of
L. curvatus strain TMW 1.1365, and D: Lysate of L. curvatus strain TMW 1.2272). In the lysates of
L. curvatus strains TMW 1.1381 and TMW 1.1447, no fully assembled virions were found. Notably, we
could not identify different morphotypes of virions within one sample, even when multiple intact prophages
were predicted in the same strain (e.g., L. curvatus TMW 1.706, and L. curvatus TMW 1.1365). While
capsids (50-65 nm in diameter) and tail width (10-15 nm) were similar in size, tail lengths varied drastically
between approximately 125-365 nm, with the shortest tail detected in the lysate of strain TMW 1.591 and
the longest in the lysate of strain TMW 1.2272. Additional micrographs of purified post-induction lysates
can be found in the supplemental data [Supplementary Figure 4].

As further proof of successful prophage induction, we extracted viral DNA from post-induction lysates after
DNase I treatment. This ensures that host DNA is degraded, while viral DNA is protected within the phage
capsids and is available for extraction and concentration. The viral DNA of all nine intact predicted phages
was isolated from their corresponding lysate samples, although the quality of the sequenced genomes varied
not only by strain but also by phage. Of the nine intact predicted phages, five were fully sequenced, three of
them were marked as circular (phage TMW 1.1365 P2, phage TMW 1.1365 P3, and phage TMW 1.1381 P1),
and two of them as linear (phage TMW 1.591 P1, phage TMW 1.706 P1). Alignments of isolated phage
DNA with respective prophages are depicted in Supplementary Figure 5. Interestingly, the genome of phage
TMW 1.1365 P2 contained a transposase that was previously not sequenced in its corresponding prophage.
Three phage genomes were fragmented after assembly (phage TMW 1.706 P2, phage TMW 1.1.1365 P1, and
phage TMW 1.2272 P1) with partially high depths of each phage-related fragment [phage TMW 1.706 P2
(depth = 5.26x to 8.02x), phage TMW 1.1.1365 P1 (depth = 235.19x to 330.19x), and phage TMW 1.2272 P1
(depth = 1238.18x to 2981.81x)]. Phage TMW 1.1447 P1 was sequenced as prophage but with a higher depth
of its contig (depth = 131.29x). Fragmented phage genomes were completed by aligning to their
corresponding prophage. A correlation between terminase genes and genome circularity, indicating
potentially different packaging mechanisms, could not be unambiguously determined [Supplementary
Figure 6].

DISCUSSION
Prophage incidence and integration
As a first step in understanding temperate phages infecting L. curvatus, we searched for prophages in openly
available genomes (accessible on the NCBI web page) as well as genomes of strains from our in-house strain
collection, where lysis was observable after an induction treatment with either UV light or mitomycin C.

A previous study by Pei et al. demonstrated that prophage distribution within the genus Lactobacillus is
extremely uneven . The authors suggested that multiple habitat species, such as L. brevis, L. plantarum,
[14]
and L. paracasei would retain more intact prophages, both in terms of absolute count and incidence, in their
genomes, than species occupying a smaller range of habitats. They identified L. plantarum as having the
highest occurrence of intact prophages (98% of 134 analysed strains), and L. paracasei as the species with the
highest “maximum number of prophage fragments” (15) and highest mean in “number of prophage
fragments” (4.39 per strain; analysed strains: 147) .
[14]