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Ambros et al. Microbiome Res Rep 2023;2:34 https://dx.doi.org/10.20517/mrr.2023.18 Page 13 of 19

L. curvatus seems to harbour more prophages than L. sakei (maximum prophage sequence count: 5; Mean:
[14]
2.43; Occurrence intact prophages: 65.9%) , a species known for sharing a phylogenetically and
phenotypically close relationship with L. curvatus to such an extent that rapid differentiation between the
[44]
two species was historically challenging . According to Pei et al. , this might be explained by a less
[14]
restricted habitat L. curvatus occupies in contrast to L. sakei, even though both species share some ecological
niches, such as meat and fermented meat products [16-20] .

In total, of the 13 different distinct chromosomal loci we identified for Latilactobacillus phages (L. sakei and
L. curvatus), intact L. curvatus prophages were integrated into twelve of them, mostly in genes known for
their conservative nature (i.e., tRNA genes , lepA coding for translation elongation factor 4 ). Conserved
[45]
[46]
genes are often targeted by phages as integration loci, and our results are therefore consistent with the
[47]
literature . It should be noted that, in some cases, an exact prediction of the att-sites was hindered by
ambiguous BLAST results or incomplete sequencing data.

Genomic composition and features of L. curvatus prophages
The intact prophages we found in L. curvatus are genomically similarly organized compared to L. sakei
[21]
prophages and share similar integrase genes, and thus also chromosomal integration loci . Next to L. sakei
prophages, the correlation between closely related integrase genes and identical chromosomal integration
loci has also been demonstrated for Levilactobacillus brevis temperate phages . When comparing closely-
[7]
related phages, highly conserved genetic regions with > 63% nucleotide similarity were mainly found in
replication and head/tail-related genes. Putatively identical prophages were only harboured by L. curvatus
strain TMW 1.706 and the type strain L. curvatus DSM 20019 . This can be explained by the overall high
T
percentage identity of the genomes of both strains (100.00% ANIb over 99.95% of aligned nucleotides). The
average nucleotide consistency of all intact predicted prophages is depicted as a pairwise comparison table
in the supplemental material [Supplementary Figure 1].

Some of the found prophages harboured features that were not ubiquitously present in all of them. Of
course, the partial presence or lack of some of these features in those prophages might be explained by poor
annotation/database entries. Here, we discuss the presence and putative function of transposases,
methyltransferases, potential modification systems (methyltransferases in combination with restriction
endonucleases), and tRNAs.

Transposases were located in five of the intact predicted prophages transposases. Whether these phage
genomes are truly intact is ambiguous; on one hand, these phage genomes still have the potential to be
intact; however, on the other hand, transposases can replace important genes for further propagation, as
shown in Neisseria meningitidis, where an IS30 transposase replaced the prophage-related head and tail
[48]
morphogenesis genes . Bobay et al. discussed the rapid deletion of prophage genes, not only as a fast
protective mechanism by the host but also as an early step to “domesticate” phage components for the host’s
usage . In the genome of phage TMW 1.1365 P2, a putative transposase was located after the sequencing of
[49]
the post-induction lysate, which was not present in the prophage. We therefore suggest that transposases
can indeed be tolerated to some extent during phage induction, with the prerequisite that no essential genes
are disrupted/lost, although they might also be an indication for the ongoing degradation of the prophage.

34% of intact predicted prophages contained tRNA genes (excluding partial tRNA genes used for
integration). The presence of tRNA genes in phage genomes was discussed to support translation during the
degradation of the host chromosome after infection with lytic vibriophage 2.275.O . Furthermore, tRNA
[50]
genes provided by the phage are discussed to potentially compensate for codon usage of the host .
[51]