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Ambros et al. Microbiome Res Rep 2023;2:34 https://dx.doi.org/10.20517/mrr.2023.18 Page 9 of 19

determinable attR-site; phage VRA_2sq_f P1 = P3 had both att-sites sequenced, hinting at integration into a
tRNA gene for serine, yet no tRNA gene was annotated as the corresponding sequence information was
missing.

Methyltransferase genes were annotated in the replication gene modules of sixteen intact predicted
prophages [Supplementary Table 5], with one to two of those genes per prophage. Of the 18 annotated
methyltransferases, seven were marked as adenine-specific, eight as cytosine-specific, and for three, no
specificity was determinable (checked via BLAST at uniprot.org). In direct proximity, only three prophages
(phage FAM25164 P1, phage MRS6 P1, and phage TMW 1.591 P1) harboured downstream genes coding for
a product with a predicted endonuclease function (tyrosine recombinase, or HNH homing endonuclease).

We analysed the phylogenetic relationship between L. curvatus phage integrase genes in a similar fashion as
[21]
previously described for L. sakei phage integrase genes , which were included as outgroups in this analysis.
This gave us additional information about the integration locus of each prophage. We adopted the
previously found insertion groups I-VI and extended them by the L. curvatus phage-specific Groups VII-
XIII [Figure 2]. L. sakei phages with group I integrases were all found integrated in tRNA genes for arginine
and leucine. Group II phage integrases facilitated integration into tmRNA genes, group III into a gene with
unknown function, group IV into a sufB-like gene, group V into a glutamine-hydrolysing GMP synthase
gene, and group VI into a glucose-6-phosphate isomerase gene.

With the exception of group III integrases, all those groups obtained new L. curvatus phage members.
Additionally, L. curvatus phages also integrated into a lepA gene (group VII; product: Translation
elongation factor 4), and tRNA genes for serine (group VIII), glutamine (group IX), and glutamic acid
(group X). Furthermore, two integrase clusters (group XI and Group XII) were found, which facilitate
integration into non-coding regions.

Three outliers were found in which the integrase position in the phylogenetic tree did not reflect the
chromosomal integration locus of the prophage. Phage TMW 1.2270 P1 contains a group II integrase, yet
integrates into a tRNA gene for serine (normally group VIII). The other two outliers were phage members
of group XIII (phage NFH-Km12 P1 and phage WiKim38 P2) integrating into a tRNA gene for leucine
(normally group I). The integrases of group XIII share only 46.69% to 47.40% nucleotide similarity to the
other integrases with leucine integration locus of group I, in contrast to 88.41% to 92.99% between group I
integrases with leucine tRNAs as integration locus.

Notably, strain L. curvatus NFH-Km12 (here used as an example for both strains) has multiple genes coding
for leucine tRNAs with percent identities ranging from 57.95% to 100% (100% when the same leucine tRNA
gene is present in multiple copies). The leucine tRNA gene chosen for integration of phage NFH-Km12 P1
only shared a nucleotide similarity of 66.28% to 67.44% with group I leucine tRNAs in which the phages
FLEC03 P1, TMW 1.1928 P1, and ZJUNIT8 P1 integrated (in contrast to 99.81% to 100.00% between the
three leucine tRNA genes of group I). Therefore, the integrases of NFH-Km12 P1 and WiKim38 P2 were
assigned to the new group XIII, despite also facilitating integration into leucine tRNA genes.

The determination of the chromosomal integration site of phage DRD-164 P1 was ambiguous. While the
phage shared the att-sites with phages of group IX (insertion into tRNA-Gln gene), its respective attR-site
was located before its lysis gene module. Another potential pair of att-sites was found (attL:
TATTCGTTGATGATATT, attR: TATTCGTTGATCATTTT). It cannot be said with certainty if the phage
is still intact.