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. 2023 Sep 8;2(4):34.
doi: 10.20517/mrr.2023.18. eCollection 2023.

Distribution, inducibility, and characteristics of Latilactobacillus curvatus temperate phages

Conrad L Ambros  1 , Matthias A Ehrmann  1
Affiliations

Distribution, inducibility, and characteristics of Latilactobacillus curvatus temperate phages

Conrad L Ambros et al. Microbiome Res Rep. .

Abstract

Aim: Temperate phages are known to heavily impact the growth of their host, be it in a positive way, e.g., when beneficial genes are provided by the phage, or negatively when lysis occurs after prophage induction. This study provides an in-depth look into the distribution and variety of prophages in Latilactobacillus curvatus (L. curvatus). This species is found in a wide variety of ecological niches and is routinely used as a meat starter culture. Methods: Fourty five L. curvatus genomes were screened for prophages. The intact predicted prophages and their chromosomal integration loci were described. Six L. curvatus lysogens were analysed for phage-mediated lysis post induction via UV light and/or mitomycin C. Their lysates were analysed for phage particles via viral DNA sequencing and transmission electron microscopy. Results: Two hundred and six prophage sequences of any completeness were detected within L. curvatus genomes. The 50 as intact predicted prophages show high levels of genetic diversity on an intraspecies level with conserved regions mostly in the replication and head/tail gene clusters. Twelve chromosomal loci, mostly tRNA genes, were identified, where intact L. curvatus phages were integrated. The six analysed L. curvatus lysogens showed strain-dependent lysis in various degrees after induction, yet only four of their lysates appeared to contain fully assembled virions with the siphovirus morphotype. Conclusion: Our data demonstrate that L. curvatus is a (pro)phage-susceptible species, harbouring multiple intact prophages and remnant sequences thereof. This knowledge provides a basis to study phage-host interaction influencing microbial communities in food fermentations.

Keywords: Latilactobacillus curvatus; induction; lysis; prophages; temperate bacteriophages.

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Conflict of interest statement

All authors declared that there are no conflicts of interest.

Figures

Figure 1
Figure 1
The 50 intact predicted L. curvatus prophages, pre-sorted by their phylogenetic relationship. Closer related phages are depicted next to each other. The phylogenetic tree is depicted as a cladogram and was constructed after whole genome alignment of those prophages. Prophages are displayed ranging from their predicted attL to their attR sites. Whole genome BLAST analysis was performed via Easyfig[42] and indicates nucleotide similarities between 63% and 100% (darker bars between the sequences indicate higher similarity). “-RC” after the name of the phage indicates that the reverse complement sequence of the found phage region is displayed. Genes were colourised for their predicted task: Lysogeny (ochre), replication (green), packaging (lilac), head (light blue), tail (dark blue), fiber (yellow; includes host recognition genes), lysis (red), hypothetical protein (grey), transposase (light yellow), unknown task (white), and tRNA (pink). L. curvatus: Latilactobacillus curvatus.
Figure 2
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 and bold lettering) and L. sakei. The groups I to VI (including phages infecting L. sakei) were described in a previous study[21]. 1,000 replicates were used in the bootstrap analysis (Jukes-Cantor model). L. curvatus: Latilactobacillus curvatus.
Figure 3
Figure 3
Growth curves of lysogenic L. curvatus strains after induction via UV light (A) or mitomycin C (B) treatment and without such treatment. Optical density was measured at 600 nm (OD600) (Y-axis; scale: 0-3.5) over the time (X-axis; scale 0-13) in hours (h). For the induction in (A), 4-min UV light exposure was used (no UV light exposure for control); For the induction in (B), different mitomycin C concentrations (20 µg/mL, 10 µg/mL, 5 µg/mL, 0.5 µg/mL, 0.2 µg/mL, and 0 µg/mL as control) were used. In (A), induction was performed directly before the measurement was started; In (B), the addition of mitomycin C to the bacterial cultures is indicated by an arrow. L. curvatus: Latilactobacillus curvatus.
Figure 4
Figure 4
TEM derived micrographs of purified post-induction lysates of different L. curvatus strains. For negative staining of the samples, 2% uranyl acetate was used. (A) L. curvatus TMW 1.591; (B) L. curvatus TMW 1.706; (C) L. curvatus TMW 1.1365; (D) L. curvatus TMW 1.2272. Scale bar: 50 nm. The microscope was operated at 80 kV in zero-loss mode. TEM: Transmission electron microscopy. L. curvatus: Latilactobacillus curvatus.
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