Closely related Lak megaphages replicate in the microbiomes of diverse animals

Abstract

Lak phages with alternatively coded ∼540 kbp genomes were recently reported to replicate in Prevotella in microbiomes of humans that consume a non-Western diet, baboons, and pigs. Here, we explore Lak phage diversity and broader distribution using diagnostic polymerase chain reaction and genome-resolved metagenomics. Lak phages were detected in 13 animal types, including reptiles, and are particularly prevalent in pigs. Tracking Lak through the pig gastrointestinal tract revealed significant enrichment in the hindgut compared to the foregut. We reconstructed 34 new Lak genomes, including six curated complete genomes, all of which are alternatively coded. An anomalously large (∼660 kbp) complete genome reconstructed for the most deeply branched Lak from a horse microbiome is also alternatively coded. From the Lak genomes, we identified proteins associated with specific animal species; notably, most have no functional predictions. The presence of closely related Lak phages in diverse animals indicates facile distribution coupled to host-specific adaptation.

Keywords: Microbiome; Omics; Virology.

Graphical abstract

Figure 1

Lak phage and Prevotella abundance differs across the pig gastrointestinal tract (A) Schematic of pig GIT with labels indicating the sites sampled: Blue labels = foregut, Red labels = hindgut (main sites of microbial fiber fermentation). For both (B) and (C), Lak phage major capsid and Prevotella 16S rRNA gene copy numbers determined by absolute quantification qPCR, with 10 ng pooled DNA from each GIT site from 6 finisher pigs; for all sites except ileal lumens, where digesta was only present in 4/6 pigs. Top and bottom whiskers = minimum and maximum values. Box width = Interquartile range (IQR). Significant differences in Lak, Prevotella and Lak: Prevotella ratio means were determined by Tukey's HSD test. (B) Difference in Lak phage abundance across pig lumen and mucosal sites coincides with Prevotella 16S rRNA gene abundance. Solid green and pink lines represent differences in abundance deemed statistically significant (P < 0.001). Standard errors ranged from 0.17–0.21 (Lak), and 0.28–0.34 (Prevotella). (C) Difference in ratios of Lak phage to Prevotella 16S rRNA gene copies (P < 0.05). Standard errors ranged from 0.28–0.35.

Figure 2

Lak phages from diverse animals are phylogenetically related Phylogeny was based on sequences from PCR, genomes, and metagenomes. The nucleotide sequences encoding the major capsid protein (MCP) were aligned and trimmed so that all lengths corresponded with that of the PCR-derived sequences. The capsid of the ~660-kbp phage is very divergent from others, thus was excluded from the tree to enable resolution of the other sequences. The tree was rooted between the GC31 group and GC26 group, according to the full phylogeny of all Lak (including the ~660-kbp one) and some other published huge phages (Figure S6). Three partial Lak phage genomes do not contain the MCP sequences thus were excluded. The names of the complete Lak genomes reported in this study are in bold. Bristol pig sequences obtained from the vaginal mucosa were identical to those found in the digestive tract. Corresponding trees for portal vertex and tail sheath monomer genes are shown in Figures S2 and S3.

Figure 3

Alternative coding is a persistent feature of the expanded Lak phage clade The maximum likelihood phylogenetic tree (iqtree (v1.6.12) using the "LG+G4" model (-bb = 1000)) was constructed using sequences for the large terminase protein sequence (see STAR Methods). The genome sizes shown are based on those of complete Lak phages in each clade. Nodes with ≥90% bootstrap support values are indicated by filled black circles and nodes with 70–90% support by open circles. Recoding of the TAG stop codon was detected through the Lak lineages but not in phages represented by the deepest branch.

Figure 4

Compressed version of the large terminase protein sequence alignment in which all positions except those with in-frame TAG codons (represented by ∗) have been deleted Background shading indicates different Lak phage lineages, as shown in Figure 3). Colors superimposed on ∗ indicate positions in which there is within-clade consensus as to the identity of the aligned amino acid. In the Lak clades with ~26% GC (bottom three groups), Q is the aligned amino acid in 77%, 75% and 85% of cases. There is insufficient information in other groups to predict how TAG is translated.

Figure 5

Lak phage genomes exhibit conserved, lineage-specific and animal-specific protein families Phylogenomic analyses of the 51 Lak phage genomes were performed. The phylogenetic tree (left) was built based on concatenated sequences of 49 single copy protein families detected in all Lak genomes and re-rooted using the sequence of the ~660-kbp horse-associated Lak phage. The protein family content heatmap (right), aligned with the phylogenetic tree, shows the presence/absence of protein families that could be detected in at least 4 genomes. The names of the 6 complete Lak genomes reported in this study are in bold. A total of 6 blocks of protein families with group-specific or animal-specific distribution patterns are highlighted in boxes and numbered.