Bacteroides thetaiotaomicron-Infecting Bacteriophage Isolates Inform Sequence-Based Host Range Predictions

Abstract

Our emerging view of the gut microbiome largely focuses on bacteria, while less is known about other microbial components, such as bacteriophages (phages). Though phages are abundant in the gut, very few phages have been isolated from this ecosystem. Here, we report the genomes of 27 phages from the United States and Bangladesh that infect the prevalent human gut bacterium Bacteroides thetaiotaomicron. These phages are mostly distinct from previously sequenced phages with the exception of two, which are crAss-like phages. We compare these isolates to existing human gut metagenomes, revealing similarities to previously inferred phages and additional unexplored phage diversity. Finally, we use host tropisms of these phages to identify alleles of phage structural genes associated with infectivity. This work provides a detailed view of the gut's "viral dark matter" and a framework for future efforts to further integrate isolation- and sequencing-focused efforts to understand gut-resident phages.

Keywords: Bacteroides; Bacteroides thetaiotaomicron; bacteriophage; comparative genomics; gut microbiome; metagenomics; microbiota; phage; phage isolation.

Figure 1.. Isolation and characterization of 27 Bacteroides thetaiotaomicron-infecting phages.

(A) Phages were isolated from wastewater from 3 locations in the United States and from 2 locations in Dhaka, Bangladesh. (B) Network phylogeny analysis of phage genomes, compared according to shared gene content using Phamerator and Splitstree (see STAR Methods). Colored ellipses indicate groups of phages according to cluster assignment, assigned by vConTACT2. The scale bar indicates 0.1 substitutions per site. (C-E) Annotated genome maps of representative members of each cluster (SJC01, DAC15, and DAC20). Genes are represented as colored boxes and conserved domains are inlaid yellow boxes within genes. If a gene has a conserved domain, it is annotated in black text. iVireons was used to predict structural genes as described in STAR Methods and are annotated in red as predicted tail, major capsid, or general structural (tail, MCP, +S, respectively). (F-H) Transmission electron micrographs of SJC01, DAC15, and DAC20 show morphological differences between these representatives of the phage clusters. See also Tables S1–S6 and Figures S1–S3.

Figure 2.. Network phylogeny of 31 Bacteroides-infecting phages based on gene content.

The genomes of 31 Bacteroides infecting phages were compared according to shared gene content using Phamerator and Splitstree, (see STAR Methods). Colored ellipses indicate groups of phages according to cluster assignment, assigned by vConTACT2. The scale bar indicates 0.1 substitutions per site. See also Table S6 and Figure S4.

Figure 3.. Identification of Phage in SearchSRA (PhiSh) related to isolated B. thetaiotaomicron-infecting phages.

Representatives of each genome cluster (SJC01, DAC15, DAC20) were used to query the entire NCBI SRA using SearchSRA (see STAR Methods). (A-C) Log₁₀-transformed coverage depth of the 100 best hits identified via SearchSRA (tDNA mode) to SJC01, DAC15, and DAC20, respectively. Hits are ranked along the y-axis based on percent coverage by reads. The x-axis represents genome positions of SJC01, DAC15, and DAC20, respectively. The percentage of SJC01, DAC15, and DAC20 genomes detected (≥ 1 read) in each metagenome is indicated by the gray shaded column on the right of each panel. (D) Network phylogeny of Bacteroides-infecting phage genomes described in Figure 2 and related genomes identified in publicly available metagenomes. Genomes were compared according to shared gene content using Phamerator and Splitstree (see STAR Methods). Colored ellipses indicate groups of phages according to cluster assignment, assigned by vConTACT2. The subset of cluster alpha phages enclosed in a rectangle is shown in greater detail at the right-hand side of the panel. Phages highlighted in panel E are in bold. The scale bars indicate 0.01 and 0.001 substitutions per site for the main tree and the cluster alpha subset, respectively. (E) Genome maps of 4 cluster α phages (SJC01, ARB25, PhiSh04, and HSC01). The genes are color-coded according to pham membership and are numbered. Pairwise nucleotide identity is represented as shading between genomes. The color of this shading represents the degree of sequence similarity with violet being the most similar (BLASTN score = 0), progressing through the color spectrum from indigo, blue, green, yellow, orange, to red, which is the least similar (BLASTN score = 10⁻⁴). Regions with no shading indicate no similarity with a BLASTN score greater than 10⁻⁴. See also Tables S6–S8.

Figure 4.. Prediction of infection-associated phams (IAPs) in Bacteroides-infecting phages.

(A) Host range of phages on strains of Bacteroides thetaiotaomicron VPI-5482 expressing a variety of CPS (WT, wild type), a single CPS (cps1-cps8 strains) or no CPS (Δcps, acapsular). Tenfold serial dilutions of phage lysates ranging from approximately 10⁶ to 10³ plaque-forming units (PFU)/mL were spotted onto top agar plates containing each of the 10 bacterial strains. Plates were then incubated overnight, and plaques on each host were counted. Phage titers (PFU/ml) were calculated for each host and normalized to the titer on the “preferred host strain” for each replicate (individual replicates are shown to highlight variation between replicates, n=3 per phage). The phages were then clustered based on their plaquing efficiencies on the different strains (see STAR Methods). Each row in the heat map corresponds to one of three individual experimental replicates with a phage, whereas each column corresponds to one of the 10 host strains. (B) Changes in the total number of phams and average pham size as a function of percent amino acid identity. (C) Partial genome maps of 4 cluster α phages (SJC01, SJC10, HNL05, and ARB25) highlighting variation in gp4, gp5, and gp8. The genes are color coded according to pham membership at standard cutoffs and are numbered. Pairwise nucleotide identity is represented as shading between genomes. The color of this shading represents the degree of sequence similarity with violet being the most similar (BLASTN score = 0), progressing through the color spectrum from indigo, blue, green, yellow, orange, to red, which is the least similar (BLASTN score = 10⁻⁴). Regions with no shading indicate no similarity with a BLASTN score greater than 10⁻⁴. The red asterisk highlights gp8 from these phages. Data corresponding to these 4 phages in panels A and E are in bold. (D) Phages containing SJC01-like gp8 were compared against phages containing the alternative allele of gp8 (85% AA identity threshold) in terms of infectivity on bacterial strains highlighted in panel A. SJC01 gp8 is associated with higher infectivity of B. thetaiotaomicron cps1, cps5, cps6, and Δcps as assessed by Mann-Whitney U Test (p<0.05 = *, p<0.01 = **, p<0.001 = ***). (E) gp8 from cluster α isolates and the gene in the same position in cluster α genomes identified from metagenomes (PhiSh01–07, and HSC01) were aligned using ClustalW and a dendrogram of these alleles was created using The Interactive Tree of Life (see STAR Methods).