Transcriptome of Epibiont Saccharibacteria Nanosynbacter lyticus Strain TM7x During the Establishment of Symbiosis

Abstract

Saccharibacteria Nanosynbacter lyticus strain TM7x is a member of the broadly distributed candidate phylum radiation. These bacteria have ultrasmall cell sizes, have reduced genomes, and live as epibionts on the surfaces of other bacteria. The mechanisms by which they establish and maintain this relationship are not yet fully understood. The transcriptomes of the epibiont TM7x and its host bacteria Schaalia odontolytica strain XH001 were captured across the establishment of symbiosis during both the initial interaction and stable symbiosis. The results showed a dynamic interaction with large shifts in gene expression for both species between the initial encounter and stable symbiosis, notably in transporter genes. During stable symbiosis, the host XH001 showed higher gene expression for peptidoglycan biosynthesis, mannosylation, cell cycle and stress-related genes, whereas it showed lower expression of chromosomal partitioning genes. This was consistent with the elongated cell shape seen in XH001 infected with TM7x and our discovery that infection resulted in thickened cell walls. Within TM7x, increased pili, type IV effector genes, and arginine catabolism/biosynthesis gene expression during stable symbiosis implied a key role for these functions in the interaction. Consistent with its survival and persistence in the human microbiome as an obligate epibiont with reduced de novo biosynthetic capacities, TM7x also showed higher levels of energy production and peptidoglycan biosynthesis, but lower expression of stress-related genes, during stable symbiosis. These results imply that TM7x and its host bacteria keep a delicate balance in order to sustain an episymbiotic lifestyle. IMPORTANCE Nanosynbacter lyticus type strain TM7x is the first cultivated member of the Saccharibacteria and the candidate phyla radiation (CPR). It was discovered to be ultrasmall in cell size with a highly reduced genome that establishes an obligate epibiotic relationship with its host bacterium. The CPR is a large, monophyletic radiation of bacteria with reduced genomes that includes Saccharibacteria. The vast majority of the CPR have yet to be cultivated, and our insights into these unique organisms to date have been derived from only a few Saccharibacteria species. Being obligate parasites, it is unknown how these ultrasmall Saccharibacteria, which are missing many de novo biosynthetic pathways, are maintained at a high prevalence within the human microbiome as well as in the environment.

Keywords: Nanosynbacter; RNAseq; Saccharibacteria; TM7x; actinomycetes; gingivitis; microbiome; oral microbiome; periodontitis; transcriptome.

FIG 1

Experimental design. (A) Schematic of culture setup, growth, and measurement for the coculture and naive cultures. Red dots represent TM7x, while the XH001 are shown as rod shaped tubes. Optical density at 600 nm (OD600) and colony forming units (CFU) were quantified at each step of the passage and are represented in panel B. (B) Growth measurements for passages using both OD600 (left and middle) and CFU (right) over seven passages (x axis). The middle panel shows the OD600 values for the dilutions as well as at the end of each passage. Three individual XH001n cultures are shown in blue circles, squares, and triangles, while cultures of XH001 with TM7x added are shown in red circles, squares, and triangles. Sampling points for the initial encounter (passage 0 at 6 h) and stable symbiosis (passage 6 at 6 h) phases for the transcriptomics sequencing are indicated by arrows. (C) Phase contrast images of XH001n and XH001/TM7x cultures at indicated sampling times are shown. Instances of TM7x infecting XH001 on the surface are indicated by red arrows. Scale bars are 10 μm.

FIG 2

Significant differences between conditions. (A) Volcano plots of the log₂ ratio of expression levels and the −log of the FDR. Shown are all three XH001 comparisons: initial encounter coculture versus naive, stable symbiosis coculture versus naive, and stable symbiosis coculture versus initial encounter coculture. Colored dots indicate genes that made the 0.05 FDR cutoff. Blue, lower in coculture; red, higher in coculture; yellow, higher in initial encounter coculture; green, higher in stable symbiosis coculture. The number of significant differences are shown on the plots. The TM7x comparison of the stable symbiosis coculture versus the initial encounter coculture is also shown. (B) Venn diagram comparing significantly different genes for the initial encounter versus naive and the stable symbiosis versus naive comparisons.

FIG 3

Clusters of orthologous groups (COG). The number of unchanged (gray) and significantly differentially expressed (colored) genes for each COG are shown for both the XH001 (top row) and TM7x (bottom row) comparisons. To prevent the large number of genes in cluster S (unknown function) from dominating the scale, only the significant differences are shown at full value. XH001 contains 621 genes annotated as S: unknown function, and TM7x contains 260.

FIG 4

Cell wall of XH001 observed by cryo-FIB milling and cryo-ET imaging. Representative section selected from the tomograms corresponding to XH001 cells in monoculture (A) and in established XH001/TM7x symbiosis coculture (B). The cell wall thickness was measured based on the density plot along the cell wall using IMOD and ImageJ. Top: Scale bars are 200 nm. Bottom: Scale bars are 50 nm.

FIG 5

DNA replication, cell cycle, and peptidoglycan biosynthesis. (A) Genes for DNA replication and the cell cycle. Heatmaps of z score-adjusted sequence counts for the three biological replicates for each condition. Bar plots show the log₂ ratio between the stable symbiosis coculture and the initial encounter coculture. Green indicates significantly increased during the stable symbiosis phase, and yellow indicates significantly increased in the initial encounter phase. (B–E) A schematic of the peptidoglycan biosynthesis pathway for XH001 and TM7x. Due to its small genome, TM7x has fewer predicted genes in these pathways. Steps with alternative pathways are shown as separate connections. Steps with multiple subunits are shown with multiple lines. Red, increased in XH001/TM7x versus XH001n; blue, decreased in XH001/TM7x versus XH001n; green, increased in stable symbiosis; yellow, increased in initial encounter; gray, statistically unchanged. (B) Initial encounter versus naive. The XH001 APY09 gene designations are given. (C) Stable symbiosis versus naive. (D) Stable symbiosis versus initial encounter. (E) TM7x stable symbiosis versus initial encounter. The TM7x gene designations are given. Steps that currently have no predicted gene but are expected to exist are shown in thin black lines.

FIG 6

Glycolysis and mannosylation substrates. A schematic of the glycolysis pathway and polyprenol phosphate mannose for XH001 and TM7x. Due to its small genome, TM7x has fewer predicted genes in these pathways. Steps with alternative pathways are shown as separate connections. Steps with multiple subunits are shown with multiple lines. Red, increased in XH001/TM7x versus XH001n; blue, decreased; green, increased in stable symbiosis; yellow, increased in initial encounter; gray, statistically unchanged. (A) Initial encounter versus naive. The XH001 APY09 gene designations are given. (B) Stable symbiosis versus naive. (C) Stable symbiosis versus initial encounter. (D) TM7x stable symbiosis versus initial encounter. The TM7x gene designations are given.

FIG 7

Arginine metabolism. A schematic of the arginine pathway for XH001 and TM7x. Red, increased in XH001/TM7x versus XH001n; blue, decreased in XH001/TM7x versus XH001n; green, increased in stable symbiosis; yellow, increased in initial encounter; gray, statistically unchanged. (A) Initial encounter versus naive. The XH001 APY09 gene designations are given. (B) Stable symbiosis versus naive. (C) Stable symbiosis versus initial encounter. (D) TM7x stable symbiosis versus initial encounter. The TM7x gene designations are given.

FIG 8

Stress and secretory systems. (A and B) Heatmaps of z score-adjusted sequence counts for the three biological replicates for each condition. Bar plots show the log₂ ratio between the stable symbiosis coculture and the initial encounter coculture. Green indicates significantly higher during stable symbiosis, and yellow indicates significantly higher in the initial encounter phase. (A) Genes involved in stress responses broken down into subcategories: chaperones, heat shock proteins, cold shock proteins, clp proteases, defense transporters, DNA repair, sodium oxide dismutase (SOD), universal stress protein (usp), and spermidine synthase (spm). (B) Genes for secretory systems, excluding the type IV system from TM7x. (C) A schematic of the type IV secretory system region of TM7x. Black outlines indicate predicted type IV component genes. Putative effector genes are backed in red. Green indicates significantly increased during stable symbiosis, and yellow indicates significantly increased in the initial encounter.

FIG 9

Transporters. The number of unchanged and significantly differentially expressed genes for the transporter groups are shown for the XH001 comparisons. Groups: amino acids, peptides, iron (Fe), noniron metal transporters, sodium/potassium (Na/K), sugar, other, and genes predicted to be transporters but without an identifiable substrate (unknown). To prevent the large number of genes in the unknown category (95) from dominating the scale, only the significant differences are shown at full value. (A) Initial encounter versus naive control. (B) Stable symbiosis versus naive control. (C) Stable symbiosis versus initial encounter.