Human-Gut Phages Harbor Sporulation Genes

Abstract

Spore-forming bacteria are prevalent in mammalian guts and have implications for host health and nutrition. The production of dormant spores is thought to play an important role in the colonization, persistence, and transmission of these bacteria. Spore formation also modifies interactions among microorganisms such as infection by phages. Recent studies suggest that phages may counter dormancy-mediated defense through the expression of phage-carried sporulation genes during infection, which can alter the transitions between active and inactive states. By mining genomes and gut-derived metagenomes, we identified sporulation genes that are preferentially carried by phages that infect spore-forming bacteria. These included genes involved in chromosome partitioning, DNA damage repair, and cell wall-associated functions. In addition, phages contained homologs of sporulation-specific transcription factors, notably spo0A, the master regulator of sporulation, which could allow phages to control the complex genetic network responsible for spore development. Our findings suggest that phages could influence the formation of bacterial spores with implications for the health of the human gut microbiome, as well as bacterial communities in other environments. IMPORTANCE Phages acquire bacterial genes and use them to alter host metabolism in ways that enhance phage fitness. To date, most auxiliary genes replace or modulate enzymes that are used by the host for nutrition or energy production. However, phage fitness is affected by all aspects of host physiology, including decisions that reduce the metabolic activity of the cell. Here, we focus on endosporulation, a complex and ancient form of dormancy found among the Bacillota that involves hundreds of genes. By coupling homology searches with host classification, we identified 31 phage-carried homologs of sporulation genes that are mostly limited to phages infecting spore-forming bacteria. Nearly one-third of the homologs recovered were regulatory genes, suggesting that phages may manipulate host genetic networks by tapping into their control elements. Our findings also suggest a mechanism by which phages can overcome the defensive strategy of dormancy, which may be involved in coevolutionary dynamics of spore-forming bacteria.

Keywords: dormancy; evolution; genomics; guts; metagenomes; microbiome; phage; sporulation.

FIG 1

Sporulation genes are enriched in phages that infect spore-forming bacteria. Homologs of both sporulation and nonsporulation genes were identified in RefSeq isolate phage genomes and in published gut viromes (18, 19) using DRAM-v. However, genes enriched among phages infecting spore-forming hosts (purple points) were mostly sporulation-related genes. The number of homologs detected for each gene is plotted on the x axis. For each of the genes, a hypergeometric enrichment test evaluated if it was found in phages that infect spore-forming hosts more than the random expectation given the number of phages infecting spore-forming hosts among all phages surveyed. The enrichment result [–log₁₀ (hypergeometric P value)] is plotted on the y axis. Purple points represent enriched genes with an adjusted P value of <10⁻⁶ (horizontal dashed line) and a sample size of >30 (vertical dashed line). Representative names of B. subtilis sporulation genes are provided for genes that were enriched and of viral origin.

FIG 2

Multiple sequence alignment of protein sequences of spo0A homologs from viromes (n = 29) and diverse bacteria (n = 57; from COG5801). (a) Summary of alignment coverage showing the percentage of nongap characters at each position, separated by the source of the sequence. Spo0A from viromes align to the C-terminal effector domain of the bacterial genes. (b) Focus on effector domain, showing all virome sequences aligned with spo0A of model spore-forming bacteria Clostridioides difficile and Bacillus subtilis. The alpha helices of the effector domain are indicated above the sequences. Sequence colors correspond to physicochemical properties of amino acids, using the Zappo coloring scheme. Information on functional and structural domains is from reference .