Conservation and Evolution of the Sporulation Gene Set in Diverse Members of the Firmicutes

Abstract

The current classification of the phylum Firmicutes (new name, Bacillota) features eight distinct classes, six of which include known spore-forming bacteria. In Bacillus subtilis, sporulation involves up to 500 genes, many of which do not have orthologs in other bacilli and/or clostridia. Previous studies identified about 60 sporulation genes of B. subtilis that were shared by all spore-forming members of the Firmicutes. These genes are referred to as the sporulation core or signature, although many of these are also found in genomes of nonsporeformers. Using an expanded set of 180 firmicute genomes from 160 genera, including 76 spore-forming species, we investigated the conservation of the sporulation genes, in particular seeking to identify lineages that lack some of the genes from the conserved sporulation core. The results of this analysis confirmed that many small acid-soluble spore proteins (SASPs), spore coat proteins, and germination proteins, which were previously characterized in bacilli, are missing in spore-forming members of Clostridia and other classes of Firmicutes. A particularly dramatic loss of sporulation genes was observed in the spore-forming members of the families Planococcaceae and Erysipelotrichaceae. Fifteen species from diverse lineages were found to carry skin (sigK-interrupting) elements of different sizes that all encoded SpoIVCA-like recombinases but did not share any other genes. Phylogenetic trees built from concatenated alignments of sporulation proteins and ribosomal proteins showed similar topology, indicating an early origin and subsequent vertical inheritance of the sporulation genes. IMPORTANCE Many members of the phylum Firmicutes (Bacillota) are capable of producing endospores, which enhance the survival of important Gram-positive pathogens that cause such diseases as anthrax, botulism, colitis, gas gangrene, and tetanus. We show that the core set of sporulation genes, defined previously through genome comparisons of several bacilli and clostridia, is conserved in a wide variety of sporeformers from several distinct lineages of Firmicutes. We also detected widespread loss of sporulation genes in many organisms, particularly within the families Planococcaceae and Erysipelotrichaceae. Members of these families, such as Lysinibacillus sphaericus and Clostridium innocuum, could be excellent model organisms for studying sporulation mechanisms, such as engulfment, formation of the spore coat, and spore germination.

Keywords: Bacillus; Clostridia; gene loss; genome analysis; phylogenetic analysis; spore coat; spores; taxonomy.

FIG 1

Distribution of sporulation genes in Spo0A⁺ and Spo0A⁻ firmicutes. The graphs show the numbers of widespread sporulation genes (out of 112) (A) and more narrowly conserved sporulation genes (out of 74) (B) encoded in sporeformers (which are all Spo0A⁺; blue columns), Spo0A⁺ nonsporeformers (orange columns), and Spo0A⁻ nonsporeformers (gray columns). “Other classes” includes Negativicutes, Tissierellia, Erysipelotrichia, and Limnochordia.

FIG 2

Organization of skin elements in 15 firmicute genomes. spoIVCB genes are shown in light green, spoIIIC in dark green, and spoIVCA in orange. The organism names are followed by the GenBank genome entries and genomic coordinates of the regions between spoIVCB and spoIIIC. The numbers above the gaps indicate the distances between spoIVCB or spoIIIC and spoIVCA (~1.5 kb less than the total length of the skin element). R. cellulolyticum Ccel_0434 is annotated as a single 7,394-nucleotide pseudogene.

FIG 3

Phylogenic trees for sporulation and ribosomal proteins from Firmicutes. Maximum-likelihood phylogenetic trees were built using IQ-TREE2 (125) with the LG+F+R9 substitution model from concatenated alignments of 41 core sporulation proteins (left panel) and 54 ribosomal proteins (right panel) encoded in the genomes of 76 spore-forming members of the Firmicutes.