Metagenome-assembled genomes (MAGs) have contributed to identifying non-culturable microorganisms and understanding their ecological functions. MAGs offer an advantage in investigating sporulation-associated genes, especially given the difficulty of isolating many species residing in the gut microbiota of multiple hosts. Bacterial sporulation is a key survival mechanism with implications for pathogenicity and biotechnology. Here, we investigate MAGs from vertebrate hosts, emphasizing taxonomic identification and identifying sporulation-associated genes in potential novel species within the
Firmicutes
phylum. We identified potential new species in the classes
Clostridia
(
Borkfalkiaceae
,
Lachnospiraceae
,
Monoglobaceae
, and
Oscillospiraceae
families) and
Bacilli
(
Bacillaceae
and
Erysipelotrichaceae
families) through phylogenetic and functional pathway analyses, highlighting their sporulation potential. Our study covers 146 MAGs, 124 of them without refined taxonomic assignments at the family level. We found that
Clostridia
and
Bacilli
have unique sporulation gene profiles in the refined family MAGs for cattle, swine, poultry, and human hosts. The presence of genes related to Spo0A regulon, engulfment, and spore cortex in MAGs underscores fundamental mechanisms in sporulation processes in currently uncharacterized species with sporulation potential from metagenomic dark matter. Furthermore, genomic analyses predict sporulation potential based on gene presence, genome size, and metabolic pathways involved in spore formation. We emphasize MAGs covering families not yet characterized through the phylogenetic analysis, and with extensive potential for spore-forming bacteria within
Clostridia
,
Bacilli
, UBA4882, and UBA994 classes. These findings contribute to exploring spore-forming bacteria, which provides evidence for novel species diversity in multiple hosts, their adaptive strategies, and potential applications in biotechnology and host health.
IMPORTANCE
Spores are essential for bacterial survival in harsh environments, facilitating their persistence and adaptation. Exploring sporulation-associated genes in metagenome-assembled genomes (MAGs) from different hosts contributes to clinical and biotechnological domains. Our study investigated the extent of genes associated with bacterial sporulation in MAGs from poultry, swine, cattle, and humans, revealing these genes in uncultivated bacteria. We identified potential novel
Firmicutes
species with sporulation capabilities through phylogenetic and functional analyses. Notably, MAGs belonging to
Clostridia
,
Bacilli
, and unknown classes, namely UBA4882 and UBA994, remained uncharacterized at the family level, which raises the hypothesis that sporulation would also be present in these genomes. These findings contribute to our understanding of microbial adaptation and have implications for microbial ecology, underlining the importance of sporulation in
Firmicutes
across different hosts. Further studies into novel species and their sporulation capability can contribute to bacterial maintenance mechanisms in various organisms and their applications in biotechnology studies.
