Endospore formation is used by members of the phylum Firmicutes to withstand extreme environmental conditions. Several recent studies have proposed endospore formation in species outside of Firmicutes, particularly in Rhodobacter johrii and Serratia marcescens, members of the phylum Proteobacteria. Here, we aimed to investigate endospore formation in these two species by using advanced imaging and analytical approaches. Examination of the phase-bright structures observed in R. johrii and S. marcescens using cryo-electron tomography failed to identify endospores or stages of endospore formation. We determined that the phase-bright objects in R. johrii cells were triacylglycerol storage granules and those in S. marcescens were aggregates of cellular debris. In addition, R. johrii and S. marcescens containing phase-bright objects do not possess phenotypic and genetic features of endospores, including enhanced resistance to heat, presence of dipicolinic acid, or the presence of many of the genes associated with endospore formation. Our results support the hypothesis that endospore formation is restricted to the phylum Firmicutes.Importance: Bacterial endospore formation is an important process that allows the formation of dormant life forms called spores. As such, organisms able to sporulate can survive harsh environmental conditions for hundreds of years. Here, we follow up on previous claims that two members of Proteobacteria, Serratia marcescens and Rhodobacter johrii, are able to form spores. We conclude that those claims were incorrect and show that the putative spores in R. johrii and S. marcescens are storage granules and cellular debris, respectively. This study concludes that endospore formation is still unique to the phylum Firmicutes.

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