Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

Abstract

Sporulation is a specialized developmental program employed by a diverse set of bacteria which culminates in the formation of dormant cells displaying increased resilience to stressors. This represents a major survival strategy for bacteria facing harsh environmental conditions, including nutrient limitation, heat, desiccation, and exposure to antimicrobial compounds. Through dispersal to new environments via biotic or abiotic factors, sporulation provides a means for disseminating genetic material and promotes encounters with preferable environments thus promoting environmental selection. Several types of bacterial sporulation have been characterized, each involving numerous morphological changes regulated and performed by non-homologous pathways. Despite their likely independent evolutionary origins, all known modes of sporulation are typically triggered by limited nutrients and require extensive membrane and peptidoglycan remodeling. While distinct modes of sporulation have been observed in diverse species, two major types are at the forefront of understanding the role of sporulation in human health, and microbial population dynamics and survival. Here, we outline endospore and exospore formation by members of the phyla Firmicutes and Actinobacteria, respectively. Using recent advances in molecular and structural biology, we point to the regulatory, genetic, and morphological differences unique to endo- and exospore formation, discuss shared characteristics that contribute to the enhanced environmental survival of spores and, finally, cover the evolutionary aspects of sporulation that contribute to bacterial species diversification.

Keywords: Actinobacteria; Firmicutes; bacterial evolution; endospore; exospore; regulation; structural biology.

FIGURE 1

Overview of sporulation. (A) Endospore formation in Firmicutes. The process begins with the formation of an asymmetric septum. Next, the larger compartment engulfs the smaller immature spore. The spore matures by the formation of protective layers and is released through lysis of the mother cell. (B) Exospore formation in Streptomyces. The process begins by aerial hyphae formation, which subsequently divide into numerous compartments. Each compartment matures into an exospore that gets released from the spore chain.

FIGURE 2

In situ structural detail of sporulation revealed by cryo electron tomography. Each image is a 20-nm slice through a tomogram. Column one (A,D,G,J) outlines major stages of endospore formation in the model organism Bacillus subtilis. Column two (B,E,H,K) outlines endospore formation in Acetonema longum, a diderm Firmicute and member of the Negativicutes. Column three (C,F,I,L) outlines exospore formation in Streptomyces albus. Cytoplasmic (CM) and inner membranes (IM) are shown in red, outer membrane (OM) is shown in blue. The inner and outer spore membranes in Firmicutes (IsM and OsM) are both colored in red to show that they are derived from the CM/IM of the mother cell.

FIGURE 3

Maximum likelihood phylogeny of sporulating and non-sporulating Firmicutes and Actinobacteria. Tree was constructed using an alignment of 120 single-copy marker gene sequences from several hundred representative genomes from each phylum using GTDB-Tk (Parks et al., 2018; Chaumeil et al., 2019). Whole genome phylogeny was determined using the concatenated marker gene alignment and IQ-TREE (v. 2.0.3), with the substitution model LG + G4 and 1000 ultrafast bootstraps (Minh et al., 2020). The tree was subsequently down-sampled and collapsed to show major families. Several representative Cyanobacteria genomes served as an outgroup. Tree was visualized using ggtree (Yu, 2020). Green dots indicate that endospore formation was likely present in the last universal ancestor of all Firmicutes, whereas exospore formation appears to have evolved after phylum differentiation. Black and gray dots represent the demonstrated ability to sporulate in all members and some members, respectively, whereas white dots represent the lack of sporulation ability. Major losses involving multiple families are shown (red x). Clades which include diderm bacteria are indicated for possession of either an LPS-containing OM (red dots) or a mycolic acid-containing OM (blue dot). Classes, as defined by the GTDB, are labeled on the right.