Deeply branching species exhibit atypical Gram-negative staining.

UNLABELLED

The Gram staining method differentiates bacteria based on their cell envelope structure, with the monoderm and diderm cell envelope types traditionally being synonymous with Gram-positive and Gram-negative stain results, respectively. Monoderms have a single phospholipid membrane surrounded by a thick layer of peptidoglycan, while diderms have a lipopolysaccharide outer membrane exterior to a thin peptidoglycan layer. The (formerly ) phylum has members with both cell wall types, and recent phylogenetic analyses have shown that monoderm evolved from diderm ancestors on multiple occasions. Here, we compiled Gram staining and ultrastructural data for species with complete genomes to further investigate the evolution of Gram-positive and Gram-negative cell wall types. The results indicate that many deeply branching lineages at the root of phylum stain Gram-negative but do not harbor genes for outer membrane protein or lipopolysaccharide biosynthesis. Phylogenetic reconstructions suggest that several deeply branching species have retained a thin peptidoglycan layer in their cell walls, which was inherited from a diderm ancestor. Taxa with this atypical Gram-negative-staining cell wall structure include the thermophilic anaerobe and members of the and . Using Gram-staining results as a proxy for cell wall thickness, our analysis indicates that several independent peptidoglycan thickening events may have occurred in the evolution of the Gram-positive cell envelope.

IMPORTANCE

In this study, we examined the evolution of bacterial cell envelopes, specifically focusing on Gram-positive and Gram-negative cell wall types in the phylum. Our results indicate that certain bacteria can stain Gram-negative despite having a monoderm cell wall structure, thus challenging the conventional interpretation of Gram-staining results. Our observations also question the assumption that Gram-negative staining is always indicative of a diderm structure. These findings have broader implications for understanding how and when cell walls thicken during the evolution of bacterial cell envelopes.