Deeply branching Bacillota species exhibit atypical Gram-negative staining.

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.