Deeply branching Bacillota species exhibit atypical Gram-negative staining

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 Bacillota (formerly Firmicutes ) phylum has members with both cell wall types, and recent phylogenetic analyses have shown that monoderm Bacillota evolved from diderm ancestors on multiple occasions. Here, we compiled Gram staining and ultrastructural data for Bacillota 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 Bacillota phylum stain Gram-negative but do not harbor genes for outer membrane protein or lipopolysacchar­ ide biosynthesis. Phylogenetic reconstructions suggest that several deeply branching Bacillota 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 Symbiobacterium thermophilum and members of the Desulfotomaculia, Syntrophamonadia, Desulfitobacteriia, Thermosedimi­ nibacteria, and Thermoanaerobacteria . 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

retains the CV-iodide precipitates during the organic solvent decolorizing step, resulting in the deep purple coloring that typifies Gram-positive bacteria.In contrast, diderms have an outer membrane (OM) located exterior to a thinner peptidoglycan cell wall (4).Decolorization disrupts diderm OM, and the thinner peptidoglycan layer is unable to retain the CV-iodide complex, resulting in the loss of the CV stain.Counter staining results in the final red/pink appearance typical of Gram-negative bacteria.The canonical OM in Gram-negative diderm bacteria is an asymmetric lipid bilayer, with a lipopolysaccharide outer membrane (LPS-OM) and outer membrane proteins (OMPs).
Because the Bacillota (previously known as the Firmicutes) members encompass both monoderm and diderm cell envelope types, there is significant interest in understanding the evolutionary history of this phylum to discern when and how the cell envelope transition occurred.Systematic and taxonomic studies from the past two decades have established that this phylum also contains Gram-negative-staining diderm members in the classes Negativicutes (5,6), Halanaerobiia (7,8), and Limnochordia (9).Like the canonical Gram-negative bacteria (e.g., the Pseudomonadota, formerly the Proteobacte ria), the cell walls of Negativicutes and Halanaerobiia are comprised of an LPS-containing OM exterior to a thin peptidoglycan layer.The genomes of Negativicutes and Halanaero biia encode complete sets of genes for the biosynthesis of an LPS-OM, including genes for the synthesis of the lipid A and lipopolysaccharide transport system for exporting LPS across the periplasm to the outer leaflet of the OM (1,10,11).Phylogenetic analyses have shown that Gram-positive monoderms evolved from Gram-negative diderm ancestors through multiple LPS-OM loss events (1,(10)(11)(12)(13).Recently, it was suggested that the thickening of the peptidoglycan layer in Gram-positive species may have been automatic following OM loss in monoderms (14); however, the mechanistic details of this transition remain unclear.We propose that the Gram-stain result of a bacterium can be used as a proxy for the thickness of the peptidoglycan cell wall.We hypothesized that among the monoderm Bacillota, representatives of intermediary cell wall types exist that lack an LPS-OM but still have a thin peptidoglycan wall, resulting in a Gram-negative stain.
We analyzed a total of 366 representative complete Bacillota genomes and identified numerous atypical Gram-negative species with multi-layered cell envelopes that lack LPS-OM biosynthesis genes (Table 1).All analysis methods are described in detail in the Supplemental materials file.Compilation of previously published data indicates that these organisms stain Gram-negative and that their cell envelopes contain one or more outer surface layers that enclose a thin peptidoglycan layer.The genomes of these atypical Gram-negative strains do not contain genes for lipid A biosynthesis (lpxABCD) or LPS transport (lptA), demonstrating that the observed outer layers are not composed of lipopolysaccharide.Their genomes also lack genes for the outer membrane protein OmpH and the OMP assembly protein BamA, further confirming that these outer layers are not diderm OMs.In certain cases, the outer layer is a protein S-layer (15)(16)(17)(18).Atypical Gram-negative species with this unusual cell wall structure include the anaerobic thermophiles Symbiobacterium thermophilum, Ammonifex degensii, Syntrophothermus lipocalidus, Thermoanaerobacterium aotearoense, Thermoanaerobacter kivui, Thermoanaerobacter tengcongensis, Pseudoclostridium thermosuccinogenes, and Novibacillus thermophilus.Others in this group include the anaerobes Syntrophomo nas wolfei, Dehalobacter restrictus, Desulfosporosinus acidiphilus, Flavonifractor plautii, Lachnoclostridium phytofermentans, Clostridium scatologenes, and Christensenella minuta.
We also found numerous examples of monoderms that stain Gram-negative (Table 1).Our data compilation indicates that these organisms stain Gram-negative even though transmission electron microscopy images show a monoderm cell wall structure.None of these organisms have genes for LpxABCD, LptA, OmpH, and BamA, further supporting the absence of an LPS-OM.The Gram-negative stain results are likely due to a thin peptidoglycan layer in the cell wall [e.g., as described in reference (19)].Examples of Gram-negative-staining monoderms are Thermanaeromonas toyohensis, Thermacetoge nium phaeum, Thermosediminibacter oceani, Caldicellulosiruptor owensensis, Syntrophobo tulus glycolicus, Oscillibacter valericigenes, Cellulosilyticum lentocellum, and Mageeibacillus indolicus.Gram-negative-staining monoderms also include the anaerobic phototrophs Heliobacterium modesticaldum and Heliorestis convoluta.In total, we found 45 Gram-neg ative Bacillota species that lack LPS-OM biosynthesis genes (Table S1) that we collectively describe as "atypical Gram-negative" bacteria.These atypical Gram-negative bacteria stain Gram-negative but lack a diderm cell envelope and/or lack LPS-OM biosynthesis genes.
Among our 366 representative genomes, we identified two with singleton lpx genes (Table S1): lpxC in Desulfotomaculum acetoxidans (20) and lpxA in Cohnella abietis (21).While an ultrastructure is not reported for either of these two species, both belong to Bacillota classes with monoderm representatives (Desulfotomaculia and Bacilli, respec tively, Table S1).D. acetoxidans stains Gram-negative while C. abietis stains Gram-posi tive (Table S1).An expanded search of 3,855 Bacillota genomes (Table S2), found a partial lpx operon (lpxACD) in the Gram-positive Paenibacillus crassostreae (22) and an LpxC homolog in Peptococcaceae bacterium DCMF (23) (also referred to as Candidatus Forminomas warabiya), a member of the monoderm class Dehalobacteriia (Table S1).A phylogenetic analysis was performed to determine if these genes were re-acquired by horizontal gene transfer or if they represent "vestigial" genes remaining after the loss of the LPS-OM.A detailed analysis with methods and an expanded discussion of results is reported in the Supplemental materials.Briefly, the lpxACD genes of P. crassostreae and the lpxA gene of C. abietis appear to have been re-acquired by a single horizontal gene transfer from members of the Terrabacteria (Fig. S1 to S4).In contrast, the lpxC genes in D. acetoxidans and Peptococcaceae bacterium DCMF may represent genuine remnants of a paralogous lpxC2 gene region found in some Bacillota (Fig. S5 and S6) that is distinct from the main LPS-OM biosynthesis gene cluster.However, as two out of four of these strains with lpx genes stain Gram-positive, we suggest that these isolated lpx genes are unlikely to contribute to the atypical Gram-negative phenotype.Based on the function of characterized lpx paralogues in other bacteria, these genes may instead have novel roles in virulence (24,25), temperature adaptation (25), and other environmental stress responses (26).
To examine the phylogeny of the atypical Gram-negative Bacillota, we generated a concatenated ribosomal protein tree (Fig. 1A) overlaid with the Gram staining data.The ancestral state of the Bacillota is a Gram-negative diderm cell envelope (1, 10-13), and our phylogenetic reconstruction indicates that the atypical Gram-negative cell wall type was inherited from the diderm ancestor with a thin cell wall peptidoglycan layer.S. thermophilum IAM 14863 is the deepest branching atypical Gram-negative species and is closely related to the diderm lineages of the Bacillota.This organism has a thin peptidoglycan cell wall (27) but lacks LPS-OM biosynthesis genes, thus demonstrating that LPS-OM gene loss occurred without subsequent cell wall thickening.We also identified several deeply branching atypical Gram-negative lineages-members of the Desulfotomaculia, Syntrophamonadia, and Desulfitobacteriia-that are closely related to the Negativicutes, a class of diderm Bacillota.Like, S. thermophilum, these deeply branching atypical Gram-negative organisms lost their LPS-OM biosynthesis genes but did not undergo subsequent thickening of their cell walls.Two later-diverging classes, the Thermosediminibacteria and Thermoanaerobacteria, also have this thin peptidogly can layer cell wall type while lacking LPS-OM biosynthesis genes.In total, among the deeper branching monoderm lineages (i.e., the Thermaerobacteria, Syntrophamonadia, Symbiobacteriia, Moorellia, Desulfotomaculia, and Dehalobacteriia) 15 out of the 17 (88%) analyzed genomes stain Gram-negative.In contrast, the Gram-positive pheno type (i.e., a thick peptidoglycan layer) is common among the later-branching Clostridia (63/84 analyzed Clostridia genomes stain Gram-positive) and Bacilli (233/242 analyzed Bacilli genomes stain Gram-positive).Interestingly, many of the atypical Gram-negative lineages across the Bacillota tree are thermophilic organisms (Table S5).Even among the Bacilli, two of the three (N.thermophilus, Thermobacillus composti) atypical Gram-negative strains are thermophilic.Prior work with the facultative thermophile Bacillus coagulans showed that growth at 55°C, compared to 37°C, resulted in lower teichoic acid content and reduced peptide cross-bridging in its peptidoglycan cell wall (28).This decreased peptide cross-bridging may have a role in limiting the thickness of the peptidoglycan cell wall in these thermophilic atypical Gram-negative lineages.
The phylogeny of Gram-positive and Gram-negative species indicate that several independent peptidoglycan thickening events have transpired in the evolutionary history of the Bacillota (Fig. 1B).These results suggest that peptidoglycan cell wall thickening did not happen automatically after OM loss in the evolution of monoderm cell envelopes.We hypothesize that the thickening of the peptidoglycan layer may have been driven primarily by changes in gene regulation in response to environmental cues (28).Future work should compare regulatory differences in cell wall formation between canonical Gram-positive monoderm Bacillota and atypical Gram-negative monoderm Bacillota.Additionally, Thermaerobacter marianensis-one of the earliest monoderms (Fig. 1)-stains weakly Gram-positive during the exponential phase of growth and Gram-negative during the stationary phase (29).The genome of this deeply branching Bacillota lacks the Lpx, Lpt, and OMP machinery required for synthesizing an LPS-OM.Because T. marianensis is one of the earliest monoderms to diverge from the ances tral Bacillota diderm, it could be a candidate representing the early evolution of the Gram-positive cell wall type.

TABLE 1
Ultrastructural data for atypical Gram-negative Bacillota that lack LPS genes a Data sources for each species are reported in TableS1.bAccessionnumbers and amino acid sequences of query proteins are shown in TableS2; a total of 3,854 complete Bacillota genomes were analyzed, and results are shown in TableS2; full white circle (○) indicate the absence of the gene, a full black circle (•) indicate the presence of the gene.Observation Microbiology Spectrum Month XXXX Volume 0 Issue 0 10.1128/spectrum.00732-243 Downloaded from https://journals.asm.org/journal/spectrum on 25 August 2024 by 2603:7081:4cf0:6530:2818:67f7:6819:9186.