Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes.

Endosporulation is a complex morphophysiological process resulting in a more resistant cellular structure that is produced within the mother cell and is called endospore. Endosporulation evolved in the common ancestor of Firmicutes, but it is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only asporogenic species, we show here that strain 11kri321, which was isolated from an oligotrophic geothermal reservoir, produces phase-bright spore-like structures. Phylogenomics of strain 11kri321 and other Kurthia strains reveals little similarity to genetic determinants of sporulation known from endosporulating Bacilli. However, morphological hallmarks of endosporulation were observed in two of the four Kurthia strains tested, resulting in spore-like structures (cryptospores). In contrast to classic endospores, these cryptospores did not protect against heat or UV damage and successive sub-culturing led to the loss of the cryptosporulating phenotype. Our findings imply that a cryptosporulation phenotype may have been prevalent and subsequently lost by laboratory culturing in other Firmicutes currently considered as asporogenic. Cryptosporulation might thus represent an ancestral but unstable and adaptive developmental state in Firmicutes that is under selection under harsh environmental conditions.

An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition.

Recent data support the hypothesis that Gram-positive bacteria (monoderms) arose from Gram-negative ones (diderms) through loss of the outer membrane (OM), but how this happened remains unknown. As tethering of the OM is essential for cell envelope stability in diderm bacteria, its destabilization may have been involved in this transition. In the present study, we present an in-depth analysis of the four known main OM-tethering systems across the Tree of Bacteria (ToB). We show that the presence of such systems follows the ToB with a bimodal distribution matching the deepest phylogenetic divergence between Terrabacteria and Gracilicutes. Whereas the lipoprotein peptidoglycan-associated lipoprotein (Pal) is restricted to the Gracilicutes, along with a more sporadic occurrence of OmpA, and Braun's lipoprotein is present only in a subclade of Gammaproteobacteria, diderm Terrabacteria display, as the main system, the OmpM protein. We propose an evolutionary scenario whereby OmpM represents a simple, ancestral OM-tethering system that was later replaced by one based on Pal after the emergence of the Lol machinery to deliver lipoproteins to the OM, with OmpA as a possible transition state. We speculate that the existence of only one main OM-tethering system in the Terrabacteria would have allowed the multiple OM losses specifically inferred in this clade through OmpM perturbation, and we provide experimental support for this hypothesis by inactivating all four ompM gene copies in the genetically tractable diderm Firmicute Veillonella parvula. High-resolution imaging and tomogram reconstructions reveal a non-lethal phenotype in which vast portions of the OM detach from the cells, forming huge vesicles with an inflated periplasm shared by multiple dividing cells. Together, our results highlight an ancient shift of OM-tethering systems in bacterial evolution and suggest a mechanism for OM loss and the multiple emergences of the monoderm phenotype from diderm ancestors.

Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition.

The transition between cell envelopes with one membrane (Gram-positive or monoderm) and those with two membranes (Gram-negative or diderm) is a fundamental open question in the evolution of Bacteria. Evidence of the presence of two independent diderm lineages, the Halanaerobiales and the Negativicutes, within the classically monoderm Firmicutes has blurred the monoderm/diderm divide and specifically anticipated that other members with an outer membrane (OM) might exist in this phylum. Here, by screening 1,639 genomes of uncultured Firmicutes for signatures of an OM, we highlight a third and deep branching diderm clade, the Limnochordia, strengthening the hypothesis of a diderm ancestor and the occurrence of independent transitions leading to the monoderm phenotype. Phyletic patterns of over 176,000 protein families constituting the Firmicutes pan-proteome identify those that strongly correlate with the diderm phenotype and suggest the existence of new potential players in OM biogenesis. In contrast, we find practically no largely conserved core of monoderms, a fact possibly linked to different ways of adapting to repeated OM losses. Phylogenetic analysis of a concatenation of main OM components totalling nearly 2,000 amino acid positions illustrates the common origin and vertical evolution of most diderm bacterial envelopes. Finally, mapping the presence/absence of OM markers onto the tree of Bacteria shows the overwhelming presence of diderm phyla and the non-monophyly of monoderm ones, pointing to an early origin of two-membraned cells and the derived nature of the Gram-positive envelope following multiple OM losses.

Outer Membrane Proteome of Veillonella parvula: A Diderm Firmicute of the Human Microbiome.

Veillonella parvula is a biofilm-forming commensal found in the lungs, vagina, mouth, and gastro-intestinal tract of humans, yet it may develop into an opportunistic pathogen. Furthermore, the presence of Veillonella has been associated with the development of a healthy immune system in infants. Veillonella belongs to the Negativicutes, a diverse clade of bacteria that represent an evolutionary enigma: they phylogenetically belong to Gram-positive (monoderm) Firmicutes yet maintain an outer membrane (OM) with lipopolysaccharide similar to classic Gram-negative (diderm) bacteria. The OMs of Negativicutes have unique characteristics including the replacement of Braun's lipoprotein by OmpM for tethering the OM to the peptidoglycan. Through phylogenomic analysis, we have recently provided bioinformatic annotation of the Negativicutes diderm cell envelope. We showed that it is a unique type of envelope that was present in the ancestor of present-day Firmicutes and lost multiple times independently in this phylum, giving rise to the monoderm architecture; however, little experimental data is presently available for any Negativicutes cell envelope. Here, we performed the first experimental proteomic characterization of the cell envelope of a diderm Firmicute, producing an OM proteome of V. parvula. We initially conducted a thorough bioinformatics analysis of all 1,844 predicted proteins from V. parvula DSM 2008's genome using 12 different localization prediction programs. These results were complemented by protein extraction with surface exposed (SE) protein tags and by subcellular fractionation, both of which were analyzed by liquid chromatography tandem mass spectrometry. The merging of proteomics and bioinformatics results allowed identification of 78 OM proteins. These include a number of receptors for TonB-dependent transport, the main component of the BAM system for OM protein biogenesis (BamA), the Lpt system component LptD, which is responsible for insertion of LPS into the OM, and several copies of the major OmpM protein. The annotation of V. parvula's OM proteome markedly extends previous inferences on the nature of the cell envelope of Negativicutes, including the experimental evidence of a BAM/TAM system for OM protein biogenesis and of a complete Lpt system for LPS transport to the OM. It also provides important information on the role of OM components in the lifestyle of Veillonella, such as a possible gene cluster for O-antigen synthesis and a large number of adhesins. Finally, many OM hypothetical proteins were identified, which are priority targets for further characterization.

Phylogenomic analysis supports the ancestral presence of LPS-outer membranes in the Firmicutes.

One of the major unanswered questions in evolutionary biology is when and how the transition between diderm (two membranes) and monoderm (one membrane) cell envelopes occurred in Bacteria. The Negativicutes and the Halanaerobiales belong to the classically monoderm Firmicutes, but possess outer membranes with lipopolysaccharide (LPS-OM). Here, we show that they form two phylogenetically distinct lineages, each close to different monoderm relatives. In contrast, their core LPS biosynthesis enzymes were inherited vertically, as in the majority of bacterial phyla. Finally, annotation of key OM systems in the Halanaerobiales and the Negativicutes shows a puzzling combination of monoderm and diderm features. Together, these results support the hypothesis that the LPS-OMs of Negativicutes and Halanaerobiales are remnants of an ancient diderm cell envelope that was present in the ancestor of the Firmicutes, and that the monoderm phenotype in this phylum is a derived character that arose multiple times independently through OM loss.