Cyclodipeptide synthases (CDPSs) constitute a family of peptide bond-forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach.

How, when and why the transition between cell envelopes with one membrane (Gram‐positives or monoderms) and two (Gram‐negative or diderms) occurred in Bacteria is a key unanswered question in evolutionary biology. Different hypotheses have been put forward, suggesting that either the monoderm or the diderm phenotype is ancestral. The existence of diderm members in the classically monoderm Firmicutes challenges the Gram‐positive/Gram‐negative divide and provides a great opportunity to tackle the issue. In this review, we present current knowledge on the diversity of bacterial cell envelopes, including these atypical Firmicutes. We discuss how phylogenomic analysis supports the hypothesis that the diderm cell envelope architecture is an ancestral character in the Firmicutes, and that the monoderm phenotype in this phylum arose multiple times independently by loss of the outer membrane. Given the overwhelming distribution of diderm phenotypes with respect to monoderm ones, this scenario likely extends to the ancestor of all bacteria. Finally, we discuss the recent development of genetic tools for Veillonella parvula, a diderm Firmicute member of the human microbiome, which indicates it as an emerging new experimental model to investigate fundamental aspects of 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.The evidence 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 multiple transitions leading to the monoderm phenotype. Phyletic patterns of over 176,000 protein families constituting the Firmicutes pan-proteome identify those that are specific to the three diderm lineages, and suggest new potential players in OM biogenesis. In contrast, we find practically no largely conserved core for monoderms, a fact possibly linked to different ways of adapting to OM loss. Phylogenetic analysis of a concatenation of main OM components totalling nearly 2000 amino acid 2 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 highlights the overwhelming presence of diderms and the non-monophyly of monoderms, pointing to an early origin of two-membraned cells and the derived nature of the Gram-positive envelope following independent OM losses. 8are among most correlated with the diderm phenotype (see below, Figure 3), strongly suggesting functional interaction and a role in OM biogenesis. Phylogenomic analysis does not support acquisition of the OM by horizontal gene transferThe presence of the OM cluster may suggest that it was acquired by HGT. We previously showed by phylogenetic analysis of a concatenation of the four core LPS proteins (LpxABCD), that the sequences from Halanaerobiales and Negativicutes are closely related, match their reference species phylogeny, and do not stem from within another diderm bacterial phylum. We interpreted this result as support that these genes (and by extension the whole OM cluster) were not acquired via HGT, but were rather inherited from the ancestor of all Firmicutes, which would therefore have been a diderm with LPS 5,10 .Although in our opinion it is unlikely, the possibility of an acquisition in either the ancestor of Halanaerobiales or Negativicutes followed by a further HGT between the ancestors of these two clades remained open. We think that its presence in Limnochordia weakens this scenario, as this would imply an additional ancient transfer event. Nevertheless, in order to investigate further the HGT hypothesis, we searched for OM gene clusters similar to the one present in diderm Firmicutes in our Firmicutes databank as well as in 377 genomes representatives of major bacterial phyla (Methods). We could confirm that no other bacterial phylum possesse...