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The epeXEPAB (formerly yydFGHIJ) locus of Bacillus subtilis encodes a minimalistic biosynthetic pathway for a linear antimicrobial epipeptide, EpeX, which is ribosomally produced and post-translationally processed by the action of the radical-SAM epimerase, EpeE, and a membrane-anchored signal 2 peptide peptidase, EpeP. The ABC transporter EpeAB provides intrinsic immunity against self-produced EpeX, without conferring resistance against extrinsically added EpeX. EpeX specifically targets, and severely perturbs the integrity of the cytoplasmic membrane, which leads to the induction of the Lia-dependent envelope stress response. Here, we provide new insights into the distribution, expression, and regulation of the minimalistic epeXEPAB locus of B. subtilis, as well as the biosynthesis and biological efficiency of the produced epipeptide EpeX*. A comprehensive comparative genomics study demonstrates that the epe-locus is restricted to but widely distributed within the phylum Firmicutes. The gene products of epeXEP are necessary and sufficient for the production of the mature antimicrobial peptide EpeX*. In B. subtilis, the epeXEPAB locus is transcribed from three different promoters, one upstream of epeX (PepeX) and two within epeP (PepeA1 and PepeA2). While the latter two are mostly constitutive, PepeX shows a growth phase-dependent induction at the onset of stationary phase. We demonstrate that this regulation is the result of the antagonistic action of two global regulators: The transition state regulator AbrB keeps the epe locus shut off during exponential growth by direct binding. This tight repression is relieved by the master regulator of sporulation, Spo0A, which counteracts the AbrB-dependent repression of epeXEPAB expression during the transition to stationary phase. The net result of these three ­promoters is an expression pattern that ensures EpeAB-dependent autoimmunity prior to EpeX* production. In the absence of EpeAB, the general envelope stress response proteins LiaIH can compensate for the loss of specific autoimmunity by providing sufficient protection against the membrane-perturbating action of EpeX*. Hence, the transcriptional regulation of epe expression and the resulting intrinsic induction of the two corresponding resistance functions, encoded by epeAB and liaIH, are well balanced to provide a need-based immunity against mature EpeX*.

International audience; Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four C-alpha-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-alpha-carbon thioether-containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by C-alpha H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an alpha,beta-dehydro-amino acid intermediate during C-alpha-thioether bridge LC-MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.

Pseudomonas aeruginosa is a pathogenic micro-organism responsible for many hospital-acquired infections. It is able to adhere to solid surfaces and develop an immobilized community or so-called biofilm. Many studies have been focusing on the use of specific materials to prevent the formation of these biofilms, but the reactivity of the bacteria in contact to surfaces remains unknown. The aim of this study was to evaluate the impact of the abiotic surface on the physiology of adherent bacteria. Three different materials, stainless steel (SS), glass (G), and polystyrene (PS) that were relevant to industrial or medical environments were characterized at the physicochemical level in terms of their hydrophobicity and roughness. We showed that SS was moderately hydrophilic and rough, potentially containing crevices, G was hydrophilic and smooth while PS was hydrophobic and smooth. We further showed that P. aeruginosa cells were more likely able to adhere to SS and G rather than PS surfaces under our experimental conditions. The physiological response of P. aeruginosa when adhering to each of these materials was then evaluated by global proteomic analysis. The abundance of 70 proteins was shown to differ between the materials suggesting that their abundance was modified as a function of the material to which bacteria adhered. Our data lead to enabling the identification of abundance patterns that appeared to be specific to a given surface. Taken together, our data showed that P. aeruginosa is capable of sensing and responding to a surface probably via specific programmes to adapt its physiological response accordingly.

International audience; Radical S-adenosylmethionine (SAM) enzymes are emerging as a major superfamily of biological catalysts involved in the biosynthesis of the broad family of bioactive peptides called ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes have been shown to catalyze unconventional reactions, such as methyl transfer to electrophilic carbon atoms, sulfur to Cα atom thioether bonds, or carbon-carbon bond formation. Recently, a novel radical SAM enzyme catalyzing the formation of a lysine-tryptophan bond has been identified in Streptococcus thermophilus, and a reaction mechanism has been proposed. By combining site-directed mutagenesis, biochemical assays, and spectroscopic analyses, we show here that this enzyme, belonging to the emerging family of SPASM domain radical SAM enzymes, likely contains three [4Fe-4S] clusters. Notably, our data support that the seven conserved cysteine residues, present within the SPASM domain, are critical for enzyme activity. In addition, we uncovered the minimum substrate requirements and demonstrate that KW cyclic peptides are more widespread than anticipated, notably in pathogenic bacteria. Finally, we show a strict specificity of the enzyme for lysine and tryptophan residues and the dependence of an eight-amino acid leader peptide for activity. Altogether, our study suggests novel mechanistic links among SPASM domain radical SAM enzymes and supports the involvement of non-cysteinyl ligands in the coordination of auxiliary clusters.

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the xyl-doc cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics., This work was supported by a grant [R2DS 2010-08] from Conseil Regional d'Ile-de-France through DIM R2DS programs (http://www.r2ds-ile-de-france.com/). Irstea (www.irstea.fr/) contributed to the funding of a PhD grant for the first author. The funders provided support in the form of salaries for author [NB], funding for consumables and laboratory equipment, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Omics Services provided support in the form of salaries for authors [VS, MD], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors [NB, VS, MD] are articulated in the 'author contributions' section., info:eu-repo/semantics/publishedVersion

Radical S-adenosylmethionine (SAM) enzymes are emerging as a major superfamily of biological catalysts involved in the biosynthesis of the broad family of bioactive peptides called ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes have been shown to catalyze unconventional reactions, such as methyl transfer to electrophilic carbon atoms, sulfur to Cα atom thioether bonds, or carbon-carbon bond formation. Recently, a novel radical SAM enzyme catalyzing the formation of a lysine-tryptophan bond has been identified in Streptococcus thermophilus, and a reaction mechanism has been proposed. By combining site-directed mutagenesis, biochemical assays, and spectroscopic analyses, we show here that this enzyme, belonging to the emerging family of SPASM domain radical SAM enzymes, likely contains three [4Fe-4S] clusters. Notably, our data support that the seven conserved cysteine residues, present within the SPASM domain, are critical for enzyme activity. In addition, we uncovered the minimum substrate requirements and demonstrate that KW cyclic peptides are more widespread than anticipated, notably in pathogenic bacteria. Finally, we show a strict specificity of the enzyme for lysine and tryptophan residues and the dependence of an eight-amino acid leader peptide for activity. Altogether, our study suggests novel mechanistic links among SPASM domain radical SAM enzymes and supports the involvement of non-cysteinyl ligands in the coordination of auxiliary clusters.

Pseudomonas aeruginosa is a pathogenic micro-organism responsible for many hospital-acquired infections. It is able to adhere to solid surfaces and develop an immobilized community or so-called biofilm. Many studies have been focusing on the use of specific materials to prevent the formation of these biofilms, but the reactivity of the bacteria in contact to surfaces remains unknown. The aim of this study was to evaluate the impact of the abiotic surface on the physiology of adherent bacteria. Three different materials, stainless steel (SS), glass (G), and polystyrene (PS) that were relevant to industrial or medical environments were characterized at the physicochemical level in terms of their hydrophobicity and roughness. We showed that SS was moderately hydrophilic and rough, potentially containing crevices, G was hydrophilic and smooth while PS was hydrophobic and smooth. We further showed that P. aeruginosa cells were more likely able to adhere to SS and G rather than PS surfaces under our experimental conditions. The physiological response of P. aeruginosa when adhering to each of these materials was then evaluated by global proteomic analysis. The abundance of 70 proteins was shown to differ between the materials suggesting that their abundance was modified as a function of the material to which bacteria adhered. Our data lead to enabling the identification of abundance patterns that appeared to be specific to a given surface. Taken together, our data showed that P. aeruginosa is capable of sensing and responding to a surface probably via specific programmes to adapt its physiological response accordingly.

Purification and analysis of YvmBHis-tag protein. (A) SDS-PAGE analysis of purified YvmBHis-tag protein from E. coli cells. (B) Fractionated chromatography on a Superdex 200 h/300 gel filtration column. The YvmBHis-tag protein eluted as a major peak with an apparent molecular mass of 45 ± 5 kDa. (PDF 164 kb)

The yvmA-yvmC-cypX locus from B. subtilis and B. licheniformis ATCC 14580. (A) For each gene product of B. subtilis and its equivalent in B. licheniformis ATCC 14580 a percentage of identity is indicated. The position of the genes on each chromosome is given. The yvmC and cypX genes encoding the enzymes involved in pulcherriminic synthesis are represented by purple arrows, the MarR-type regulators YvmB by brown arrows, and the MFS-like transporters YvmA by white arrows. The conserved palindromic motif, palindrome I, is indicated by blue boxes. (B) Alignment of the 42-bp palindromic motifs from the promoter regions of B. subtilis yvmB and B. licheniformis yvmA genes. Stars indicate the seven nucleotides, which are not conserved between the two palindromic sequences. The 14Â bp core region highly conserved in the two sequences is boxed. (PDF 147 kb)

Heatmap representation of proteins that were found differentially abundant between the Î yvmB mutant and the wild type strain in the cytosolic fraction. Heatmap shows the change in protein levels among four independent samples. Protein levels are indicated by yellow to red colouring, a red shade indicates a higher abundance level. (PDF 7 kb)

Proteins differing significantly in Î yvmB cells from proteome analysis of the cytosolic fraction. A Kruskal-Wallis one-way analysis of variance (ANOVA) was done on the whole proteomic data. (PDF 20 kb)

International audience; AlbA is a radical SAM enzyme catalyzing the formation of three unusual thioether bonds in the antibiotic subtilosin A. We demonstrate here that AlbA catalyzes direct Cα H-atom abstraction and likely contains three essential [4Fe-4S] centers. This leads us to propose novel mechanistic perspectives for thioether bond catalysis by radical SAM enzymes.

Promoter regions of the divergent yvnA-yvnB genes and of the yisI gene. (A) The −10 and −35 regions of yvnA (in purple) and the −10 and −35 regions of yvnB (in brown) are indicated according to Nicolas et al. [33]. The predicted YvmB box is indicated in red and a degenerated YvmB box motif is indicated in orange. (B) The −10 and −35 regions of yisI (in brown) is indicated according to Nicolas et al. [33]. The prdicted YvmB box is indicated in red. (PDF 84 kb)