Enterolyin S, a Polythiazole-containing Hemolytic Peptide from Enterococcus caccae.

The ß-hemolytic factor streptolysin S (SLS) is an important linear azol(in)e-containing peptide (LAP) that contributes significantly to the virulence of Streptococcus pyogenes. Despite its discovery 85 years ago, SLS has evaded structural characterizing owing to its notoriously problematic physicochemical properties. Here, we report the discovery and characterization of a structurally analogous hemolytic peptide from Enterococcus caccae, termed enterolysin S (ELS). Through heterologous expression, site-directed mutagenesis, chemoselective modification, and high-resolution mass spectrometry, we found that ELS contains an intriguing contiguous octathiazole moiety. The discovery of ELS expands our knowledge of hemolytic LAPs by adding a new member to this virulence-promoting family of modified peptides.

Enzymatic reconstitution of ribosomal peptide backbone thioamidation.

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

Bioinformatic mining for RiPP biosynthetic gene clusters in Bacteroidales reveals possible new subfamily architectures and novel natural products.

The Bacteroidales order, widely distributed among diverse human populations, constitutes a key component of the human microbiota. Members of this Gram-negative order have been shown to modulate the host immune system, play a fundamental role in the gut's microbial food webs, or be involved in pathogenesis. Bacteria inhabiting such a complex environment as the human microbiome are expected to display social behaviors and, hence, possess factors that mediate cooperative and competitive interactions. Different types of molecules can mediate interference competition, including non-ribosomal peptides (NRPs), polyketides, and bacteriocins. The present study investigates the potential of Bacteroidales bacteria to biosynthesize class I bacteriocins, which are ribosomally synthesized and post-translationally modified peptides (RiPPs). For this purpose, 1,136 genome-sequenced strains from this order were mined using BAGEL4. A total of 1,340 areas of interest (AOIs) were detected. The most commonly identified enzymes involved in RiPP biosynthesis were radical S-adenosylmethionine (rSAM), either alone or in combination with other biosynthetic enzymes such as YcaO. A more comprehensive analysis of a subset of 9 biosynthetic gene clusters (BGCs) revealed a consistent association in Bacteroidales BGCs between peptidase-containing ATP-binding transporters (PCATs) and precursor peptides with GG-motifs. This finding suggests a possibly shared mechanism for leader peptide cleavage and transport of mature products. Notably, human metagenomic studies showed a high prevalence and abundance of the RiPP BGCs from Phocaeicola vulgatus and Porphyromonas gulae. The mature product of P. gulae BGC is hypothesized to display γ-thioether linkages and a C-terminal backbone amidine, a potential new combination of post-translational modifications (PTM). All these findings highlight the RiPP biosynthetic potential of Bacteroidales bacteria, as a rich source of novel peptide structures of possible relevance in the human microbiome context.

Global Genome Mining Reveals the Distribution of Diverse Thioamidated RiPP Biosynthesis Gene Clusters.

Thioamidated ribosomally synthesized and post-translationally modified peptides (RiPPs) are recently characterized natural products with wide range of potent bioactivities, such as antibiotic, antiproliferative, and cytotoxic activities. These peptides are distinguished by the presence of thioamide bonds in the peptide backbone catalyzed by the YcaO-TfuA protein pair with its genes adjacent to each other. Genome mining has facilitated an in silico approach to identify biosynthesis gene clusters (BGCs) responsible for thioamidated RiPP production. In this work, publicly available genomic data was used to detect and illustrate the diversity of putative BGCs encoding for thioamidated RiPPs. AntiSMASH and RiPPER analysis identified 613 unique TfuA-related gene cluster families (GCFs) and 797 precursor peptide families, even on phyla where the presence of these clusters have not been previously described. Several additional biosynthesis genes are colocalized with the detected BGCs, suggesting an array of possible chemical modifications. This study shows that thioamidated RiPPs occupy a widely unexplored chemical landscape.

Enzymatic thioamidation of peptide backbones.

Thioamides are found in a few natural products and two known protein assemblies: the Escherichia coli ribosome and methyl-coenzyme M reductase (MCR) from methane-metabolizing archaea. Compared to an amide, thioamides alter the physical and chemical properties of peptide backbones, including the conformation dynamics, proteolytic stability, hydrogen-bonding capabilities, and possibly reactivity of a protein when installed. Recently, there has been significant progress in elucidating enzymatic post-translational thioamide installation, with most work leveraging the archaeal MCR-modifying enzymes. This chapter describes the protocols used for the in vitro enzymatic thioamidation of MCR-derived peptides, including polypeptide overexpression, purification, reaction reconstitution, and mass spectrometry-based product analysis. In addition, we highlight the protocols used for the biochemical, kinetics, and binding studies using recombinant enzymes obtained heterologously from E. coli. We anticipate that these methods will serve to guide future studies on peptide post-translational thioamidation, as well as other peptide backbone modifications using a parallel workflow.

Novel Mode of Molybdate Inhibition of Desulfovibrio vulgaris Hildenborough.

Sulfate-reducing microorganisms (SRM) are found in multiple environments and play a major role in global carbon and sulfur cycling. Because of their growth capabilities and association with metal corrosion, controlling the growth of SRM has become of increased interest. One such mechanism of control has been the use of molybdate (MoO4 2-), which is thought to be a specific inhibitor of SRM. The way in which molybdate inhibits the growth of SRM has been enigmatic. It has been reported that molybdate is involved in a futile energy cycle with the sulfate-activating enzyme, sulfate adenylyl transferase (Sat), which results in loss of cellular ATP. However, we show here that a deletion of this enzyme in the model SRM, Desulfovibrio vulgaris Hildenborough, remained sensitive to molybdate. We performed several subcultures of the ∆sat strain in the presence of increasing concentrations of molybdate and obtained a culture with increased resistance to the inhibitor (up to 3 mM). The culture was re-sequenced and three single nucleotide polymorphisms (SNPs) were identified that were not present in the parental strain. Two of the SNPs seemed unlikely candidates for molybdate resistance due to a lack of conservation of the mutated residues in homologous genes of closely related strains. The remaining SNP was located in DVU2210, a protein containing two domains: a YcaO-like domain and a tetratricopeptide-repeat domain. The SNP resulted in a change of a serine residue to arginine in the ATP-hydrolyzing motif of the YcaO-like domain. Deletion mutants of each of the three genes apparently enriched with SNPs in the presence of inhibitory molybdate and combinations of these genes were generated in the Δsat and wild-type strains. Strains lacking both sat and DVU2210 became more resistant to molybdate. Deletions of the other two genes in which SNPs were observed did not result in increased resistance to molybdate. YcaO-like proteins are distributed across the bacterial and archaeal domains, though the function of these proteins is largely unknown. The role of this protein in D. vulgaris is unknown. Due to the distribution of YcaO-like proteins in prokaryotes, the veracity of molybdate as a specific SRM inhibitor should be reconsidered.

Translation-Targeting RiPPs and Where to Find Them.

Prokaryotic translation is among the major targets of diverse natural products with antibacterial activity including several classes of clinically relevant antibiotics. In this review, we summarize the information about the structure, biosynthesis, and modes of action of translation inhibiting ribosomally synthesized and post-translationally modified peptides (RiPPs). Azol(in)e-containing RiPPs are known to target translation, and several new compounds inhibiting the ribosome have been characterized recently. We performed a systematic search for biosynthetic gene clusters (BGCs) of azol(in)e-containing RiPPs. This search uncovered several groups of clusters that likely direct the synthesis of novel compounds, some of which may be targeting the ribosome.