Bacteriocins represent a large family of ribosomally produced peptide antibiotics. Here we describe the discovery of a widely conserved biosynthetic gene cluster for the synthesis of thiazole and oxazole heterocycles on ribosomally produced peptides. These clusters encode a toxin precursor and all necessary proteins for toxin maturation and export. Using the toxin precursor peptide and heterocycle-forming synthetase proteins from the human pathogen Streptococcus pyogenes, we demonstrate the in vitro reconstitution of streptolysin S activity. We provide evidence that the synthetase enzymes, as predicted from our bioinformatics analysis, introduce heterocycles onto precursor peptides, thereby providing molecular insight into the chemical structure of streptolysin S. Furthermore, our studies reveal that the synthetase exhibits relaxed substrate specificity and modifies toxin precursors from both related and distant species. Given our findings, it is likely that the discovery of similar peptidic toxins will rapidly expand to existing and emerging genomes.antibiotics ͉ bacteriocin ͉ bioinformatics ͉ hemolytic ͉ streptolysin

The application of synthetic biology requires characterized tools to precisely control gene expression. This toolbox of genetic parts previously did not exist for the industrially promising cyanobacterium, Synechococcus sp. strain PCC 7002. To address this gap, two orthogonal constitutive promoter libraries, one based on a cyanobacterial promoter and the other ported from Escherichia coli, were built and tested in PCC 7002. The libraries demonstrated 3 and 2.5 log dynamic ranges, respectively, but correlated poorly with E. coli expression levels. These promoter libraries were then combined to create and optimize a series of IPTG inducible cassettes. The resultant induction system had a 48-fold dynamic range and was shown to out-perform Ptrc constructs. Finally, a RBS library was designed and tested in PCC 7002. The presented synthetic biology toolbox will enable accelerated engineering of PCC 7002.

Here we report on a novel thiazole/oxazole-modified microcin (TOMM) from Bacillus amyloliquefaciens FZB42, a Gram-positive soil bacterium. This organism is well known for stimulating plant growth and biosynthesizing complex small molecules that suppress the growth of bacterial and fungal plant pathogens. Like microcin B17 and streptolysin S, the TOMM from B. amyloliquefaciens FZB42 undergoes extensive posttranslational modification to become a bioactive natural product. Our data show that the modified peptide bears a molecular mass of 1,335 Da and displays antibacterial activity toward closely related Gram-positive bacteria. A cluster of 12 genes that covers ϳ10 kb is essential for the production, modification, export, and self-immunity of this natural product. We have named this compound plantazolicin (PZN), based on the association of several producing organisms with plants and the incorporation of azole heterocycles, which derive from Cys, Ser, and Thr residues of the precursor peptide.Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant growth-promoting bacterium with an impressive capacity to produce secondary metabolites with antimicrobial activity (7). The nonribosomal syntheses of polyketides (bacillaene, difficidin, and macrolactin), lipopeptides (surfactin, fengycin, and bacillomycin D), and siderophores (bacillibactin and the product of the nrs cluster) are carried out by large gene clusters distributed over the entire genome of B. amyloliquefaciens FZB42. While these compounds are biosynthesized in a 4Ј-phosphopantetheine transferase (Sfp)-dependent fashion, the production of the antibacterial dipeptide bacilysin is independent of Sfp (8,9). In total, 8.5% of the entire genomic capacity of B. amyloliquefaciens FZB42 is devoted to the nonribosomal synthesis of secondary metabolites, exceeding that of the model Gram-positive bacterium Bacillus subtilis 168 by more than 2-fold (6). Prophage sequences that often harbor biosynthetic gene clusters of ribosomally synthesized peptides (microcins, lantibiotics/lantipeptides), which are common in B. subtilis strains, were not previously detected within the FZB42 genome. However, the presence of an antimicrobial compound(s) active against sigW mutant strain HB0042 of B. subtilis has been reported. SigW is an extracytoplasmic sigma factor that provides intrinsic resistance to antimicrobial compounds produced by other Bacilli (4).The driving force for the current report was the finding that FZB42 mutant RS6, which is deficient in the Sfp-dependent synthesis of lipopeptides and polyketides and in Sfp-independent bacilysin production (9), still produced an antibacterial substance active against Bacillus subtilis HB0042. This finding underscores the diversity of biosynthetic strategies employed by FZB42 and offers new possibilities for discovering novel natural products with biomedically relevant activities. Recent genomic analysis of FZB42 revealed a ribosomally encoded biosynthetic gene cluster that is conserved among many species across two domains of life ...

The human pathogen Streptococcus pyogenes secretes a highly cytolytic toxin known as streptolysin S (SLS). SLS is a key virulence determinant and responsible for the ␤-hemolytic phenotype of these bacteria. Despite over a century of research, the chemical structure of SLS remains unknown. Recent experiments have revealed that SLS is generated from an inactive precursor peptide that undergoes extensive post-translational modification to an active form. In this work, we address outstanding questions regarding the SLS biosynthetic process, elucidating the features of substrate recognition and sites of posttranslational modification to the SLS precursor peptide. Further, we exploit these findings to guide the design of artificial cytolytic toxins that are recognized by the SLS biosynthetic enzymes and others that are intrinsically cytolytic. This new structural information has ramifications for future antimicrobial therapies.

Trans-acting regulators provide novel opportunities to study essential genes and regulate metabolic pathways. We have adapted the clustered regularly interspersed palindromic repeats (CRISPR) system from Streptococcus pyogenes to repress genes in trans in the cyanobacterium Synechococcus sp. strain PCC 7002 (hereafter PCC 7002). With this approach, termed CRISPR interference (CRISPRi), transcription of a specific target sequence is repressed by a catalytically inactive Cas9 protein recruited to the target DNA by base-pair interactions with a single guide RNA that is complementary to the target sequence. We adapted this system for PCC 7002 and achieved conditional and titratable repression of a heterologous reporter gene, yellow fluorescent protein. Next, we demonstrated the utility of finely tuning native gene expression by downregulating the abundance of phycobillisomes. In addition, we created a conditional auxotroph by repressing synthesis of the carboxysome, an essential component of the carbon concentrating mechanism cyanobacteria use to fix atmospheric CO2. Lastly, we demonstrated a novel strategy for increasing central carbon flux by conditionally downregulating a key node in nitrogen assimilation. The resulting cells produced 2-fold more lactate than a baseline engineered cell line, representing the highest photosynthetically generated productivity to date. This work is the first example of titratable repression in cyanobacteria using CRISPRi, enabling dynamic regulation of essential processes and manipulation of flux through central carbon metabolism. This tool facilitates the study of essential genes of unknown function and enables groundbreaking metabolic engineering capability, by providing a straightforward approach to redirect metabolism and carbon flux in the production of high-value chemicals.

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Through elaboration of its botulinum toxins, Clostridium botulinum produces clinical syndromes of infant botulism, wound botulism, and other invasive infections. Using comparative genomic analysis, an orphan nine-gene cluster was identified in C. botulinum and the related foodborne pathogen Clostridium sporogenes that resembled the biosynthetic machinery for streptolysin S, a key virulence factor from group A Streptococcus responsible for its hallmark ␤-hemolytic phenotype. Genetic complementation, in vitro reconstitution, mass spectral analysis, and plasmid intergrational mutagenesis demonstrate that the streptolysin S-like gene cluster from Clostridium sp. is responsible for the biogenesis of a novel post-translationally modified hemolytic toxin, clostridiolysin S.Microbial virulence and survival are often defined by metabolic output. Among the many molecular species used to give a competitive advantage are hydrogen peroxide, genetically encoded small molecules, siderophores, proteins, and bacteriocins (1, 2). Streptolysin S (SLS) 2 is a well known hemolytic/ cytolytic, ribosomally encoded bacteriocin and virulence factor group A Streptococcus (GAS) (3)(4)(5). To this day, the characteristic ␤-hemolytic phenotype observed on blood agar plates is used as a clinical diagnostic tool for GAS identification. GAS is best known as the agent of acute pharyngitis (strep throat) but also may cause invasive infections, including necrotizing fasciitis and toxic shock syndrome. In the 100-year history of our knowledge of streptococcal bacteria, the precise chemical structure of this toxin has remained elusive, although the discovery of its biosynthetic gene cluster more than a decade ago (4) has guided investigations into its post-translational modification and likely heterocyclic nature (6, 7).Through recent comparative genomic analysis, an SLS-type gene cluster was identified in clostridia species including Clostridium botulinum and Clostridium sporogenes, two diseasecausing bacteria known to endanger food supplies (8 -10). Similar gene clusters were found in Staphylococcus aureus RF122, Bacillus thuringiensis, Streptococcus iniae, and Listeria monocytogenes 4b. The L. monocytogenes 4b strain is the primary serotype and causative agent for outbreaks of listeriosis (11). The S. aureus RF122 strain is responsible for bovine mastitis. S. iniae is a cytotoxic fish pathogen. This suggests that a shared metabolic output of these pathogens including SLS-like toxins may contribute to their pathogenic potential (11).Each of these SLS family gene clusters contains a similar set of genes. For instance, in C. botulinum and C. sporogenes, the closA-I genes are related by sequence to sagA-I from GAS (see Fig. 1A). Of these genes, ClosF is a protein of unknown function. ClosG, -H, and -I are ABC transporters and therefore are likely to be responsible for exporting the mature hemolytic product. ClosA is the prepropeptide (structural peptide) that is post-translationally modified to form the propeptide. ClosE has been annotated as an i...

Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).