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Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool.

ABSTRACT: The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, which are proposed to be introduced by YcaO-TfuA proteins. Using RiPPER, we show that previously undescribed RiPP gene clusters encoding YcaO and TfuA proteins are widespread in Actinobacteria and encode a highly diverse landscape of precursor peptides that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new structural class of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

SUBMITTER: Santos-Aberturas J

PROVIDER: S-EPMC6511847 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool.

Santos-Aberturas Javier J Chandra Govind G Frattaruolo Luca L Lacret Rodney R Pham Thu H TH Vior Natalia M NM Eyles Tom H TH Truman Andrew W AW

Nucleic acids research 20190501 9

The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we descr ...[more]

PMID: 30916321

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Project description:The search for novel bioactive metabolites continues to be of much importance around the world for pharmaceutical, agricultural, and industrial applications. Actinobacteria constitute one of the extremely interesting groups of microorganisms widely used as important biological contributors for a wide range of novel secondary metabolites. This study focused on the assessment of antimicrobial and antioxidant activity of crude extracts of actinobacterial strains. Western Ghats of India represents unique regions of biologically diverse areas called "hot spots". A total of 32 isolates were obtained from soil samples of different forest locations of Bisle Ghat and Virjapet situated in Western Ghats of Karnataka, India. The isolates were identified as species of Streptomyces, Nocardiopsis, and Nocardioides by cultural, morphological, and molecular studies. Based on preliminary screening, seven isolates were chosen for metabolites extraction and to determine antimicrobial activity qualitatively (disc diffusion method) and quantitatively (micro dilution method) and scavenging activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS(2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals. Crude extracts of all seven isolates exhibited fairly strong antibacterial activity towards MRSA strains (MRSA ATCC 33591, MRSA ATCC NR-46071, and MRSA ATCC 46171) with MIC varying from 15.62 to 125 μg/mL, whereas showed less inhibition potential towards Gram-negative bacteria Salmonella typhi (ATCC 25241) and Escherichia coli (ATCC 11775) with MIC of 125-500 μg/mL. The isolates namely S1A, SS5, SCA35, and SCA 11 inhibited Fusariummoniliforme (MTCC 6576) to a maximum extent with MIC ranging from 62.5 to 250 μg/mL. Crude extract of SCA 11 and SCA 13 exhibited potent scavenging activities against DPPH and ABTS radicals. The results from this study suggest that actinobacterial strains of Western Ghats are an excellent source of natural antimicrobial and antioxidant compounds. Further research investigations on purification, recovery, and structural characterization of the active compounds are to be carried out.

Project description:Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their host's chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria.

Project description:BACKGROUND: Predicting the function of a protein from its sequence is a long-standing challenge of bioinformatic research, typically addressed using either sequence-similarity or sequence-motifs. We employ the novel motif method that consists of Specific Peptides (SPs) that are unique to specific branches of the Enzyme Commission (EC) functional classification. We devise the Data Mining of Enzymes (DME) methodology that allows for searching SPs on arbitrary proteins, determining from its sequence whether a protein is an enzyme and what the enzyme's EC classification is. RESULTS: We extract novel SP sets from Swiss-Prot enzyme data. Using a training set of July 2006, and test sets of July 2008, we find that the predictive power of SPs, both for true-positives (enzymes) and true-negatives (non-enzymes), depends on the coverage length of all SP matches (the number of amino-acids matched on the protein sequence). DME is quite different from BLAST. Comparing the two on an enzyme test set of July 2008, we find that DME has lower recall. On the other hand, DME can provide predictions for proteins regarded by BLAST as having low homologies with known enzymes, thus supplying complementary information. We test our method on a set of proteins belonging to 10 bacteria, dated July 2008, establishing the usefulness of the coverage-length cutoff to determine true-negatives. Moreover, sifting through our predictions we find that some of them have been substantiated by Swiss-Prot annotations by July 2009. Finally we extract, for production purposes, a novel SP set trained on all Swiss-Prot enzymes as of July 2009. This new set increases considerably the recall of DME. The new SP set is being applied to three metagenomes: Sargasso Sea with over 1,000,000 proteins, producing predictions of over 220,000 enzymes, and two human gut metagenomes. The outcome of these analyses can be characterized by the enzymatic profile of the metagenomes, describing the relative numbers of enzymes observed for different EC categories. CONCLUSIONS: Employing SPs for predicting enzymatic activity of proteins works well once one utilizes coverage-length criteria. In our analysis, L >or= 7 has led to highly accurate results.

Dataset Information

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool.

Publications

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool.

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