Nonribosomal Peptide Synthetase Gene Clusters Research Articles

Orfamide production in Pseudomonas protegens CHA0T promotes rhizospheric colonization and influences assemblage of the bacterial community of wheat roots in soil.

Orfamides (Ofa) are a family of closely related cyclic lipopeptides (cLPs) produced by different soil-inhabiting and plant beneficial strains of the Pseudomonas protegens subgroup. Orfamides are required for swarming motility, they mediate toxicity on oomycetes, algae, and insects, and they can act as ISR elicitors. Ofa biosynthesis depends on a non-ribosomal peptide synthetase gene cluster whose expression is tightly controlled by the post-transcriptional regulatory cascade Gac-Rsm. Although the biological properties and physiological role of Ofa have been well characterized in vitro, the role of Ofa in the fitness of the producing bacterium in its niche has not been studied so far. Here, we used an Ofa biosynthesis-deficient mutant (ΔofaABC) derived from the biocontrol model strain P. protegens CHA0T to explore the relevance of this cLP in soil for the survival of strain CHA0T, its contribution to the bacterial competitiveness for colonization of wheat roots, and its impact on the corresponding rhizobacterial community structure. To facilitate CFU counts in non-sterile soil and root samples we used chromosomally tagged versions of CHA0T and its ΔofaABC mutant with mini-Tn7 constructs carrying antibiotic resistance markers. We confirmed that the tagged Ofa− mutant does not show drop-collapse activity neither it can swarm in semi-solid medium. We also confirmed that Ofa can be exploited in vitro as a social good by the Ofa− mutant to resume swarming in the presence of CHA0T cells. We found that Ofa production did not confer any advantage for bacterial survival along a period of 5 weeks after soil inoculation. However, we observed that Ofa production was associated with a higher competitiveness for colonization of the wheat rhizosphere. Expression of the ofa biosynthetic operon, by means of a transcriptional Pofa-gfp reporter fusion, was detected in the rhizoplane of colonized wheat roots, suggesting that Ofa is produced in situ on the surface of colonized wheat roots. Finally, we observed that seed inoculation with the wild type Ofa producer strain CHA0T resulted in a differential assemblage of the bacterial community of the wheat rhizosphere with respect to that of seeds bacterized with the Ofa deficient mutant. Overall, we provide evidence that Ofa is important for the competitiveness of P. protegens CHA0T cells to colonize the surface of wheat roots and that Ofa production in the rhizosphere can impact the structure of the assembled bacterial microbiome.

Decoding the chemical language of Suillus fungi: genome mining and untargeted metabolomics uncover terpene chemical diversity.

Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) potentially involved in fungal competition and communication. However, the functionality of these BGCs is not known. This study employed co-culture techniques to activate BGC expression and then used metabolomics to investigate the diversity of metabolic products produced by three Suillus species (Suillus hirtellus EM16, Suillus decipiens EM49, and Suillus cothurnatus VC1858), core members of the pine microbiome. After 28 days of growth on solid media, liquid chromatography-tandem mass spectrometry identified a diverse range of extracellular metabolites (exometabolites) along the interaction zone between Suillus co-cultures. Prenol lipids were among the most abundant chemical classes. Out of the 62 unique terpene BGCs predicted by genome mining, 41 putative prenol lipids (includes 37 putative terpenes) were identified across the three Suillus species using metabolomics. Notably, some terpenes were significantly more abundant in co-culture conditions. For example, we identified a metabolite matching to isomers isopimaric acid, sandaracopimaric acid, and abietic acid, which can be found in pine resin and play important roles in host defense mechanisms and Suillus spore germination. This research highlights the importance of combining genomics and metabolomics to advance our understanding of the chemical diversity underpinning fungal signaling and communication.IMPORTANCEUsing a combination of genomics and metabolomics, this study's findings offer new insights into the chemical diversity of Suillus fungi, which serve a critical role in forest ecosystems.

Complete Genome Sequence and Pan-Genome Analysis of Shewanella oncorhynchi Z-P2, a Siderophore Putrebactin-Producing Bacterium.

In this study, we reported the complete genome sequence of Shewanella oncorhynchi for the first time. S. oncorhynchi Z-P2 is a bacterium that produces the siderophore putrebactin. Its genome consists of a circular chromosome of 5,034,612 bp with a G + C content of 45.4%. A total of 4544 protein-coding genes, 109 tRNAs and 31 rRNAs were annotated by the RAST. Five non-ribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) gene clusters were identified by the antiSMASH analysis. The pan-genome analysis of Z-P2 and 10 Shewanella putrefaciens revealed 9228 pan-gene clusters and 2681 core gene clusters, with Z-P2 having 618 unique gene clusters. Additionally, the gene cluster involved in putrebactin biosynthesis in Z-P2 was annotated, and the mechanism of putrebactin biosynthesis was analyzed. The putrebactin produced by Z-P2 was detected using UPLC-MS analysis, with an [M + H]+ molecular ion at m/z 373.21. These findings provide valuable support for further research on the genetic engineering of putrebactin biosynthetic genes of Z-P2 and their potential applications.

A small step to discover candidate biological control agents from preexisting bioresources by using novel nonribosomal peptide synthetases hidden in activated sludge metagenomes.

Biological control agents (BCAs), beneficial organisms that reduce the incidence or severity of plant disease, have been expected to be alternatives to replace chemical pesticides worldwide. To date, BCAs have been screened by culture-dependent methods from various environments. However, previously unknown BCA candidates may be buried and overlooked because this approach preferentially selects only easy-to-culture microbial lineages. To overcome this limitation, as a small-scale test case, we attempted to explore novel BCA candidates by employing the shotgun metagenomic information of the activated sludge (AS) microbiome, which is thought to contain unutilized biological resources. We first performed genome-resolved metagenomics for AS taken from a municipal sewage treatment plant and obtained 97 nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS)-related gene sequences from 43 metagenomic assembled bins, most of which were assigned to the phyla Proteobacteria and Myxococcota. Furthermore, these NRPS/PKS-related genes are predicted to be novel because they were genetically dissimilar to known NRPS/PKS gene clusters. Of these, the condensation domain of the syringomycin-related NRPS gene cluster was detected in Rhodoferax- and Rhodocyclaceae-related bins, and its homolog was found in previously reported AS metagenomes as well as the genomes of three strains available from the microbial culture collections, implying their potential BCA ability. Then, we tested the antimicrobial activity of these strains against phytopathogenic fungi to investigate the potential ability of BCA by in vitro cultivation and successfully confirmed the actual antifungal activity of three strains harboring a possibly novel NRPS gene cluster. Our findings provide a possible strategy for discovering novel BCAs buried in the environment using genome-resolved metagenomics.

Profile of PKS and NRPS Gene Clusters in the Genome of Streptomyces cellostaticus NBRC 12849T

Polyketides and nonribosomal peptides are major secondary metabolites in members of the genus Streptomyces. Streptomyces cellostaticus is a validly recognized species and the type strain produces cellostatin. However, little is known about whether it has the potential to produce diverse polyketides and nonribosomal peptides. Here, we sequenced the whole genome of S. cellostaticus NBRC 12849T and surveyed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters in the genome. The genome encoded 12 PKS, one NRPS and eight hybrid PKS/NRPS gene clusters. Among the 21 gene clusters, products of 10 gene clusters were annotated to be an annimycin congener, fuelimycins, lankamycin, streptovaricin, spore pigment, flaviolin, foxicin, blasticidin, lankacidin and an incarnatapeptine congener via our bioinformatic analysis. Although the other clusters were orphan and their products were unknown, five of them were predicted to be compounds derived from two independent diketides, a tridecaketide, a triketide and a tetraketide with a cysteine residue, respectively. These results suggest that S. cellostaticus is a source of diverse polyketides and hybrid polyketide/nonribosomal peptides, including unknown and new secondary metabolites.

A Staphylococcus capitis strain with unusual bacteriocin production.

Staphylococcus capitis is a member of the human and mammal skin microbiomes and is considered less harmful than Staphylococcus aureus. S. capitis subsp. urealyticus BN2 was isolated from a cat and expressed strong antibacterial activity against a range of Gram-positive species, most notably including S. aureus strains with resistance to methicillin (MRSA) and strains with intermediate resistance to vancomycin (VISA). These latter strains are normally relatively resistant to bacteriocins, due to cell wall and cell membrane modifications. Genomic sequencing showed that the strain harboured at least two complete gene clusters for biosynthesis of antagonistic substances. The complete biosynthetic gene cluster of the well-known lantibiotic gallidermin was encoded on a large plasmid and the mature peptide was present in isopropanol cell extracts. In addition, a chromosomal island contained a novel non-ribosomal peptide synthetase (NRPS) gene cluster. Accidental deletion of two NRPS modules and partial purification of the anti-VISA activity showed that this novel bacteriocin represents a complex of differently decorated, non-ribosomal peptides. Additionally, a number of phenol-soluble modulins (PSMs) was detected by mass spectrometry of whole cells. Producing these compounds, the strain was able to outcompete several S. aureus strains, including MRSA and VISA, in tube cultures.

Brevibacillus brevis HNCS-1: a biocontrol bacterium against tea plant diseases.

As a biocontrol bacteria, Brevibacillus has been the subject of extensive research for agricultural applications. Antibacterial peptides (AMPs) are the main antibacterial products of Brevibacillus. This study isolated a strain of Br. brevis HNCS-1 from tea garden soil, and the strain has an antagonistic effect against five types of pathogens of tea diseases, namely Gloeosporium theae-sinensis, Elsinoe leucospira, Phyllosticta theaefolia, Fusarium sp., and Cercospora theae. To determine the genetic characteristics implicated in the biocontrol mechanism, the genome sequence of the HNCS-1 strain was obtained and analyzed further, and the data are deposited in the GenBank repository (No. CP128411). Comparative genomics analyses revealed that the HNCS-1 strain and 17 public Br. brevis share a core genome composed of 3,742 genes. Interestingly, only one non-ribosomal peptide synthetase (NRPS) gene cluster annotated as edeine is present in the core genome. And UHPLC-MS/MS detection results showd that edeine B and edeine A were the principal antibacterial peptides in the HNCS-1 strain. This study proves that edeine is the main antibacterial peptide of Br. brevis, and provides a new strategy for the identification of antibacterial products from other biocontrol bacteria.

Cyclic Peptides from the Opportunistic Pathogen Basidiobolus meristosporus.

Three new cyclic peptides, meristosporins A, B, and C (1-3), one of which features an unusual amino acid, were isolated from the opportunistic pathogen Basidiobolus meristosporus and identified by 1D, 2D NMR, MS/MS, and Marfey's analysis. The biosynthetic pathway of the hexapeptide meristosporin A (1) was deduced based on nonribosomal peptide synthetase gene clusters analysis. Compounds 1 and 2 showed cytotoxicity to RAW264.7 and 293T cells, respectively. These compounds may be involved in the fungal injury caused to human cells.

Detection of non-ribosomal and polyketide biosynthetic genes in bacteria from green mud crab Scylla serrata gut microbiome and their antagonistic activities

Multi-modular enzyme complexes known as non-ribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) have been widely reported in bacteria that produce secondary bioactive metabolites such as non-ribosomal peptides (NRPs) and polyketides (PKs), respectively. These NRPS/PKS pathways contribute to synthesizing several antibiotics, such as vancomycin, rifamycin, and bleomycin, which are vital in human medicine. The present study aimed to isolate gut-associated bacteria from mud crab Scylla serrata, and detect NRPS and PKS gene clusters associated with it. This study included 36 bacterial isolates from five mud crab gut samples. Biosynthetic gene clusters (NRPS and PKS), were detected by PCR using degenerative primers specific to these genes. Three isolates (FKP2–4, FKP4–1, and FKP2–16) were positive for NRPS and two for PKS (FKP2–4 and FKP4–1) genes. The isolates were subjected to 16S rRNA gene amplification and sequenced. In silico analysis of the sequences using the Basic Local Alignment Search Tool (BLAST) identified the isolates FKP2–4, FKP4–1, and FKP2–16 as Acinetobacter variabilis, Vagococcus fluvialis, and Staphylococcus arlettae, respectively, after comparing with the existing sequences available in the National Center for Biotechnology Information (NCBI) database. Compared to the control, it was observed that these isolates exhibited intriguing antagonistic activities against Escherichia coli and Staphylococcus aureus. However, these isolates failed to show significant activity against Candida albicans. Exopolysaccharide production by the isolated organisms was tested using Zobell marine agar (ZMA) with 5% sucrose, but none of the colonies were mucoid or slimy.

Taxonogenomic Analysis of Marine-Derived Streptomyces sp. N11-50 and the Profile of NRPS and PKS Gene Clusters

Streptomyces sp. N11-50 was isolated from deep-sea water and found to produce diketopiperazine (DKP) compounds such as albonoursin and cyclo(Phe-Leu). This study aimed to reveal the potential to synthesize diverse nonribosomal peptide and polyketide compounds as the other secondary metabolites different from DKP after clarifying the taxonomic position. Strain N11-50 was identified as Streptomyces albus, as it showed 100% 16S rRNA gene sequence similarities and 95.5% DNA–DNA relatedness to S. albus NBRC 13014T. We annotated the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene clusters in the genome. Consequently, five NRPS, one hybrid PKS/NRPS, five type-I PKS and one type-II PKS gene clusters were observed, of which we predicted the products through bioinformatic analysis. These gene clusters were well conserved in already whole-genome sequence (WGS)-published strains belonging to S. albus. On the other hand, our taxonogenomic analysis revealed that three WGS-published S. albus strains were not S. albus. Two of the three should be classified as Streptomyces albidoflavus, and the remaining one was likely a new genomospecies. After reclassifying these appropriately, we demonstrated species-specific profiles of the NRPS and PKS gene clusters with little strain-level diversities.

Genome-Guided Discovery of the Myxobacterial Thiolactone-Containing Sorangibactins.

In this study, an unprecedented myxobacterial siderophore termed sorangibactin was discovered by heterologous expression of a coelibactin-like nonribosomal peptide synthetase (NRPS) gene cluster from the Sorangiineae strain MSr11367 in the host Myxococcus xanthus DK1622. De novo structure elucidation uncovered a linear polycyclic structure consisting of an N-terminal phenol group, an oxazole, tandem N-methyl-thiazolidines, and an unusual C-terminal γ-thiolactone moiety. Except for the unprecedented oxazoline dehydrogenation to form an oxazole, which we show to be catalyzed by a cytochrome P450-dependent enzyme, other tailoring steps were found necessary for efficient downstream processing. The unusual thioesterase (TE) domain is proposed to select homocysteine or methionine for offloading involving an intramolecular γ-thiolactone formation. Its active site comprises a rare cysteine, which was found essential for product formation by point mutation to alanine or serine, which both abolished its activity. This unusual release mechanism and the resulting rare thiolactone structure can serve as a starting point for detailed biochemical investigations.

Classification and Secondary Metabolite-Biosynthetic Gene Clusters of Marine Streptomyces Strains Including a Lobophorin- and Divergolide-Producer

Two Streptomyces strains, named N11-26 and DC10-5, were isolated from deep-sea and non-photosynthetic stony coral, respectively. Strain N11-26 produces lobophorin C and divergolides, which are antimicrobial substances. This study aimed to classify these strains and reveal their cryptic potential to synthesize other secondary metabolites, such as polyketides and nonribosomal peptides. Strains N11-26 and DC10-5 showed 16S rRNA gene sequence similarities of 100% and 99.9% to Streptomyces olivaceus NRRL B-3009T, respectively. By digital DNA–DNA hybridization using whole-genome sequences, these strains were classified as Streptomyces olivaceus. Strain N11-26 was closer to the type strain of S. olivaceus than strain DC10-5 and possessed 17 clusters of polyketide synthase (PKS) and/or nonribosomal peptide synthetases (NRPS) genes, whereas strain DC10-5 harbored 19 clusters. Putative products by these gene clusters were predicted by bioinformatic analyses. Although 15 clusters were conserved between the two strains, two and four clusters were specific in strains N11-26 and DC10-5, respectively. This represents a diversity of potential polyketide and nonribosomal peptide compounds between strains of S. olivaceus. To the best of our knowledge, this is the first report annotating all the PKS and NRPS gene clusters in S. olivaceus strains with their putative products to provide useful information for genome mining.

Description of two novel anaerobic members in the family Clostridiaceae, Anaeromonas gelatinilytica gen.nov., sp. nov., and Anaeromonas frigoriresistens sp. nov., isolated from saline lake sediment.

Cells of members of the family Clostridiaceae, phylum Firmicutes, are generally obligate anaerobic rods. Strains D2Q-14T and D2Q-11T were isolated from sediment of the saline lake Manisi in the Xinjiang Uygur Autonomous Region, PR China. In this study, we employed a polyphasic approach and whole genome analysis of the two isolates. Cells of both isolates were Gram-stain-positive rods that were motile by means of flagella and could utilize sulphate, thiosulphate, elemental sulphur and nitrate as electron acceptors. Phylogenetic analyses based on 16S rRNA gene and whole genome sequences indicated that strains D2Q-14T and D2Q-11T constituted a coherent cluster affiliated to the family Clostridiaceae. In addition, genome analysis revealed that strain D2Q-14Tharboured one nonribosomal peptide synthetase gene cluster, making up 1.4 % of the entire genome. The genome-based analysis, including average nucleotide identity, average amino acid identity and in silico DNA-DNA hybridization, biochemical, phenotypic and chemotaxonomic characterization, indicated that strains D2Q-14T and D2Q-11T represented two novel species of a novel genus in the family Clostridiaceae, for which we propose the names Anaeromonas gelatinilytica gen. nov., sp. nov. and Anaeromonas frigoriresistens sp. nov., with the type strains D2Q-14T (=KCTC 15986T=MCCC 1K04634T) and D2Q-11T (=KCTC 15985T=MCCC 1K04391T), respectively.

Taxonomic Positions of a Nyuzenamide-Producer and Its Closely Related Strains.

Streptomyces sp. N11-34 is a producer of bicyclic peptides named nyuzenamides A and B. We elucidated its taxonomic position and surveyed its nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene clusters by whole genome analysis. Streptomyces sp. N11-34 showed 16S rRNA gene sequence similarities of 99.9% and 99.8% to Streptomyces hygroscopicus NBRC 13472T and Streptomyces demainii NRRL B-1478T, respectively. Although these members formed a clade in a phylogenetic tree based on 16S rRNA gene sequences, the clade split into two closely related subclades in multilocus sequence analysis (MLSA). One included Streptomyces sp. N11-34, S. demainii NRRL B-1478T, S. hygroscopicus NBRC 100766, S. hygroscopicus NBRC 16556 and S. hygroscopicus TP-A0867 and the other comprised S. hygroscopicus NBRC 13472T and S. hygroscopicus NBRC 12859. These phylogenetic relationships were supported by phylogenomic analysis. Although Streptomyces sp. N11-34 was classified to S. hygroscopicus at the species level based on MLSA evolutionary distances and DNA–DNA relatedness, these distances and relatedness of members between the two subclades were comparatively far (0.004–0.006) and low (75.4–76.4%), respectively. Streptomyces sp. N11-34 possessed six NRPS, seven PKS and four hybrid PKS/NRPS gene clusters in the genome. Among the seventeen, ten were identified to be biosynthetic gene clusters (BGCs) of nyuzenamide, echoside, coelichelin, geldanamycin, mediomycin, nigericin, azalomycin, spore pigment, alchivemycin and totopotensamide, whereas the remaining seven were orphan in our bioinformatic analysis. All seventeen are conserved in S. hygroscopicus NBRC 100766, S. hygroscopicus NBRC 16556 and S. hygroscopicus TP-A0867. In contrast, S. hygroscopicus NBRC 13472T and S. hygroscopicus NBRC 12859 lacked the BGCs of alchivemycin, totopotensamide, a nonribosomal peptide and a hybrid polyketide/nonribosomal peptide compound. This difference was in a good accordance with the abovementioned phylogenetic relationship. Based on phenotypic differences in addition to phylogenetic relationship, DNA–DNA relatedness and BGCs, strains of S. hygroscopicus should be reclassified to two subspecies: S. hygroscopicus subsp. hygroscopicus and a new subspecies, for which we proposed S. hygroscopicus subsp. sporocinereus subsp. nov. The type strain is NBRC 100766T (=ATCC 43692T = DSM 41460T = INMI 32T = JCM 9093T = NRRL B-16376T = VKM Ac-312T). S. demainii was classified in this subspecies.

Mining the Genome of Bacillus velezensis VB7 (CP047587) for MAMP Genes and Non-Ribosomal Peptide Synthetase Gene Clusters Conferring Antiviral and Antifungal Activity.

Chemical pesticides have an immense role in curbing the infection of plant viruses and soil-borne pathogens of high valued crops. However, the usage of chemical pesticides also contributes to the development of resistance among pathogens. Hence, attempts were made in this study to identify a suitable bacterial antagonist for managing viral and fungal pathogens infecting crop plants. Based on our earlier investigations, we identified Bacillus amyloliquefaciens VB7 as a potential antagonist for managing Sclerotinia sclerotiorum infecting carnation, tobacco streak virus infecting cotton and groundnut bud necrosis infecting tomato. Considering the multifaceted action of B. amyloliquefaciens VB7, attempts were made for whole-genome sequencing to assess the antiviral activity against tomato spotted wilt virus infecting chrysanthemum and antifungal action against Fusarium oxysporum f. sp. cubense (Foc). Genome annotation of the isolate B. amyloliquefaciens VB7 was confirmed as B. velezensis VB7 with accession number CP047587. Genome analysis revealed the presence of 9,231,928 reads with an average read length of 149 bp. Assembled genome had 1 contig, with a total length of 3,021,183 bp and an average G+C content of 46.79%. The protein-coding sequences (CDS) in the genome was 3090, transfer RNA (tRNA) genes were 85 with 29 ribosomal RNA (rRNA) genes and 21 repeat regions. The genome of B. velezensis VB7 had 506 hypothetical proteins and 2584 proteins with functional assignments. VB7 genome had the presence of flagellin protein FlaA with 987 nucleotides and translation elongation factor TU (Ef-Tu) with 1191 nucleotides. The identified ORFs were 3911 with 47.22% GC content. Non ribosomal pepide synthetase cluster (NRPS) gene clusters in the genome of VB7, coded for the anti-microbial peptides surfactin, butirosin A/butirosin B, fengycin, difficidin, bacillibactin, bacilysin, and mersacidin the Ripp lanthipeptide. Antiviral action of VB7 was confirmed by suppression of local lesion formation of TSWV in the local lesion host cowpea (Co-7). Moreover, combined application of B. velezensis VB7 with phyto-antiviral principles M. Jalapa and H. cupanioides increased shoot length, shoot diameter, number of flower buds per plant, flower diameter, and fresh weight of chrysanthemum. Further, screening for antifungal action of VB7 expressed antifungal action against Foc in vitro by producing VOC/NVOC compounds, including hexadecanoic acid, linoelaidic acid, octadecanoic acid, clindamycin, formic acid, succinamide, furanone, 4H-pyran, nonanol and oleic acid, contributing to the total suppression of Foc apart from the presence of NRPS gene clusters. Thus, our study confirmed the scope for exploring B. velezensis VB7 on a commercial scale to manage tomato spotted wilt virus, groundnut bud necrosis virus, tobacco streak virus, S. sclerotiorum, and Foc causing panama wilt of banana.

Differences at Species Level and in Repertoires of Secondary Metabolite Biosynthetic Gene Clusters among Streptomyces coelicolor A3(2) and Type Strains of S. coelicolor and Its Taxonomic Neighbors

Streptomyces coelicolor A3(2) is used worldwide for genetic studies, and its complete genome sequence was published in 2002. However, as the whole genome of the type strain of S. coelicolor has not been analyzed, the relationship between S. coelicolor A3(2) and the type strain is not yet well known. To clarify differences in their biosynthetic potential, as well as their taxonomic positions, we sequenced whole genomes of S. coelicolor NBRC 12854T and type strains of its closely related species—such as Streptomyces daghestanicus, Streptomyces hydrogenans, and Streptomyces violascens—via PacBio. Biosynthetic gene clusters for polyketides and non-ribosomal peptides were surveyed by antiSMASH, followed by bioinformatic analyses. Type strains of Streptomyces albidoflavus, S. coelicolor, S. daghestanicus, S. hydrogenans, and S. violascens shared the same 16S rDNA sequence, but S. coelicolor A3(2) did not. S. coelicolor A3(2) and S. coelicolor NBRC 12854T can be classified as Streptomycesanthocyanicus and S. albidoflavus, respectively. In contrast, S. daghestanicus, S. hydrogenans, and S. violascens are independent species, despite their identical 16S rDNA sequences. S. coelicolor A3(2), S. coelicolor NBRC 12854T, S. daghestanicus NBRC 12762T, S. hydrogenans NBRC 13475T, and S. violascens NBRC 12920T each harbor specific polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) gene clusters in their genomes, whereas PKS and NRPS gene clusters are well conserved between S. coelicolor A3(2) and S. anthocyanicus JCM 5058T, and between S. coelicolor NBRC 12854T and S. albidoflavus DSM 40455T, belonging to the same species. These results support our hypothesis that the repertoires of PKS and NRPS gene clusters are different between different species.

Comprehensive Genomic Analysis of the Endophytic Bacillus altitudinis Strain GLB197, a Potential Biocontrol Agent of Grape Downy Mildew.

Bacillus has been extensively studied for agricultural application as a biocontrol agent. B. altitudinis GLB197, an endophytic bacterium isolated from grape leaves, exhibits distinctive inhibition to grape downy mildew based on unknown mechanisms. To determine the genetic traits involved in the mechanism of biocontrol and host-interaction traits, the genome sequence of GLB197 was obtained and further analyzed. The genome of B. altitudinis GLB197 consisted of one plasmid and a 3,733,835-bp circular chromosome with 41.56% G + C content, containing 3,770 protein-coding genes. Phylogenetic analysis of 17 Bacillus strains using the concatenated 1,226 single-copy core genes divided into different clusters was conducted. In addition, average nucleotide identity (ANI) values indicate that the current taxonomy of some B. pumilus group strains is incorrect. Comparative analysis of B. altitudinis GLB197 proteins with other B. altitudinis strains identified 3,157 core genes. Furthermore, we found that the pan-genome of B. altitudinis is open. The genome of B. altitudinis GLB197 contains one nonribosomal peptide synthetase (NRPS) gene cluster which was annotated as lichenysin. Interestingly, the cluster in B. altitudinis has two more genes than other Bacillus strains (lgrD and lgrB). The two genes were probably obtained via horizontal gene transfer (HGT) during the evolutionary process from Brevibacillus. Taken together, these observations enable the future application of B. altitudinis GLB197 as a biocontrol agent for control of grape downy mildew and promote our understanding of the beneficial interactions between B. altitudinis GLB197 and plants.

Bioactivities of endophytic actinobacteria inhabiting Artemisia herba-alba emphasizing differences from free-living strains.

The endophytic actinobacteria associated with Artemisia herba-alba (synonym: Seriphidium herba-alba) are highly diverse. This study aimed to illustrate the extent of their differences from the free-living actinobacteria in the surrounding environment. A selection of eighteen actinobacteria inhabiting A. herba-alba were compared with twenty and ten actinobatceria isolates from the surrounding desert and groundwater, respectively, representing six genera. Antagonistic and enzymatic activities, plant growth-promoting traits, and the occurrence of biosynthetic genes were compared among the isolates. Data were analyzed statistically using principal component analysis (PCA) and were visualized using heat map. Endophytic strains showed higher antimicrobial activity and production of plant growth promoters compared to desert and groundwater strains. Polyketide synthase and non-ribosomal peptide synthetase gene clusters were detected at higher frequencies in the endophytic strains (8 and 11 strains, respectively) than the desert strains (1 and 2 strains, respectively). In contrast, both gene clusters were not detected in the groundwater strains. The PCA revealed unique metabolic characteristics of the endophytes. The heatmap clustered the endophytic strains apart from the free-living strains, indicating distinctive qualitative and quantitative bioactivities. Analysis of 16S rRNA genes confirmed the chemotaxonomic identity of all but two strains, with > 94.5% similarity. Six endophytes displayed < 99.5% similarity with their closest type strains, which might indicate species novelty. This study provides an evidence of functional differences and possible species novelty of the endophytic actinobacteria inhabiting A. herba-alba, compared with the free-living species.

Identification of Novel Rotihibin Analogues in Streptomyces scabies, Including Discovery of Its Biosynthetic Gene Cluster.

ABSTRACTStreptomyces scabies is a phytopathogen associated with common scab disease. This is mainly attributed to its ability to produce the phytotoxin thaxtomin A, the biosynthesis of which is triggered by cellobiose. During a survey of other metabolites released in the presence of cellobiose, we discovered additional compounds in the thaxtomin-containing extract from Streptomyces scabies. Structural analysis by mass spectrometry (MS) and nuclear magnetic resonance (NMR) revealed that these compounds are amino acid sequence variants of the TOR (target of rapamycin) kinase (TORK) pathway-inhibitory lipopeptide rotihibin A, and the main compounds were named rotihibins C and D. In contrast to thaxtomin, the production of rotihibins C and D was also elicited in the presence of glucose, indicating different regulation of their biosynthesis. Through a combination of shotgun and targeted proteomics, the putative rotihibin biosynthetic gene cluster rth was identified in the publicly available genome of S. scabies 87-22. This cluster spans 33 kbp and encodes 2 different nonribosomal peptide synthetases (NRPSs) and 12 additional enzymes. Homologous rth biosynthetic gene clusters were found in other publicly available and complete actinomycete genomes. Rotihibins C and D display herbicidal activity against Lemna minor and Arabidopsis thaliana at low concentrations, shown by monitoring the effects on growth and the maximal photochemistry efficiency of photosystem II.IMPORTANCE Rotihibins A and B are plant growth inhibitors acting on the TORK pathway. We report the isolation and characterization of new sequence analogues of rotihibin from Streptomyces scabies, a major cause of common scab in potato and other tuber and root vegetables. By combining proteomics data with genomic analysis, we found a cryptic biosynthetic gene cluster coding for enzyme machinery capable of rotihibin production. This work may lead to the biotechnological production of variants of this lipopeptide to investigate the exact mechanism by which it can target the plant TORK pathway in Arabidopsis thaliana. In addition, bioinformatics revealed the existence of other variants in plant-associated Streptomyces strains, both pathogenic and nonpathogenic species, raising new questions about the actual function of this lipopeptide. The discovery of a module in the nonribosomal peptide synthetase (NRPS) that incorporates the unusual citrulline residue may improve the prediction of peptides encoded by cryptic NRPS gene clusters.

Nonribosomal peptide synthetases and nonribosomal cyanopeptides synthesis in Microcystis: A comparative genomics study

Microcystis, the most prevalent bloom-forming cyanobacterial genus, can synthesize a variety of toxic cyanopeptides, such as hepatotoxic microcystins, protease and phosphatase-inhibitory aeruginosins, and cyanopeptolins. Most toxic cyanopeptides are nonribosomal peptides (NRPs) synthesized via nonribosomal peptide synthetase (NRPS) or hybrid NRPS/polyketide synthase pathways. In NRPS modules, adenylation domains (A-domains) can selectively recognize and activate specificity substrates. The various toxicity and enzyme-inhibiting activities of cyanopeptides are mainly decided by the molecules' structure and composition, which can be deduced according to the order and specificity of NRPS modules in NRPS gene clusters using bioinformatics approaches. Here, we performed comparative genomic analyses mainly focused on the distribution of NRPS gene clusters in association with phylogenomic relationships, and on substrate and structural predictions of A-domains in the NRPS modules for nonribosomal cyanopeptide synthesis in 36 Microcystis strains. Aeruginosin, cyanopeptolin, and microcystin biosynthesis gene clusters are the most frequently observed in the Microcystis genomes we analyzed. Phylogenomic affiliation analysis of NRPS gene cluster profiles indicates that Microcystis phylogenomic marker genes and nonribosomal cyanopeptide synthesis genes have coevolved during evolution. The NRPS A-domains for toxic cyanopeptide synthesis were characterized based on NRPS gene clusters analysis. Substrate and structure prediction indicates varied substrates of the A-domains. Selection analysis demonstrates that all A-domains encoded by aer, mcn, and mcy gene clusters are subjected to purifying selection, and positive selection has acted on some residues in core motifs and substrate binding pockets of the A-domains. In summary, our study reveals that the distribution of NRPS gene clusters in Microcystis and the diversity of the nonribosomal cyanopeptides produced relate to an evolutionary history of environmental adaptations.