Peptide Synthetase Clusters Research Articles

The pan-genome of Mycobacterium avium subsp. paratuberculosis (Map) confirms ancestral lineage and reveals gene rearrangements within Map Type S

BackgroundTo date genomic studies on Map have concentrated on Type C strains with only a few Type S strains included for comparison. In this study the entire pan-genome of 261 Map genomes (205 Type C, 52 Type S and 4 Type B) and 7 Mycobacterium avium complex (Mac) genomes were analysed to identify genomic similarities and differences between the strains and provide more insight into the evolutionary relationship within this Mycobacterial species.ResultsOur analysis of the core genome of all the Map isolates identified two distinct lineages, Type S and Type C Map that is consistent with previous phylogenetic studies of Map. Pan-genome analysis revealed that Map has a larger accessory genome than Mycobacterium avium subsp. avium (Maa) and Type C Map has a larger accessory genome than Type S Map. In addition, we found large rearrangements within Type S strains of Map and little to none in Type C and Type B strains. There were 50 core genes identified that were unique to Type S Map and there were no unique core genes identified between Type B and Type C Map strains. In Type C Map we identified an additional CE10 CAZyme class which was identified as an alpha/beta hydrolase and an additional polyketide and non-ribosomal peptide synthetase cluster. Consistent with previous analysis no plasmids and only incomplete prophages were identified in the genomes of Map. There were 45 hypothetical CRISPR elements identified with no associated cas genes.ConclusionThis is the most comprehensive comparison of the genomic content of Map isolates to date and included the closing of eight Map genomes. The analysis revealed that there is greater variation in gene synteny within Type S strains when compared to Type C indicating that the Type C Map strain emerged after Type S. Further analysis of Type C and Type B genomes revealed that they are structurally similar with little to no genetic variation and that Type B Map may be a distinct clade within Type C Map and not a different strain type of Map. The evolutionary lineage of Maa and Map was confirmed as emerging after M. hominissuis.

GPCR affecting fatty acid amide produced by non-ribosomal peptide synthetase cluster

GPCR affecting fatty acid amide produced by non-ribosomal peptide synthetase cluster

Identification, phylogenetic analysis, and genome mining of the tetracycline-resistant Bacillus thuringiensis strain m401 reveal its potential for biotechnological and biocontrol applications

Bacillus thuringiensis is an entomopathogen belonging to the Bacillus cereus clade. We isolated a tetracycline-resistant strain called m401, recovered it from honey, and identified it as Bacillus thuringiensis sv. kumamotoensis based on the average nucleotide identity calculations (ANIb) comparison and the analysis of the gyrB gene sequences of different B. thuringiensis serovars. Sequences with homology to virulence factors [cytK, nheA, nheB, nheC, hblA, hblB, hblC, hblD, entFM, and inhA] and tetracycline resistance genes [tet(45), tet(V), and tet(M)/tet(W)/tet(O)/tet(S) family] were identified in the bacterial chromosome. The prediction of plasmid-coding regions revealed homolog sequences to the MarR and TetR/AcrR family of transcriptional regulators, toxins, and lantipeptides. The genome mining analysis revealed 12 regions of biosynthetic gene clusters responsible for synthesizing secondary metabolites. We identified biosynthetic gene clusters coding for bacteriocins, siderophores, ribosomally synthesized post-translationally modified peptide products, and non-ribosomal peptide synthetase clusters that provide evidence for the possible use of Bt m401 as a biocontrol agent. Furthermore, Bt m401 showed high inhibition against all Paenibacillus larvae genotypes tested in vitro. In conclusion, Bt m401 owns various genes involved in different biological processes, such as transductional regulators associated with antibiotic resistance, toxins, and antimicrobial peptides with potential biotechnological and biocontrol applications.

Genome analysis of secondary metabolite‑biosynthetic gene clusters of Photorhabdus akhurstii subsp. akhurstii and its antibacterial activity against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.

Complete genome sequence of the deep South China Sea-derived Streptomyces niveus SCSIO 3406, the producer of cytotoxic and antibacterial marfuraquinocins.

Streptomyces niveus SCSIO 3406 was isolated from a sediment sample collected from South China Sea at a depth of 3536 m. Four new sesquiterpenoid naphthoquinones, marfuraquinocins A-D, and two new geranylated phenazines, i. e. phenaziterpenes A and B, were isolated from the fermentation broth of the strain. Here, we present its genome sequence, which contains 7,990,492 bp with a G+C content of 70.46% and harbors 7088 protein-encoding genes. The genome sequence analysis revealed the presence of a 28,787 bp gene cluster encoding for 24 open reading frames including 1,3,6,8-tetrahydroxynaphthalene synthase and monooxygenase, seven phenazine biosynthesis proteins, two prenyltransferases and a squalene-hopene cyclase. These genes are known to be necessary for the biosynthesis of both marfuraquinocins and phenaziterpenes. Outside the gene cluster (and scattered around the genome), there are seven genes belonging to the methylerythritol phosphate pathway for the biosynthesis of the essential primary metabolite, isopentenyl diphosphate, as well as six geranyl diphosphate/farnesyl diphosphate synthase genes. The strain S. niveus SCSIO 3406 showed type I PKS, type III PKS and nonribosomal peptide synthetase cluster. The sequence will provide the genetic basis for better understanding of biosynthesis mechanism of the above mentioned six compounds and for the construction of improved strain for the industrial production of antimicrobial agents.

The complete genome sequence of the nitrile biocatalyst Rhodococcus rhodochrous ATCC BAA-870

BackgroundRhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst.ResultsThe genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase.Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci.ConclusionsThe sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.

Pseudomonas sp. COW3 Produces New Bananamide-Type Cyclic Lipopeptides with Antimicrobial Activity against Pythium myriotylum and Pyricularia oryzae.

Pseudomonas species are metabolically robust, with capacity to produce secondary metabolites including cyclic lipopeptides (CLPs). Herein we conducted a chemical analysis of a crude CLP extract from the cocoyam rhizosphere-derived biocontrol strain Pseudomonas sp. COW3. We performed in silico analyses on its whole genome, and conducted in vitro antagonistic assay using the strain and purified CLPs. Via LC-MS and NMR, we elucidated the structures of four novel members of the bananamide group, named bananamides D-G. Besides variability in fatty acid length, bananamides D-G differ from previously described bananamides A-C and MD-0066 by the presence of a serine and aspartic acid at position 6 and 2, respectively. In addition, bananamide G has valine instead of isoleucine at position 8. Kendrick mass defect (KMD) allowed the assignment of molecular formulae to bananamides D and E. We unraveled a non-ribosomal peptide synthetase cluster banA, banB and banC which encodes the novel bananamide derivatives. Furthermore, COW3 displayed antagonistic activity and mycophagy against Pythium myriotylum, while it mainly showed mycophagy on Pyricularia oryzae. Purified bananamides D-G inhibited the growth of P. myriotylum and P. oryzae and caused hyphal distortion. Our study shows the complementarity of chemical analyses and genome mining in the discovery and elucidation of novel CLPs. In addition, structurally diverse bananamides differ in their antimicrobial activity.

Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

Genome Sequence of Dickeya solani, a New soft Rot Pathogen of Potato, Suggests its Emergence May Be Related to a Novel Combination of Non-Ribosomal Peptide/Polyketide Synthetase Clusters

Soft rot Enterobacteria in the genera Pectobacterium and Dickeya cause rotting of many crop plants. A new Dickeya isolate has been suggested to form a separate species, given the name Dickeya solani. This bacterium is spreading fast and replacing the closely related, but less virulent, potato pathogens. The genome of D. solani isolate D s0432-1 shows highest similarity at the nucleotide level and in synteny to D. dadantii strain 3937, but it also contains three large polyketide/fatty acid/non-ribosomal peptide synthetase clusters that are not present in D. dadantii 3937. These gene clusters may be involved in the production of toxic secondary metabolites, such as oocydin and zeamine. Furthermore, the D. solani genome harbors several specific genes that are not present in other Dickeya and Pectobacterium species and that may confer advantages for adaptation to new environments. In conclusion, the fast spreading of D. solani may be related to the acquisition of new properties that affect its interaction with plants and other microbes in the potato ecosystem.

Genomic analysis and secondary metabolite production in Bacillus amyloliquefaciens AS 43.3: A biocontrol antagonist of Fusarium head blight

The complete genome of the biocontrol antagonist Bacillus amyloliquefaciens AS 43.3 is reported. B. amyloliquefaciens AS 43.3 has previously been shown to be effective in reducing Fusarium head blight in wheat. The 3.9Mbp genome was sequenced, assembled, and annotated. Genomic analysis of the strain identified 9 biosynthetic gene clusters encoding secondary metabolites associated with biocontrol activity. The analysis identified five non-ribosomal peptide synthetase clusters encoding three lipopeptides (surfactin, iturin, and fengycin), a siderophore (bacillibactin), and the antibiotic dipeptide bacilysin. In addition, three polyketide synthetase clusters were identified which encoded for the antibacterials: bacillaene, difficidin, and macrolactin. In addition to the non-ribosomal mediated biosynthetic clusters discovered, we identified a ribosomally encoded biosynthetic cluster that produces the antibiotic plantazolicin. To confirm the gene clusters were functional, cell-free culture supernatant was analyzed using LC–MS/MS. The technique confirmed the presence of all nine metabolites or their derivatives. The study suggests the strain is most likely a member of the B. amyloliquefaciens subsp. plantarium clade. Comparative genomics of eight completed genomes of B. amyloliquefaciens identify the core and pan-genomes for the species, including identifying genes unique to the biocontrol strains. This study demonstrates the growing importance of applying genomic-based studies to biocontrol organisms of plant pathogens which can enable the rapid identification of bioactive metabolites produced by a prospective biological control organism. In addition, this work provides a foundation for a mechanistic understanding of the B. amyloliquefaciens AS 43.3/Fusarium head blight biocontrol interaction.

Diversity of Microcystis aeruginosa in the Klamath River and San Francisco Bay delta, California, USA

Blooms of the toxin-producing cyanobacterium Microcystis aeruginosa have recently appeared in the Klamath River (KR) and San Francisco Bay delta (SFBD), California, USA. We investigated Microcystis diversity in these systems by targeting cpcBA (phycocyanin gene intergenic spacer and flanking regions) and mcyA gene (encodes part of a peptide synthetase cluster for production of the toxin microcystin). Distinct differences in Microcystis populations in the KR reservoirs (Copco and Iron Gate reservoirs) and SFBD were found in both gene loci, and diversity in the mcyA gene discriminated the populations in the 2 ecosystems entirely. The cpcBA sequences from KR fell into 2 main clusters, and were closely similar to sequences from North and South America, Europe, Asia, and Africa. The majority of the cpcBA sequences in populations from SFBD formed a unique group, while the remaining sequences were closely similar to those from KR. Salinity, soluble reactive phosphorus concentration, pH, water transparency, and NH 4 + and NO 3 - + NO 2 - concentrations were significantly different in the 2 systems. The consistent differences in the 2 genetic markers between KR and SFBD populations suggest that Microcystis populations in the 2 watersheds have had limited connectivity or a different initial source population, or that environmental selection is creating distinct Microcystis populations in the eutrophic KR freshwater reservoirs and the saltwater influenced SFBD. Although Microcystis is globally distributed in temporal and subtropical climates, this study suggests local microdiversity exists and may be linked with environmental regulation.

Type II thioesterase from Streptomyces coelicolor A3(2).

Type I polyketide synthases (PKSs) are complexes of large, multimodular enzymes that catalyse biosynthesis of polyketide compounds via repetitive reaction sequences, during which each step is catalysed by a separate enzymic domain. Many type I PKSs, and also non-ribosomal peptide synthetase clusters, contain additional thioesterase genes located adjacent to PKS genes. These are discrete proteins called type II thioesterases (TE IIs) to distinguish them from chain-terminating thioesterase (TE I) domains that are usually fused to the terminal PKS module. A gene of a new TE II, scoT, associated with the cluster of putative type I PKS genes from Streptomyces coelicolor A3(2), was found. The deduced amino acid sequence of the gene product shows extensive similarity to other authentic thioesterase enzymes, including conservation of characteristic motifs and residues involved in catalysis. When expressed in the heterologous host Streptomyces fradiae, scoT successfully complemented the resident TE II gene (tylO), and, by restoring a significant level of macrolide production, proved to be catalytically equivalent to the TylO protein. S1 nuclease mapping of scoT revealed a single potential transcription start point with expression being switched on for a short period of time during a transition phase of growth.