Cyanopeptolins are nonribosomally produced heptapetides showing a highly variable composition. The cyanopeptolin synthetase operon has previously been investigated in three strains from the genera Microcystis, Planktothrix and Anabaena. Cyanopeptolins are displaying protease inhibitor activity, but the biological function(s) is (are) unknown. Cyanopeptolin gene cluster variability and biological functions of the peptide variants are likely to be interconnected.
We have investigated two cyanopeptolin gene clusters from highly similar, but geographically remote strains of the same genus. Sequencing of a nonribosomal peptide synthetase (NRPS) cyanopeptolin gene cluster from the Japanese strain Planktothrix NIES 205 (205-oci), showed the 30 kb gene cluster to be highly similar to the oci gene cluster previously described in Planktothrix NIVA CYA 116, isolated in Norway. Both operons contained seven NRPS modules, a sulfotransferase (S) and a glyceric acid loading (GA)-domain. Sequence analyses showed a high degree of conservation, except for the presence of an epimerase domain in NIES 205 and the regions around the epimerase, showing high substitution rates and Ka/Ks values above 1. The two strains produce almost identical cyanopeptolins, cyanopeptolin-1138 and oscillapeptin E respectively, but with slight differences regarding the production of minor cyanopeptolin variants. These variants may be the result of relaxed adenylation (A)-domain specificity in the nonribosomal enzyme complex. Other genetic markers (16S rRNA, ntcA and the phycocyanin cpcBA spacer) were identical, supporting that these geographically separated Planktothrix strains are closely related.
A horizontal gene transfer event resulting in exchange of a whole module-encoding region was observed. Nucleotide statistics indicate that both purifying selection and positive selection forces are operating on the gene cluster. The positive selection forces are acting within and around the epimerase insertion while purifying selection conserves the remaining (major) part of the gene cluster. The presence of an epimerase in the gene cluster is in line with the D-configuration of Htyr, determined experimentally in oscillapeptin E in a previous study.
Besides the most prominent peptide toxin, microcystin, the cyanobacteria Microcystis spp. have been shown to produce a large variety of other bioactive oligopeptides. We investigated for the first time the oligopeptide diversity within a natural Microcystis population by analyzing single colonies directly with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The results demonstrate a high diversity of known cyanobacterial peptides such as microcystins, anabaenopeptins, microginins, aeruginosins, and cyanopeptolins, but also many unknown substances in the Microcystis colonies. Oligopeptide patterns were mostly related to specific Microcystis taxa. Microcystis aeruginosa (Kütz.) Kütz. colonies contained mainly microcystins, occasionally accompanied by aeruginosins. In contrast, microcystins were not detected in Microcystis ichthyoblabe Kütz.; instead, colonies of this species contained anabaenopeptins and/or microginins or unknown peptides. Within a third group, Microcystis wesenbergii (Kom.) Kom. in Kondr., chiefly a cyanopeptolin and an unknown peptide were found. Similar patterns, however, were also found in colonies which could not be identified to species level. The significance of oligopeptides as a chemotaxonomic tool within the genus Microcystis is discussed. It could be demonstrated that the typing of single colonies by MALDI-TOF MS may be a valuable tool for ecological studies of the genus Microcystis as well as in early warning of toxic cyanobacterial blooms.
Aeruginosins are bioactive oligopeptides that are produced in high structural diversity by strains of the bloom-forming cyanobacterial genera Microcystis and Planktothrix. A hallmark of aeruginosins is the unusual Choi moiety central to the tetrapeptides, while other positions are occupied by variable moieties in individual congeners. Here we report on three aeruginosin synthetase gene clusters (aer) of Microcystis aeruginosa (strains PCC 7806, NIES-98, and NIES-843). The analysis and comparison the aer gene clusters provide the first insight into the molecular basis of biosynthetic and structural plasticity in aeruginosin pathways. Major parts of the aer gene clusters are highly similar in all strains, particularly the genes coding for the first three nonribosomal peptide synthetase (NRPS) modules except for the region coding for the second adenylation domain. However, the gene clusters differ largely in genes coding for tailoring enzymes such as halogenases and sulfotransferases, reflecting structural peculiarities in aeruginosin congeners produced by the individual strains. Significant deviations were further observed in the C-terminal NRPS modules, suggesting two distinct release mechanisms. The architecture of the gene clusters is in agreement with the particular aeruginosin variants that are produced by individual strains, the structures of two of which (aeruginosins 686 A and 686 B) were elucidated. The aer gene clusters of Microcystis and Planktothrix are proposed to originate from a common ancestor and to have evolved to their present-day diversity largely through horizontal gene transfer and recombination events.
Cyanobacteria of the genus Microcystis are known to produce secondary metabolites of large structural diversity by nonribosomal peptide synthetase (NRPS) pathways. For a number of such compounds, halogenated congeners have been reported along with nonhalogenated ones. In the present study, chlorinated cyanopeptolin- and/or aeruginosin-type peptides were detected by mass spectrometry in 17 out of 28 axenic strains of Microcystis. In these strains, a halogenase gene was identified between 2 genes coding for NRPS modules in respective gene clusters, whereas it was consistently absent when the strains produced only nonchlorinated corresponding congeners. Nucleotide sequences were obtained for 12 complete halogenase genes and 14 intermodule regions of gene clusters lacking a halogenase gene or containing only fragments of it. When a halogenase gene was found absent, a specific, identical excision pattern was observed for both synthetase gene clusters in most strains. A phylogenetic analysis including other bacterial halogenases showed that the NRPS-related halogenases of Microcystis form a monophyletic group divided into 2 subgroups, corresponding to either the cyanopeptolin or the aeruginosin peptide synthetases. The distribution of these peptide synthetase gene clusters, among the tested Microcystis strains, was found in relative agreement with their phylogeny reconstructed from 16S–23S rDNA intergenic spacer sequences, whereas the distribution of the associated halogenase genes appears to be sporadic. The presented data suggest that in cyanobacteria these prevalent halogenase genes originated from an ancient horizontal gene transfer followed by duplication in the cyanobacterial lineage. We propose an evolutionary scenario implying repeated gene losses to explain the distribution of halogenase genes in 2 NRPS gene clusters that subsequently defines the seemingly erratic production of halogenated and nonhalogenated aeruginosins and cyanopeptolins among Microcystis strains.
halogenase; cyanopeptolin; aeruginosin; DNA rearrangement; secondary peptide metabolite; chlorination; internal transcribed spacer; phylogeny
Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and d-Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from Planktothrix agardhii strain CYA126/8 consisting of five genes (apnA to apnE) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of apnC. In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven Planktothrix strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A1 domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A1 enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids in vitro. Phylogenetic analysis of apn A domain sequences revealed that strains with a promiscuous ApnA A1 domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the apnA A1 gene, suggesting that this remarkable promiscuity has evolved from point mutations only.
The major cyclic peptide cyanopeptolin 1138, produced by Planktothrix strain NIVA CYA 116, was characterized and shown to be structurally very close to the earlier-characterized oscillapeptin E. A cyanopeptolin gene cluster likely to encode the corresponding peptide synthetase was sequenced from the same strain. The 30-kb oci gene cluster contains two novel domains previously not detected in nonribosomal peptide synthetase gene clusters (a putative glyceric acid-activating domain and a sulfotransferase domain), in addition to seven nonribosomal peptide synthetase modules. Unlike in two previously described cyanopeptolin gene clusters from Anabaena and Microcystis, a halogenase gene is not present. The three cyanopeptolin gene clusters show similar gene and domain arrangements, while the binding pocket signatures deduced from the adenylation domain sequences and the additional tailoring domains vary. This suggests loss and gain of tailoring domains within each genus, after the diversification of the three clades, as major events leading to the present diversity. The ABC transporter genes associated with the cyanopeptolin gene clusters form a monophyletic clade and accordingly are likely to have evolved as part of the functional unit. Phylogenetic analyses of adenylation and condensation domains, including domains from cyanopeptolins and microcystins, show a closer similarity between the Planktothrix and Microcystis cyanopeptolin domains than between these and the Anabaena domain. No clear evidence of recombination between cyanopeptolins and microcystins could be detected. There were no strong indications of horizontal gene transfer of cyanopeptolin gene sequences across the three genera, supporting independent evolution within each genus.
The filamentous cyanobacteria Planktothrix spp. occur in the temperate region of the Northern hemisphere. The red-pigmented Planktothrix rubescens bacteria occur in deep, physically stratified, and less eutrophic lakes. Planktothrix is a known producer of the toxic heptapeptide microcystin (MC), which is produced nonribosomally by a large enzyme complex consisting of peptide synthetases and polyketide synthases encoded by a total of nine genes (mcy genes). Planktothrix spp. differ in their cellular MC contents as well as the production of MC variants; however, the mechanisms favoring this diversity are not understood. Recently, the occurrence of Planktothrix strains containing all mcy genes but lacking MC has been reported. In this study, 29 such strains were analyzed to find out if mutations of the mcy genes lead to the inability to synthesize MC. Two deletions, spanning 400 bp (in mcyB; one strain) and 1,869 bp (in mcyHA; three strains), and three insertions (IS), spanning 1,429 bp (in mcyD; eight strains), 1,433 bp (in mcyEG; one strain), and 1,433 bp (in mcyA; one strain), were identified. Though found in different genes and different isolates and transcribed in opposite directions, IS were found to be identical and contained conserved domains assigned to transposable elements. Using mutation-specific primers, two insertions (in mcyD and mcyA) and one deletion (in mcyHA) were found regularly in populations of P. rubescens in different lakes. The results demonstrate for the first time that different mutations resulting in inactivation of MC synthesis do occur frequently and make up a stable proportion of the mcy gene pool in Planktothrix populations over several years.
Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a “protein-first” method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nature Biotechnology, 2009, 27, 951 – 956), e.g. improved de novo peptide sequencing, have enabled the discovery of ten NRPS/PKS gene clusters from six strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously ‘orphan’ gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2–4 Mbp genomes) used previously.
Nonribosomal peptide; polyketide; natural product biosynthesis; Actinobacteria; de novo sequencing; Fourier-Transform Mass Spectrometry
Lysobactor enzymogenes strain OH11 is an emerging biological control agent of fungal and bacterial diseases. We recently completed its genome sequence and found it contains a large number of gene clusters putatively responsible for the biosynthesis of nonribosomal peptides and polyketides, including the previously identified antifungal dihydromaltophilin (HSAF). One of the gene clusters contains two huge open reading frames, together encoding 12 modules of nonribosomal peptide synthetases (NRPS). Gene disruption of one of the NRPS led to the disappearance of a metabolite produced in the wild type and the elimination of its antibacterial activity. The metabolite and antibacterial activity were also affected by the disruption of some of the flanking genes. We subsequently isolated this metabolite and subjected it to spectroscopic analysis. The mass spectrometry and nuclear magnetic resonance data showed that its chemical structure is identical to WAP-8294A2, a cyclic lipodepsipeptide with potent anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and currently in phase I/II clinical trials. The WAP-8294A2 biosynthetic genes had not been described previously. So far, the Gram-positive Streptomyces have been the primary source of anti-infectives. Lysobacter are Gram-negative soil/water bacteria that are genetically amendable and have not been well exploited. The WAP-8294A2 synthetase represents one of the largest NRPS complexes, consisting of 45 functional domains. The identification of these genes sets the foundation for the study of the WAP-8294A2 biosynthetic mechanism and opens the door for producing new anti-MRSA antibiotics through biosynthetic engineering in this new source of Lysobacter.
The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere. We sampled sequenced the genome of FZB42 and identified 2,947 genes with >50% identity on the amino acid level to the corresponding genes of Bacillus subtilis 168. Six large gene clusters encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) occupied 7.5% of the whole genome. Two of the PKS and one of the NRPS encoding gene clusters were unique insertions in the FZB42 genome and are not present in B. subtilis 168. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis revealed expression of the antibiotic lipopeptide products surfactin, fengycin, and bacillomycin D. The fengycin (fen) and the surfactin (srf) operons were organized and located as in B. subtilis 168. A large 37.2-kb antibiotic DNA island containing the bmy gene cluster was attributed to the biosynthesis of bacillomycin D. The bmy island was found inserted close to the fen operon. The responsibility of the bmy, fen, and srf gene clusters for the production of the corresponding secondary metabolites was demonstrated by cassette mutagenesis, which led to the loss of the ability to produce these peptides. Although these single mutants still largely retained their ability to control fungal spread, a double mutant lacking both bacillomycin D and fengycin was heavily impaired in its ability to inhibit growth of phytopathogenic fungi, suggesting that both lipopeptides act in a synergistic manner.
Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.
The oral biofilm community consists of >800 microbial species, among which Streptococcus mutans is considered a primary pathogen for dental caries. The genomic island TnSmu2 of S. mutans comprises >2% of the genome. In this study, we demonstrate that TnSmu2 harbors a gene cluster encoding nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and accessory proteins and regulators involved in nonribosomal peptide (NRP) and polyketide (PK) biosynthesis. Interestingly, the sequences of these genes and their genomic organizations and locations are highly divergent among different S. mutans strains, yet each TnSmu2 region encodes NRPS/PKS and accessory proteins. Mutagenesis of the structural genes and putative regulatory genes in strains UA159, UA140, and MT4653 resulted in colonies that were devoid of their yellow pigmentation (for strains UA140 and MT4653). In addition, these mutant strains also displayed retarded growth under aerobic conditions and in the presence of H2O2. High-performance liquid chromatography profiling of cell surface extracts identified unique peaks that were missing in the mutant strains, and partial characterization of the purified product from UA159 demonstrated that it is indeed a hybrid NRP/PK, as predicted. A genomic survey of 94 clinical S. mutans isolates suggests that the TnSmu2 gene cluster may be more prevalent than previously recognized.
The filamentous cyanobacterium Planktothrix rubescens frequently occurs in deep and stratified lakes in the temperate region of the northern hemisphere and is a known producer of the hepatotoxic secondary metabolite microcystin. These cyclic heptapepids are synthesized non-ribosomally via large enzyme complexes encoded by the microcystin (mcy) synthetase gene cluster. The occurrence of cyanobacterial strains lacking microcystin but containing the mcy gene cluster has been reported repeatedly; it was shown that this inactivation is due to mutations such as gene deletion events and the insertion of transposable elements. In the present study, twelve lakes in Austria, Germany, and Switzerland were sampled from July 2005 to October 2007, and the proportion of inactive mcy genotypes was quantified in relation to the total population of the red-pigmented filamentous cyanobacterium Planktothrix by means of quantitative PCR. In total, four different mutations were quantified, namely two insertions affecting mcyD, one insertion affecting mcyA, and a deletion within mcyH and mcyA. The mutations occurred over a wide range of the population density (40 – 570,000 filaments L−1) and their abundance was found to be positively correlated with population density. However, on average, all nontoxic mutants were found in a low proportion only (min 0%, mean 6.5% ± 1.1 (SE), max 52% of the total population). The genotype containing the mcyHA deletion had a significantly higher proportion (min 0%, mean 3.7% ± 1, max 52%) when compared with all the genotypes containing insertions within the mcy gene cluster (min 0%, mean 2.8% ± 0.7, max 24%). The results demonstrate that the occurrence of inactive mcy genotypes is linearly related to the population density and selective sweeps of nontoxic mutants did not occur during the transition from prebloom to bloom conditions.
Toxicity; microcystin; real-time PCR; gene loss; Planktothrix; transposable elements; microevolution
We isolated a novel gram-positive bacterium, Brevibacillus texasporus, that produces an antibiotic, BT. BT is a group of related peptides that are produced by B. texasporus cells in response to nutrient limitation. We report here purification and determination of the structure of the most abundant BT isomer, BT1583. Amino acid composition and tandem mass spectrometry experiments yielded a partial BT1583 structure. The presence of ornithine and d-form residues in the partial BT1583 structure indicated that the peptide is synthesized by a nonribosomal peptide synthetase (NRPS). The BT NRPS operon was rapidly and accurately identified by using a novel in silico NRPS operon hunting strategy that involved direct shotgun genomic sequencing rather than the unreliable cosmid library hybridization scheme. Sequence analysis of the BT NRPS operon indicated that it encodes a colinear modular NRPS with a strict correlation between the NRPS modules and the amino acid residues in the peptide. The colinear nature of the BT NRPS enabled us to utilize the genomic information to refine the BT1583 peptide sequence to Me2-4-methyl-4-[(E)-2-butenyl]-4,N-methyl-threonine-L-dO-I-V-V-dK-V-dL-K-dY-L-V-CH2OH. In addition, we report the discovery of novel NRPS codons (sets of the substrate specificity-conferring residues in NRPS modules) for valine, lysine, ornithine, and tyrosine.
The cluster of microcystin synthetase genes from Anabaena strain 90 was sequenced and characterized. The total size of the region is 55.4 kb, and the genes are organized in three putative operons. The first operon (mcyA-mcyB-mcyC) is transcribed in the opposite direction from the second operon (mcyG-mcyD-mcyJ-mcyE-mcyF-mcyI) and the third operon (mcyH). The genes mcyA, mcyB, and mcyC encode nonribosomal peptide synthetases (NRPS), while mcyD codes for a polyketide synthase (PKS), and mcyG and mcyE are mixed NRPS-PKS genes. The genes mcyJ, mcyF, and mcyI are similar to genes coding for a methyltransferase, an aspartate racemase, and a d-3-phosphoglycerate dehydrogenase, respectively. The region in the first module of mcyB coding for the adenylation domain was found to be 96% identical with the corresponding part of mcyC, suggesting a recent duplication of this fragment and a replacement in mcyB. In Anabaena strain 90, the order of the domains encoded by the genes in the two sets (from mcyG to mcyI and from mcyA to mcyC) is colinear with the hypothetical order of the enzymatic reactions for microcystin biosynthesis. The order of the microcystin synthetase genes in Anabaena strain 90 differs from the arrangement found in two other cyanobacterial species, Microcystis aeruginosa and Planktothrix agardhii. The average sequence match between the microcystin synthetase genes of Anabaena strain 90 and the corresponding genes of the other species is 74%. The identity of the individual proteins varies from 67 to 81%. The genes of microcystin biosynthesis from three major producers of this toxin are now known. This makes it possible to design probes and primers to identify the toxin producers in the environment.
Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis.
nonribosomal peptide synthetase; NRPS; marine sponge; Porifera; metagenomics
NRPS-PKS is web-based software for analysing large multi-enzymatic, multi-domain megasynthases that are involved in the biosynthesis of pharmaceutically important natural products such as cyclosporin, rifamycin and erythromycin. NRPS-PKS has been developed based on a comprehensive analysis of the sequence and structural features of several experimentally characterized biosynthetic gene clusters. The results of these analyses have been organized as four integrated searchable databases for elucidating domain organization and substrate specificity of nonribosomal peptide synthetases and three types of polyketide synthases. These databases work as the backend of NRPS-PKS and provide the knowledge base for predicting domain organization and substrate specificity of uncharacterized NRPS/PKS clusters. Benchmarking on a large set of biosynthetic gene clusters has demonstrated that, apart from correct identification of NRPS and PKS domains, NRPS-PKS can also predict specificities of adenylation and acyltransferase domains with reasonably high accuracy. These features of NRPS-PKS make it a valuable resource for identification of natural products biosynthesized by NRPS/PKS gene clusters found in newly sequenced genomes. The training and test sets of gene clusters included in NRPS-PKS correlate information on 307 open reading frames, 2223 functional protein domains, 68 starter/extender precursors and their specific recognition motifs, and also the chemical structure of 101 natural products from four different families. NRPS-PKS is a unique resource which provides a user-friendly interface for correlating chemical structures of natural products with the domains and modules in the corresponding nonribosomal peptide synthetases or polyketide synthases. It also provides guidelines for domain/module swapping as well as site-directed mutagenesis experiments to engineer biosynthesis of novel natural products. NRPS-PKS can be accessed at http://www.nii.res.in/nrps-pks.html.
Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms.
Amino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Salinispora arenicola CNS-205 is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters.
In order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, we cloned and identified the putative gene sare0718 annotated “amino acid adenylation domain”. Firstly, the general features and possible functions of sare0718 were predicted by bioinformatics analysis, which suggested that Sare0718 is a soluble protein with an AMP-binding domain contained in the sequence and its cognate substrate is L-Val. Then, a GST-tagged fusion protein was expressed and purified to further explore the exact adenylation activity of Sare0718 in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that L-Ala but not L-Val is the actual activated amino acid substrate and the basic kinetic parameters of Sare0718 for it are Km = 0.1164±0.0159 (mM), Vmax = 3.1484±0.1278 (µM/min), kcat = 12.5936±0.5112 (min−1).
By revealing the biochemical role of sare0718 gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic nonribosomal peptide synthetase (NRPS)-related gene cluster covering Sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on.
Fungal natural products containing benzodiazepinone- and quinazolinone-fused ring systems can be assembled by nonribosomal peptide synthetases (NRPS) using the conformationally restricted β-amino acid anthranilate as one of the key building blocks. We validated that the first module of the acetylaszonalenin synthetase of Neosartorya fischeri NRRL 181 activates anthranilate to anthranilyl-AMP. With this as starting point, we then used bioinformatic predictions about fungal adenylation domain selectivities to identify and confirm an anthranilate-activating module in the fumiquinazoline A producer Aspergillus fumigatus Af293 as well as a second anthranilate-activating NRPS in N. fischeri. This establishes an anthranilate adenylation domain code for fungal NRPS and should facilitate detection and cloning of gene clusters for benzodiazepine- and quinazoline-containing polycyclic alkaloids with a wide range of biological activities.
Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricrocin. No structural or immunologically relevant NRP products have been identified in the organism. We investigated the function of the largest gene in A. fumigatus, which encodes the NRP synthetase Pes3 (AFUA_5G12730), by targeted gene deletion and extensive phenotypic analysis. It was observed that in contrast to other NRP synthetases, deletion of pes3 significantly increases the virulence of A. fumigatus, whereby the pes3 deletion strain (A. fumigatus Δpes3) exhibited heightened virulence (increased killing) in invertebrate (P < 0.001) and increased fungal burden (P = 0.008) in a corticosteroid model of murine pulmonary aspergillosis. Complementation restored the wild-type phenotype in the invertebrate model. Deletion of pes3 also resulted in increased susceptibility to the antifungal, voriconazole (P < 0.01), shorter germlings, and significantly reduced surface β-glucan (P = 0.0325). Extensive metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP in A. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate that Δpes3 heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus. Proteome alterations in A. fumigatus Δpes3 strongly suggest impaired germination capacity. Uniquely, our data strongly indicate a structural role for the Pes3-encoded NRP, a finding that appears to be novel for an NRP synthetase.
Cyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. The npn gene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of the proteins are colinear in arrangement, with the putative order of the biosynthetic assembly of the cyclic heptapeptide. NpnB contains an embedded monooxygenase domain linking nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) catalytic domains and predicted here to hydroxylate the nostophycin during assembly. Expression of the adenylation domains and subsequent substrate specificity assays support the involvement of this cluster in nostophycin biosynthesis. Biochemical analyses suggest that the loading substrate of NpnA is likely to be a phenylpropanoic acid necessitating deletion of a carbon atom to explain the biosynthesis of nostophycin. Biosyntheses of nostophycin and microcystin resemble each other, but the phylogenetic analyses suggest that they are distantly related to one another.
The biosynthetic gene cluster for the glycopeptide-derived antitumor antibiotic zorbamycin (ZBM) was cloned by screening a cosmid library of Streptomyces flavoviridis ATCC 21892. Sequence analysis revealed 40 ORFs belonging to the ZBM biosynthetic gene cluster. However, only 23 and 22 ORFs showed striking similarities to the biosynthetic gene clusters for the bleomycins (BLMs) and tallysomycins (TLMs), respectively; the remaining ORFs do not show significant homology to ORFs from the related BLM and TLM clusters. The ZBM gene cluster consists of 16 nonribosomal peptide synthetase (NRPS) genes encoding eight complete NRPS modules, three incomplete didomain NRPS modules, and eight freestanding single NRPS domains or associated enzymes, a polyketide synthase (PKS) gene encoding one PKS module, six sugar biosynthesis genes, as well as genes encoding other biosynthesis and resistance proteins. A genetic system using Escherichia coli-Streptomyces flavoviridis intergeneric conjugation was developed to enable ZBM gene cluster boundary determinations and biosynthetic pathway manipulations.
Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Despite conservation of gene sequence, gene order is substantially different between the N. lolii, C. purpurea, and A. fumigatus ergot alkaloid gene clusters. Southern analysis indicated that the N. lolii cluster was linked with previously identified ergovaline biosynthetic genes dmaW and lpsA. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.
Albicidin is a pathotoxin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald disease of sugarcane. Albicidin is synthesized by a nonribosomal pathway via modular polyketide synthase and nonribosomal peptide synthetase (NRPS) megasynthases, and NRPS adenylation (A) domains are responsible for the recognition and activation of specific amino acid substrates. DNA fragments (0.5 kb) encoding the regions responsible for the substrate specificities of six albicidin NRPS A domains from 16 strains of X. albilineans representing the known diversity of this pathogen were amplified and sequenced. Polymorphism analysis of these DNA fragments at different levels (DNA, protein, and NRPS signature) showed that these pathogenicity loci were highly conserved. The conservation of these loci most likely reflects purifying selective pressure, as revealed by a comparison with the variability of nucleotide and amino acid sequences of two housekeeping genes (atpD and efp) of X. albilineans. Nevertheless, the 16 strains of X. albilineans were differentiated into several groups by a phylogenetic analysis of the nucleotide sequences corresponding to the NRPS A domains. One of these groups was representative of the genetic diversity previously found within the pathogen by random fragment length polymorphism and amplified fragment length polymorphism analyses. This group, which differed by three single synonymous nucleotide mutations, contained only four strains of X. albilineans that were all involved in outbreaks of sugarcane leaf scald. The amount of albicidin produced in vitro in agar and liquid media varied among the 16 strains of X. albilineans. However, no relationship among the amount of albicidin produced in vitro and the pathotypes and genetic diversity of the pathogen was found. The NRPS loci contributing to the synthesis of the primary structure of albicidin apparently are not involved in the observed pathogenicity differences among strains of X. albilineans.