Identification of promoter elements in the Dolichospermum circinale AWQC131C saxitoxin gene cluster and the experimental analysis of their use for heterologous expression

Background Dolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin (sxt) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology. Results In D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5′ RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, PsxtPER1 and PsxtPER2. In E. coli, strong expression of lux from PsxtP, PsxtD and PsxtPER1 was observed while expression from Porf24 and PsxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from PsxtP, PsxtPER1, and Porf24 promoters was statistically higher compared to the non-promoter control, while PsxtD showed poor activity under the described conditions. Conclusions Both of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that the majority of the native sxt promoters appear active in different heterologous hosts, simplifying initial cloning efforts. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable first option for producing PSTs for industrial or biomedical purposes

Cyanobacteria : health and research possibilities

Cyanobacteria are a subset of prokaryotic bacteria (also known as blue-green algae) possessing a cell wall and chlorophyll A, contributing about 35% of global photosynthesis. Cyanobacteria are found on all continents in soils and fresh, brackish and salt water, living independently or in symbiosis. As cyanobacteria are found in all water bodies, they have the potential to affect the quality of drinking and recreational water and pose a potential health risk to the public

Current knowledge of paralytic shellfish toxin biosynthesis, molecular detection and evolution

Harmful algal blooms (HABs) occur when microalgae rapidly proliferate in a water supply and detrimentally impact humans or the environment (Hudnell 2008, Anderson et al. 2012). HAB forming species are usually specific for a particular environment. For example, in marine environments, HABs predominantly consist of eukaryotic dinoflagellates, while in freshwater environments, they are composed of prokaryotic cyanobacteria (also referred to as harmful cyanobacterial blooms)

Comparative proteomics reveals that a saxitoxin-producing and a nontoxic strain of Anabaena circinalis are two different ecotypes

In Australia, saxitoxin production is restricted to the cyanobacterial species Anabaena circinalis and is strain-dependent. We aimed to characterize a saxitoxin-producing and nontoxic strain of A. circinalis at the proteomic level using iTRAQ. Seven proteins putatively involved in saxitoxin biosynthesis were identified within our iTRAQ experiment for the first time. The proteomic profile of the toxic A. circinalis was significantly different from the nontoxic strain, indicating that each is likely to inhabit a unique ecological niche. Under control growth conditions, the saxitoxin-producing A. circinalis displayed a higher abundance of photosynthetic, carbon fixation and nitrogen metabolic proteins. Differential abundance of these proteins suggests a higher intracellular C:N ratio and a higher concentration of intracellular 2-oxoglutarate in our toxic strain compared with the nontoxic strain. This may be a novel site for posttranslational regulation because saxitoxin biosynthesis putatively requires a 2-oxoglutarate-dependent dioxygenase. The nontoxic A. circinalis was more abundant in proteins, indicating cellular stress. Overall, our study has provided the first insight into fundamental differences between a toxic and nontoxic strain of A. circinalis, indicating that they are distinct ecotypes

Environmental conditions that influence toxin biosynthesis in cyanobacteria

Over the past 15 years, the genetic basis for production of many cyanobacterial bioactive compounds has been described. This knowledge has enabled investigations into the environmental factors that regulate the production of these toxins at the molecular level. Such molecular or systems level studies are also likely to reveal the physiological role of the toxin and contribute to effective water resource management. This review focuses on the environmental regulation of some of the most relevant cyanotoxins, namely the microcystins, nodularin, cylindrospermopsin, saxitoxins, anatoxins and jamaicamides

Genome mining for natural product biosynthetic gene clusters in the Subsection V cyanobacteria

Background: Cyanobacteria are well known for the production of a range of secondary metabolites. Whilst recent genome sequencing projects has led to an increase in the number of publically available cyanobacterial genomes, the secondary metabolite potential of many of these organisms remains elusive. Our study focused on the 11 publically available Subsection V cyanobacterial genomes, together with the draft genomes of Westiella intricata UH strain HT-29-1 and Hapalosiphon welwitschii UH strain IC-52-3, for their genetic potential to produce secondary metabolites. The Subsection V cyanobacterial genomes analysed in this study are reported to produce a diverse range of natural products, including the hapalindole-family of compounds, microcystin, hapalosin, mycosporine-like amino acids and hydrocarbons. Results: A putative gene cluster for the cyclic depsipeptide hapalosin, known to reverse P-glycoprotein multiple drug resistance, was identified within three Subsection V cyanobacterial genomes, including the producing cyanobacterium H. welwitschii UH strain IC-52-3. A number of orphan NRPS/PKS gene clusters and ribosomally-synthesised and post translationally-modified peptide gene clusters (including cyanobactin, microviridin and bacteriocin gene clusters) were identified. Furthermore, gene clusters encoding the biosynthesis of mycosporine-like amino acids, scytonemin, hydrocarbons and terpenes were also identified and compared. Conclusions: Genome mining has revealed the diversity, abundance and complex nature of the secondary metabolite potential of the Subsection V cyanobacteria. This bioinformatic study has identified novel biosynthetic enzymes which have not been associated with gene clusters of known classes of natural products, suggesting that these cyanobacteria potentially produce structurally novel secondary metabolites

Draft genome sequence of the fungus Lecanicillium psalliotae strain HWLR35, isolated from a wheat leaf infected with leaf rust (caused by Puccinia triticina)

Lecanicillium psalliotae is an entomopathogenic, mycoparasitical, and nematophagous fungus known to produce antibiotic and antifungal compounds. Here, we report the first 36-Mb draft genome sequence of L. psalliotae strain HWLR35. The draft genome contains 197 scaffolds and is predicted to have 11,009 protein-coding genes

Comparative analysis of hapalindole, ambiguine and welwitindolinone gene clusters and reconstitution of indole-isonitrile biosynthesis from cyanobacteria

Background: The hapalindole-type family of natural products is a group of hybrid isoprenoid-indole alkaloids, produced solely by members of the Subsection V cyanobacterial strains. This family broadly includes the hapalindoles, welwitindolinones, fisherindoles and ambiguines amongst others, all of which have an isonitrile- or isothiocyanate-containing indole alkaloid skeleton, with a cyclized isoprene unit. The hapalindoles are diversified into the welwitindolinones, fischerindoles and ambiguines through the employment of tailoring oxygenase, methyltransferase and prenyltransferase enzymes. We compare the genetic basis for the biosynthesis of this diverse group of natural products and identify key early biosynthetic intermediates. Results: Whole genome sequencing of freshwater and terrestrial cyanobacteria Westiella intricata UH strain HT-29-1, Hapalosiphon welwitschii UH strain IC-52-3, Fischerella ambigua UTEX 1903 and Fischerella sp. ATCC 43239 led to the identification of a candidate hapalindole-type gene cluster in each strain. These were compared with the recently published ambiguine and welwitindolinone gene clusters and four unpublished clusters identified within publicly available genomes. We present detailed comparative bioinformatic analysis of the gene clusters and the biosynthesis of a pivotal indole-isonitrile intermediate resulting in both cis and trans geometrical isomers. Enzyme analyses and metabolite extractions from two hapalindole-producing Fischerella strains indicate the presence of cis and trans indole-isonitriles as biosynthetic intermediates in the early steps of the pathway. Conclusions: Interestingly, the organization of the welwitindolinone gene cluster is conserved in all producing strains but distinct from the hapalindole and ambiguine clusters. Enzymatic assays using WelI1 and WelI3 from Westiella intricata UH strain HT-29-1 demonstrated the ability to catalyze the formation of both cis and trans geometrical isomers when using a cell lysate. The enzymatic and metabolic characterization of both cis and trans indole-isonitrile intermediates implies conservation of their stereochemical integrity towards members of the ambiguine and welwitindolinone products. In summary, we present data that supports a unified biosynthetic pathway towards hapalindoles in nine individual species of cyanobacteria. Diversification of the pathway occurs later through the employment of specialized enzymatic steps towards fischerindoles, ambiguines and welwitindolinones

Exploring cyanobacterial genomes for natural product biosynthesis pathways

Cyanobacteria produce a vast array of natural products, some of which are toxic to human health, while others possess potential pharmaceutical activities. Genome mining enables the identification and characterisation of natural product gene clusters; however, the current number of cyanobacterial genomes remains low compared to other phyla. There has been a recent effort to rectify this issue by increasing the number of sequenced cyanobacterial genomes. This has enabled the identification of biosynthetic gene clusters for structurally diverse metabolites, including non-ribosomal peptides, polyketides, ribosomal peptides, UV-absorbing compounds, alkaloids, terpenes and fatty acids. While some of the identified biosynthetic gene clusters correlate with known metabolites, genome mining also highlights the number and diversity of clusters for which the product is unknown (referred to as orphan gene clusters). A number of bioinformatic tools have recently been developed in order to predict the products of orphan gene clusters; however, in some cases the complexity of the cyanobacterial pathways makes the prediction problematic. This can be overcome by the use of mass spectrometry-guided natural product genome mining, or heterologous expression. Application of these techniques to cyanobacterial natural product gene clusters will be explored

Investigating hyperparasites as potential biological control agents of rust pathogens on cereal crops

Rust pathogens cause damage to cereal crops around the world, leading to reduced yield and profit. Current methods of rust control include fungicides, resistant cultivars, and preventative agronomic practices. Some hyperparasites are antagonists of plant pathogens and may provide a potential method of biocontrol against increasingly virulent strains of rust. Very little is known about the mechanism of inhibition of rust growth by hyperparasites, however, isolation of new strains and subsequent characterisation may reveal new treatment strategies for the control of rust. Here we report the isolation of six new fungal hyperparasites and their effects on the development of three Puccinia rust pathogens were examined in vitro, to determine the potential of each as biocontrol agents of rust. Cut-leaf sections of rust-susceptible wheat, barley and oat cultivars were treated with fungal hyperparasite conidia prior to infection with the rust species; Puccinia triticina, P. hordei, and P. coronata f. sp. avenae, respectively. Inhibition of rust spore germination tests were also performed on water agar plates co-inoculated with the isolates. In leaf sections, rust pustule number was significantly (P < 0.01) lower for all six isolates tested: Penicillium brevicompactum, Clonostachys rosea, Simpicillium aogashimaense, Neoascochyta sp., Lecanicillium psalliotae and Epicoccum nigrum. The results of these experiments suggest that the mechanisms underlying the reduction in pustule number could include one or more of the processes of direct parasitism, antagonism by antibiosis, competition, and/or induction of host plant resistance