Critical steps in an environmental metaproteomics workflow.

Environmental metaproteomics is a rapidly advancing field that provides insights into the structure, dynamics, and metabolic activity of microbial communities. As the field is still maturing, it lacks consistent workflows, making it challenging for non-expert researchers to navigate. This review aims to introduce the workflow of environmental metaproteomics. It outlines the standard practices for sample collection, processing, and analysis, and offers strategies to overcome the unique challenges presented by common environmental matrices such as soil, freshwater, marine environments, biofilms, sludge, and symbionts. The review also highlights the bottlenecks in data analysis that are specific to metaproteomics samples and provides suggestions for researchers to obtain high-quality datasets. It includes recent benchmarking studies and descriptions of software packages specifically built for metaproteomics analysis. The article is written without assuming the reader's familiarity with single-organism proteomic workflows, making it accessible to those new to proteomics or mass spectrometry in general. This primer for environmental metaproteomics aims to improve accessibility to this exciting technology and empower researchers to tackle challenging and ambitious research questions. While it is primarily a resource for those new to the field, it should also be useful for established researchers looking to streamline or troubleshoot their metaproteomics experiments.

Climate dictates microbial community composition and diversity in Australian biological soil crusts (biocrusts).

The soil surface of drylands can typically be colonized by cyanobacteria and other microbes, forming biological soil crusts or 'biocrusts'. Biocrusts provide critical benefits to ecosystems and are a common component of the largely arid and semi-arid Australian continent. Yet, their distribution and the parameters that shape their microbial composition have not been investigated. We present here the first detailed description of Australia's biocrust microbiome assessed from 15 sites across the continent using 16S rRNA sequencing. The most abundant bacterial phyla from all sites were Cyanobacteria, Proteobacteria, Actinobacteria, Chloroflexi and Bacteroidetes. Cyanobacterial communities from northern regions were more diverse and unclassified cyanobacteria were a noticeable feature of northern biocrusts. Segregation between northern and southern regions was largely due to the differential abundance of Microcoleus spp., with M. paludosus dominating in the north and M. vaginatus dominating in the south. The geographical shifts in bacterial composition and diversity were correlated to seasonal temperatures and summer rainfall. Our findings provide an initial reference for sampling strategies to maximize access to bacterial genetic diversity. As hubs for essential ecosystem services, further investigation into biocrusts in arid and semi-arid regions may yield discoveries of genetic mechanisms that combat increases in warming due to climate change.

Tailoring Enzyme Stringency Masks the Multispecificity of a Lyngbyatoxin (Indolactam Alkaloid) Nonribosomal Peptide Synthetase.

Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.

Heterologous Expression and Biochemical Analysis Reveal a Schizokinen-Based Siderophore Pathway in Leptolyngbya (Cyanobacteria).

Siderophores are low molecular weight iron-chelating molecules that many organisms secrete to scavenge ferric iron from the environment. While cyanobacteria inhabit a wide range of environments with poor iron availability, only two siderophore families have been characterized from this phylum. Herein, we sought to investigate siderophore production in the marine genus, Leptolyngbya. A 12 open reading frame (14.5 kb) putative nonribosomal peptide synthetase-independent siderophore biosynthesis gene cluster, identified in the genome of Leptolyngbya sp. PCC 7376, was cloned and heterologously expressed in Escherichia coli. Under iron-limiting conditions, expression strains harboring the first seven genes (lidA to lidF), produced a potent siderophore, which was subsequently identified via UPLC-MS/MS and NMR as schizokinen. The enzymes encoded by the remaining genes (lidG1 to lidG5) did not appear to be active in E. coli, therefore their function could not be determined. Bioinformatic analysis revealed gene clusters with high homology to lidA to lidF in phylogenetically and biogeographically diverse cyanobacteria, suggesting that schizokinen-based siderophore production is widespread in this phylum. Siderophore yields in E. coli expression strains were significantly higher than those achieved by Leptolyngbya, highlighting the potential of this platform for producing siderophores of industrial value. IMPORTANCE Iron availability limits the growth of many microorganisms, particularly those residing in high nutrient-low chlorophyll aquatic environments. Therefore, characterizing iron acquisition pathways in phytoplankton is essential for understanding nutrient cycling in our oceans. The results of this study suggest that Leptolyngbya sp. PCC 7376, and many other cyanobacteria, use schizokinen-based iron chelators (siderophores) to scavenge iron from the environment. We have shown that these pathways are amenable to heterologous expression in E. coli, which expands the limited arsenal of known cyanobacterial siderophores and is advantageous for the downstream overproduction of relevant siderophores of ecological and industrial value.

Comparative proteomics of the toxigenic diazotroph Raphidiopsis raciborskii (cyanobacteria) in response to iron.

Raphidiopsis raciborskii is an invasive bloom-forming cyanobacteria with the flexibility to utilize atmospheric and fixed nitrogen. Since nitrogen-fixation has a high requirement for iron as an ezyme cofactor, we hypothesize that iron availability would determine the success of the species under nitrogen-fixing conditions. This study compares the proteomic response of cylindrospermopsin-producing and non-toxic strains of R. racibroskii to reduced iron concentrations, under nitrogen-fixing conditions, to examine any strain-specific adaptations that might increase fitness under these conditions. We also compared their proteomic responses at exponential and stationary growth phases to capture the changes throughout the growth cycle. Overall, the toxic strain was more competitive under Fe-starved conditions during exponential phase, with upregulated growth and transport-related proteins. The non-toxic strain showed reduced protein expression across multiple primary metabolism pathways. We propose that the increased expression of porin proteins during the exponential growth phase enables toxic strains to persist under Fe-starved conditions with this ability providing a potential explanation for the increased fitness of cylindrospermoipsin-producing strains during unfavourable environmental conditions.

Heterologous Expression of an Unusual Ketosynthase, SxtA, Leads to Production of Saxitoxin Intermediates in Escherichia coli.

Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage-gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4-amino-3-oxo-guanidinoheptane (ethyl ketone) by an unusual polyketide synthase-like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity in vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC-ESI-HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity in vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled-precursor feeding with [guanidino-15 N2 ] arginine and [1,2-13 C2 ] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.

A Pseudoalteromonas Clade with Remarkable Biosynthetic Potential.

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here, we report the biochemical and genomic analysis of Pseudoalteromonas sp. strain HM-SA03, isolated from the blue-ringed octopus, Hapalochlaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides, and four novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through the phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of 10 biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the underexplored potential of Pseudoalteromonas as a source of new natural products.IMPORTANCE This study demonstrates that the Pseudoalteromonas strain HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products.

Quantitative detection of human- and canine-associated Bacteroides genetic markers from an urban coastal lagoon.

The contamination of water catchments by nonpoint source faecal pollution is a major issue affecting the microbial quality of receiving waters and is associated with the occurrence of a range of enteric illnesses in humans. The potential sources of faecal pollution in surface waters are diverse, including urban sewage leaks, surface runoff and wildlife contamination originating from a range of hosts. The major contributing hosts require identification to allow targeted management of this public health concern. In this study, two high-performing Microbial Source Tracking (MST) assays, HF183/Bac242 and BacCan-UCDmodif, were used for their ability to detect host-specific Bacteroides 16Sr RNA markers for faecal pollution in a 12-month study on an urban coastal lagoon in Sydney, Australia. The lagoon was found to contain year-round high numbers of human and canine faecal markers, as well as faecal indicator bacteria counts, suggesting considerable human and animal faecal pollution. The high sensitivity and specificity of the HF183/Bac242 and BacCan-UCDmodif assays, together with the manageable levels of PCR inhibition and high level DNA extraction efficiency obtained from lagoon water samples make these markers candidates for inclusion in an MST 'toolbox' for investigating host origins of faecal pollution in urban surface waters.

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.

Heterologous expression and biochemical characterisation of cyanotoxin biosynthesis pathways.

Covering: up to 2018 Marine and freshwater cyanobacteria produce a variety of toxic compounds that pose a threat to the health of humans, livestock and natural ecosystems world-wide. Significant research efforts have been directed towards understanding the biosynthesis of these cyanotoxins in an attempt to reduce their deleterious effects on water quality and, more recently, to harness their biotechnological potential. While a variety of complementary methods (such as bioinformatic analyses and isotope feeding studies) have been employed over the last three decades to address knowledge gaps in this field, this review focuses on the utility of heterologous expression and biochemical studies, including emerging technologies for engineering and expressing complete cyanotoxin gene clusters.

Physiological responses of the freshwater N2 -fixing cyanobacterium Raphidiopsis raciborskii to Fe and N availabilities.

The cyanobacterium Raphidiopsis raciborskii is of environmental and social concern in view of its toxicity, bloom-forming characteristics and increasingly widespread occurrence. However, while availability of macronutrients and micronutrients such as N and Fe are critically important for the growth and metabolism of this organism, the physiological response of toxic and non-toxic strains of R. raciborskii to varying Fe and N availabilities remains unclear. By determining physiological parameters as a function of Fe and N availability, we demonstrate that R. raciborskii growth and N2 -fixing activity are facilitated at higher Fe availability under N2 -limited conditions with faster growth of the CS-506 (cylindrospermopsin-producing) strain compared with that of CS-509 (the non-toxic) strain. Radiolabelled Fe uptake assays indicated that R. raciborskii acclimated under Fe-limited conditions acquires Fe at significantly higher rates than under Fe replete conditions, principally via unchelated Fe(II) generated as a result of photoreduction of complexed Fe(III). While N2 -fixation of both strains occurred during both day and night, the CS-506 strain overall exhibited higher N2 -fixing and Fe uptake rates than the CS-509 strain under N-deficient and Fe-limited conditions. The findings of this study highlight that Fe availability is of significance for the ecological advantage of CS-506 over CS-509 in N-deficient freshwaters.

Bioinformatic, phylogenetic and chemical analysis of the UV-absorbing compounds scytonemin and mycosporine-like amino acids from the microbial mat communities of Shark Bay, Australia.

Shark Bay, Western Australia is a World Heritage area with extensive microbial mats and stromatolites. Microbial communities that comprise these mats have developed a range of mitigation strategies against changing levels of photosynthetically active and ultraviolet radiation, including the ability to biosynthesise the UV-absorbing natural products scytonemin and mycosporine-like amino acids (MAAs). To this end, the distribution of photoprotective pigments within Shark Bay microbial mats was delineated in the present study. This involved amplicon sequencing of bacterial 16S rDNA from communities at the surface and subsurface in three distinct mat types (smooth, pustular and tufted), and correlating this data with the chemical and molecular distribution of scytonemin and MAAs. Employing UV spectroscopy and MS/MS fragmentation, mycosporine-glycine, asterina and an unknown MAA were identified based on typical fragmentation patterns. Marker genes for scytonemin and MAA production (scyC and mysC) were amplified from microbial mat DNA and placed into phylogenetic context against a broad screen throughout 363 cyanobacterial genomes. Results indicate that occurrence of UV screening compounds is associated with the upper layer of Shark Bay microbial mats, and the occurrence of scytonemin is closely dependent on the abundance of cyanobacteria.

Lack of Methylated Hopanoids Renders the Cyanobacterium Nostoc punctiforme Sensitive to Osmotic and pH Stress.

To investigate the function of 2-methylhopanoids in modern cyanobacteria, the hpnP gene coding for the radical S-adenosyl methionine (SAM) methylase protein that acts on the C-2 position of hopanoids was deleted from the filamentous cyanobacterium Nostoc punctiforme ATCC 29133S. The resulting ΔhpnP mutant lacked all 2-methylhopanoids but was found to produce much higher levels of two bacteriohopanepentol isomers than the wild type. Growth rates of the ΔhpnP mutant cultures were not significantly different from those of the wild type under standard growth conditions. Akinete formation was also not impeded by the absence of 2-methylhopanoids. The relative abundances of the different hopanoid structures in akinete-dominated cultures of the wild-type and ΔhpnP mutant strains were similar to those of vegetative cell-dominated cultures. However, the ΔhpnP mutant was found to have decreased growth rates under both pH and osmotic stress, confirming a role for 2-methylhopanoids in stress tolerance. Evidence of elevated photosystem II yield and NAD(P)H-dependent oxidoreductase activity in the ΔhpnP mutant under stress conditions, compared to the wild type, suggested that the absence of 2-methylhopanoids increases cellular metabolic rates under stress conditions.IMPORTANCE As the first group of organisms to develop oxygenic photosynthesis, Cyanobacteria are central to the evolutionary history of life on Earth and the subsequent oxygenation of the atmosphere. To investigate the origin of cyanobacteria and the emergence of oxygenic photosynthesis, geobiologists use biomarkers, the remnants of lipids produced by different organisms that are found in geologic sediments. 2-Methylhopanes have been considered indicative of cyanobacteria in some environmental settings, with the parent lipids 2-methylhopanoids being present in many contemporary cyanobacteria. We have created a Nostoc punctiforme ΔhpnP mutant strain that does not produce 2-methylhopanoids to assess the influence of 2-methylhopanoids on stress tolerance. Increased metabolic activity in the mutant under stress indicates compensatory alterations in metabolism in the absence of 2-methylhopanoids.

Industrial robustness linked to the gluconolactonase from Zymomonas mobilis.

The physiological characteristics and the potential gluconolactone production of the gluconolactonase-deficient strain, Zymomonas mobilis ZM4 gnlΔ, were investigated via growth inhibitory assay and biotransformation of glucose and fructose into gluconolactone and sorbitol, respectively. The results of ethanol fermentation studies performed in the presence of high concentration of glucose (>200 g l-1) under fermentative or aerobic conditions indicated that a significant reduction of volumetric ethanol productivity from the strain of ZM4 gnlΔ was noticeable due to the reduced rates of specific growth, sugar uptake, and biomass yield as compared with those of the parental strain ZM4. The biotransformation prepared at pH 6.0 using the permeabilized cell indicated that gluconic acid from ZM4 gnlΔ was still produced as a major product (67 g l-1) together with sorbitol (65 g l-1) rather than gluconolactone after 24 h. Only small amount of gluconolactone was transiently overproduced up to 9 g l-1, but at the end of biotransformation, all gluconolactone were oxidized into gluconic acid. This indicated that autolysis of gluconolactone at the pH led to such results despite under gluconolactonase inactivation conditions. The physiological characteristics of ZM4 gnlΔ was further investigated under various stress conditions, including suboptimal pH (3.5~6.0), temperature (25~40 °C), and presence of growth inhibitory molecules including hydrogen peroxide, ethanol, acetic acid, furfural, and so forth. The results indicated that ZM4 gnlΔ was more susceptible at high glucose concentration, low pH of 3.5, and high temperature of 40 °C and in the presence of 4 mM H2O2 comparing with ZM4. Therefore, the results were evident that gluconolactonase in Z. mobilis contributed to industrial robustness and anti-stress regulation.

Elevated Na(+) and pH influence the production and transport of saxitoxin in the cyanobacteria Anabaena circinalis AWQC131C and Cylindrospermopsis raciborskii†T3.

Saxitoxins (STX), neurotoxic alkaloids, fall under the umbrella of paralytic shellfish toxins produced by marine dinoflagellates and freshwater cyanobacteria. The genes responsible for the production of STX have been proposed, but factors that influence their expression and induce toxin efflux remain unclear. Here we characterize the putative STX NorM-like MATE transporters SxtF and SxtM. Complementation of the antibiotic-sensitive strain Escherichia coli KAM32 with these transporters decreased fluoroquinolone sensitivity, indicating that while becoming evolutionary specialized for STX transport these transporters retain relaxed specificity typical of this class. The transcriptional response of STX biosynthesis (sxtA) along with that of the STX transporters (sxtM and sxtF from Cylindrospermopsis raciborskii T3, and sxtM from Anabaena circinalis AWQC131C) were assessed in response to ionic stress. These data, coupled with a measure of toxin intracellular to extracellular ratios, provide an insight into the physiology of STX export. Cylindrospermopsis raciborskii and Anabaena circinalis exhibited opposing responses under conditions of ionic stress. High Na(+) (10 mM) induced moderate alterations of transcription and STX localization, whereas high pH (pH 9) stimulated the greatest physiological response. Saxitoxin production and cellular localization are responsive to ionic strength, indicating a role of this molecule in the maintenance of cellular homeostasis.

Proteogenomics of a saxitoxin-producing and non-toxic strain of Anabaena circinalis (cyanobacteria) in response to extracellular NaCl and phosphate depletion.

In Australia, saxitoxin production is strain dependent within the bloom-forming freshwater cyanobacterium Anabaena circinalis. Freshwater cyanobacteria are exposed to rapid fluctuations in environmental nutrient concentrations, and their adaption is vital for competition, succession and dominance. Two elements of environmental significance, phosphorus and sodium chloride, are proposed to play a role in bloom development and saxitoxin biosynthesis respectively. The aim of our study was to comparatively analyse the model saxitoxin-producing A. circinalis AWQC131C and non-toxic A. circinalis AWQC310F at the genomic level and proteomic level, in response to phosphate depletion and increased extracellular NaCl. When challenged, photosynthesis, carbon/nitrogen metabolisms, transcription/translation, oxidative stress and nutrient transport functional categories demonstrated the largest changes in protein abundance. In response to increased NaCl, SxtC, a protein conserved in all known saxitoxin biosynthetic pathways, was downregulated. Additionally, toxin quantification revealed a decrease in total saxitoxin and decarbomoyl-gonyautoxin2/3 content in response to the NaCl treatment. In response to phosphate depletion, the toxic and non-toxic strain displayed similar proteomic profiles, although the toxic strain did not alter the abundance of as many proteins as the non-toxic strain. These findings have important implications for the future, since response and adaption mechanisms are directly related to in situ dominance of cyanobacteria.

Specific global responses to N and Fe nutrition in toxic and non-toxic Microcystis aeruginosa.

The bloom-forming cyanobacteria species Microcystis aeruginosa includes toxic and non-toxic (microcystin-producing) strains. Certain stress conditions stimulate synthesis of microcystin (MCYST) and enhance the binding of the MCYST molecule to proteins. In this quantitative proteomic study, we compared the response of a wild-type toxic strain PCC 7806, an mcyH(-) knockout non-toxic strain, and a naturally occurring non-toxic strain, PCC 7005, after 8 days in low iron (Fe) and nitrogen (N) starvation in order to assess the benefit of MCYST synthesis in non-optimal conditions. Fe limitation increased MCYST synthesis and caused an accumulation of phycobilisome proteins and the ferric iron transporter FutA only in the toxic PCC 7806 but not the non-toxic strains. In N starvation, photosynthetic, C and N metabolism proteins were more abundant in the non-toxic strains, as were chaperones and proteases. Significant interaction between nutrient availability and toxicity existed for thioredoxin peroxidase and several thioredoxin-regulated proteins. We propose a competition of MCYST for binding sites in thioredoxin-regulated proteins during oxidative stress (low Fe) but not in growth-limiting conditions (low N). This then leads to differences in the regulation of C:N metabolism in toxic and non-toxic M. aeruginosa in nutrient-replete and nutrient-limited conditions.

Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli.

Mycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacterium Cylindrospermum stagnale PCC 7417 revealed a new gene cluster with homology to MAA synthase from Nostoc punctiforme This newly identified gene cluster is unusual because it has five biosynthesis genes (mylA to mylE), compared to the four found in other MAA gene clusters. Heterologous expression of mylA to mylE in Escherichia coli resulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced in E. coli and structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods. IMPORTANCE: Mycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs in E. coli is also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens.

Comparative Profiling and Discovery of Novel Glycosylated Mycosporine-Like Amino Acids in Two Strains of the Cyanobacterium Scytonema cf. crispum.

UNLABELLED: The mycosporine-like amino acids (MAAs) are a group of small molecules with a diverse ecological distribution among microorganisms. MAAs have a range of physiological functions, including protection against UV radiation, making them important from a biotechnological perspective. In the present study, we identified a putative MAA (mys) gene cluster in two New Zealand isolates of Scytonema cf. crispum (UCFS10 and UCFS15). Homology to "Anabaena-type" mys clusters suggested that this cluster was likely to be involved in shinorine biosynthesis. Surprisingly, high-performance liquid chromatography analysis of S cf. crispum cell extracts revealed a complex MAA profile, including shinorine, palythine-serine, and their hexose-bound variants. It was hypothesized that a short-chain dehydrogenase (UCFS15_00405) encoded by a gene adjacent to the S cf. crispum mys cluster was responsible for the conversion of shinorine to palythine-serine. Heterologous expression of MysABCE and UCFS15_00405 in Escherichia coli resulted in the exclusive production of the parent compound shinorine. Taken together, these results suggest that shinorine biosynthesis in S cf. crispum proceeds via an Anabaena-type mechanism and that the genes responsible for the production of other MAA analogues, including palythine-serine and glycosylated analogues, may be located elsewhere in the genome. IMPORTANCE: Recently, New Zealand isolates of S cf. crispum were linked to the production of paralytic shellfish toxins for the first time, but no other natural products from this species have been reported. Thus, the species was screened for important natural product biosynthesis. The mycosporine-like amino acids (MAAs) are among the strongest absorbers of UV radiation produced in nature. The identification of novel MAAs is important from a biotechnology perspective, as these molecules are able to be utilized as sunscreens. This study has identified two novel MAAs that have provided several new avenues of future research related to MAA genetics and biosynthesis. Further, we have revealed that the genetic basis of MAA biosynthesis may not be clustered on the genome. The identification of the genes responsible for MAA biosynthesis is vital for future genetic engineering.

Physiological and Proteomic Responses of Continuous Cultures of Microcystis aeruginosa PCC 7806 to Changes in Iron Bioavailability and Growth Rate.

UNLABELLED: The hepatotoxin microcystin (MCYST) is produced by a variety of freshwater cyanobacterial species, including Microcystis aeruginosa Interestingly, MCYST-producing M. aeruginosa strains have been shown to outcompete their nontoxic counterparts under iron-limiting conditions. However, the reasons for this are unclear. Here we examined the proteomic response of M. aeruginosa PCC 7806 continuous cultures under different iron and growth regimes. Iron limitation was correlated with a global reduction in levels of proteins associated with energy metabolism and photosynthesis. These proteomic changes were consistent with physiological observations, including reduced chlorophyll a content and reduced cell size. While levels of MCYST biosynthesis proteins did not fluctuate during the study period, both intra- and extracellular toxin quotas were significantly higher under iron-limiting conditions. Our results support the hypothesis that intracellular MCYST plays a role in protecting the cell against oxidative stress. Further, we propose that extracellular MCYST may act as a signaling molecule, stimulating MCYST production under conditions of iron limitation and enhancing the fitness of bloom populations. IMPORTANCE: Microcystin production in water supply reservoirs is a global public health problem. Understanding the ecophysiology of hepatotoxic cyanobacteria, including their responses to the presence of key micronutrient metals such as iron, is central to managing harmful blooms. To our knowledge, this was the first study to examine proteomic and physiological changes occurring in M. aeruginosa continuous cultures under conditions of iron limitation at different growth rates.