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.

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.

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.

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.

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 genomics of Cylindrospermopsis raciborskii strains with differential toxicities.

BACKGROUND: Cylindrospermopsis raciborskii is an invasive filamentous freshwater cyanobacterium, some strains of which produce toxins. Sporadic toxicity may be the result of gene deletion events, the horizontal transfer of toxin biosynthesis gene clusters, or other genomic variables, yet the evolutionary drivers for cyanotoxin production remain a mystery. Through examining the genomes of toxic and non-toxic strains of C. raciborskii, we hoped to gain a better understanding of the degree of similarity between these strains of common geographical origin, and what the primary differences between these strains might be. Additionally, we hoped to ascertain why some cyanobacteria possess the cylindrospermopsin biosynthesis (cyr) gene cluster and produce toxin, while others do not. It has been hypothesised that toxicity or lack thereof might confer a selective advantage to cyanobacteria under certain environmental conditions. RESULTS: In order to examine the fundamental differences between toxic and non-toxic C. raciborskii strains, we sequenced the genomes of two closely related isolates, CS-506 (CYN+) and CS-509 (CYN-) sourced from different lakes in tropical Queensland, Australia. These genomes were then compared to a third (reference) genome from C. raciborskii CS-505 (CYN+). Genome sizes were similar across all three strains and their G + C contents were almost identical. At least 2,767 genes were shared among all three strains, including the taxonomically important rpoc1, ssuRNA, lsuRNA, cpcA, cpcB, nifB and nifH, which exhibited 99.8-100% nucleotide identity. Strains CS-506 and CS-509 contained at least 176 and 101 strain-specific (or non-homologous) genes, respectively, most of which were associated with DNA repair and modification, nutrient uptake and transport, or adaptive measures such as osmoregulation. However, the only significant genetic difference observed between the two strains was the presence or absence of the cylindrospermopsin biosynthesis gene cluster. Interestingly, we also identified a cryptic secondary metabolite gene cluster in strain CS-509 (CYN-) and a second cryptic cluster common to CS-509 and the reference strain, CS-505 (CYN+). CONCLUSIONS: Our results confirm that the most important factor contributing to toxicity in C. raciborskii is the presence or absence of the cyr gene cluster. We did not identify any other distally encoded genes or gene clusters that correlate with CYN production. The fact that the additional genomic differences between toxic and non-toxic strains were primarily associated with stress and adaptation genes suggests that CYN production may be linked to these physiological processes.

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.

Bacterial community structure and metabolic potential in microbialite-forming mats from South Australian saline lakes.

Microbialites are sedimentary rocks created in association with benthic microorganisms. While they harbour complex microbial communities, Cyanobacteria perform critical roles in sediment stabilisation and accretion. Microbialites have been described from permanent and ephemeral saline lakes in South Australia; however, the microbial communities that generate and inhabit these biogeological structures have not been studied in detail. To address this knowledge gap, we investigated the composition, diversity and metabolic potential of bacterial communities from different microbialite-forming mats and surrounding sediments in five South Australian saline coastal lakes using 16S rRNA gene sequencing and predictive metagenome analyses. While Proteobacteria and Bacteroidetes were the dominant phyla recovered from the mats and sediments, Cyanobacteria were significantly more abundant in the mat samples. Interestingly, at lower taxonomic levels, the mat communities were vastly different across the five lakes. Comparative analysis of putative mat and sediment metagenomes via PICRUSt2 revealed important metabolic pathways driving the process of carbonate precipitation, including cyanobacterial oxygenic photosynthesis, ureolysis and nitrogen fixation. These pathways were highly conserved across the five examined lakes, although they appeared to be performed by distinct groups of bacterial taxa found in each lake. Stress response, quorum sensing and circadian clock were other important pathways predicted by the in silico metagenome analysis. The enrichment of CRISPR/Cas and phage shock associated genes in these cyanobacteria-rich communities suggests that they may be under selective pressure from viral infection. Together, these results highlight that a very stable ecosystem function is maintained by distinctly different communities in microbialite-forming mats in the five South Australian lakes and reinforce the concept that 'who' is in the community is not as critical as their net metabolic capacity.

Cyanobacterial blooms in wastewater treatment facilities: Significance and emerging monitoring strategies.

Municipal wastewater treatment facilities (WWTFs) are prone to the proliferation of cyanobacterial species which thrive in stable, nutrient-rich environments. Dense cyanobacterial blooms frequently disrupt treatment processes and the supply of recycled water due to their production of extracellular polymeric substances, which hinder microfiltration, and toxins, which pose a health risk to end-users. A variety of methods are employed by water utilities for the identification and monitoring of cyanobacteria and their toxins in WWTFs, including microscopy, flow cytometry, ELISA, chemoanalytical methods, and more recently, molecular methods. Here we review the literature on the occurrence and significance of cyanobacterial blooms in WWTFs and discuss the pros and cons of the various strategies for monitoring these potentially hazardous events. Particular focus is directed towards next-generation metagenomic sequencing technologies for the development of site-specific cyanobacterial bloom management strategies. Long-term multi-omic observations will enable the identification of indicator species and the development of site-specific bloom dynamics models for the mitigation and management of cyanobacterial blooms in WWTFs. While emerging metagenomic tools could potentially provide deep insight into the diversity and flux of problematic cyanobacterial species in these systems, they should be considered a complement to, rather than a replacement of, quantitative chemoanalytical approaches.

Recent developments in quantitative PCR for monitoring harmful marine microalgae.

Marine microalgae produce a variety of specialised metabolites that have toxic effects on humans, farmed fish, and marine wildlife. Alarmingly, many of these compounds bioaccumulate in the tissues of shellfish and higher trophic organisms, including species consumed by humans. Molecular methods are emerging as a potential alternative and complement to the conventional microscopic diagnosis of toxic or otherwise harmful microalgal species. Quantitative PCR (qPCR) in particular, has gained popularity over the past decade as a sensitive, rapid, and cost-effective method for monitoring harmful microalgae. Assays targeting taxonomic marker genes provide the opportunity to identify and quantify (or semi-quantify) microalgal species and importantly to pre-empt bloom events. Moreover, the discovery of paralytic shellfish toxin biosynthesis genes in dinoflagellates has enabled researchers to directly monitor toxigenic species in coastal waters and fisheries. This review summarises the recent developments in qPCR detection methods for harmful microalgae, with emphasis on emerging toxin gene monitoring technologies.

The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk.

Toxic cyanobacteria pose a significant hazard to human health and the environment. The recent characterisation of cyanotoxin synthetase gene clusters has resulted in an explosion of molecular detection methods for these organisms and their toxins. Conventional polymerase chain reaction (PCR) tests targeting cyanotoxin biosynthesis genes provide a rapid and sensitive means for detecting potentially toxic populations of cyanobacteria in water supplies. The adaptation of these simple PCR tests into quantitative methods has additionally enabled the monitoring of dynamic bloom populations and the identification of particularly problematic species. More recently, DNA microarray technology has been applied to cyanobacterial diagnostics offering a high-throughput option for detecting and differentiating toxic genotypes in complex samples. Together, these molecular methods are proving increasingly important for monitoring water quality.

Mutagenesis of the Microcystin Tailoring and Transport Proteins in a Heterologous Cyanotoxin Expression System.

The microcystins are a large group of cyclic peptide hepatotoxins produced by several genera of freshwater cyanobacteria. The genes responsible for microcystin biosynthesis are encoded within a large (∼55 kbp) gene cluster, mcyA-J. The recent establishment of a cyanotoxin heterologous expression system in Escherichia coli has provided the means to study microcystin biosynthesis in a genetically tractable, rapidly growing host. Using this system, we demonstrate that deletion of the ABC-transporter, mcyH, and dehydrogenase, mcyI, abolishes microcystin production, while deletion of the O-methyltransferase, mcyJ, results in the production of the demethylated (DM) toxin [d-Asp3, DMAdda5]microcystin-LR. Both methylated and DM toxin variants were heterologously produced at high titers and efficiently exported into the extracellular medium, enabling easy purification. The results show that the mcy gene cluster can be engineered in E. coli to study the function of its individual components and direct the synthesis of particular microcystin variants. This technology could potentially be applied to other natural products of ecological and biomedical significance.

The genetics, biosynthesis and regulation of toxic specialized metabolites of cyanobacteria.

The production of toxic metabolites by cyanobacterial blooms represents a significant threat to the health of humans and ecosystems worldwide. Here we summarize the current state of the knowledge regarding the genetics, biosynthesis and regulation of well-characterized cyanotoxins, including the microcystins, nodularin, cylindrospermopsin, saxitoxins and anatoxins, as well as the lesser-known marine toxins (e.g. lyngbyatoxin, aplysiatoxin, jamaicamides, barbamide, curacin, hectochlorin and apratoxins).

Molecular and cellular characterisation of the zinc uptake (Znu) system of Nostoc punctiforme.

Metal homoeostasis in cyanobacteria is based on uptake and export systems that are controlled by their own regulators. This study characterises the zinc uptake (Znu) system in Nostoc punctiforme. The system was found to comprise of three subunits in an ACB operon: a Zn(2+)-binding protein (ZnuA18), a transmembrane domain (ZnuB) and an ATPase (ZnuC). These proteins are encoded within the znu operon regulated by a zinc uptake transcription repressor (Zur). Interestingly, a second Zn(2+)-binding protein (ZnuA08) was also identified at a distal genomic location. Interactions between components of the ZnuACB system were investigated using knockouts of the individual genes. The znuA08(-), znuA18(-), znuB(-) and znuC(-) mutants displayed overall reduced znuACB transcript levels, suggesting that all system components are required for normal expression of znu genes. Zinc uptake assays in the Zn(2+)-binding protein mutant strains showed that the disruption of znuA18 had a greater negative effect on zinc uptake than disruption of znuA08. Complementation studies in Escherichia coli indicated that both znuA08 and znuA18 were able to restore zinc uptake in a znuA(-) mutant, with znuA18 permitting the highest zinc uptake rate. The N. punctiforme zur was also able to complement the E. coli zur(-) mutant.

Increased incidence of Cylindrospermopsis raciborskii in temperate zones--is climate change responsible?

The bloom-forming, toxic cyanobacterium, Cylindrospermopsis raciborskii exhibits global distribution. In recent years both the occurrence and dominance of this species, particularly in temperate regions, has increased. Whilst this may be due to increased sensitivity of analytical detection methods or more rigorous sampling routines, it is possible that this expansion has been assisted by a number of changing conditions in these environments. The geographical expansion of both the organism and toxin production can be attributed to phenomena such as eutrophication and climate change. In this review, we discuss the occurrence of C. raciborskii with respect to current literature against the backdrop of increasing global temperatures. Critically, we identify a concerning trend between the geographical spread of this organism and global climate change.

Characterization of the 2-hydroxy-acid dehydrogenase McyI, encoded within the microcystin biosynthesis gene cluster of Microcystis aeruginosa PCC7806.

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of d-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce alpha-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.

Inactivation of an ABC transporter gene, mcyH, results in loss of microcystin production in the cyanobacterium Microcystis aeruginosa PCC 7806.

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. Microcystin is synthesized nonribosomally by the thiotemplate function of a large, modular enzyme complex encoded within the 55-kb microcystin synthetase (mcy) gene cluster. Also encoded within the mcy gene cluster is a putative ATP binding cassette (ABC) transporter, McyH. This study details the bioinformatic and mutational analyses of McyH and offers functional predictions for the hypothetical protein. The transporter is putatively comprised of two homodimers, each with an N-terminal hydrophobic domain and a C-terminal ATPase. Phylogenetically, McyH was found to cluster with members of the ABC-A1 subgroup of ABC ATPases, suggesting an export function for the protein. Two mcyH null mutant (DeltamcyH) strains were constructed by partial deletion of the mcyH gene. Microcystin production was completely absent in these strains. While the mcyH deletion had no apparent effect on the transcription of other mcy genes, the complete microcystin biosynthesis enzyme complex could not be detected in DeltamcyH mutant strains. Finally, expression levels of McyH in the wild type and in DeltamcyA, DeltamcyB, and DeltamcyH mutants were investigated by using immunoblotting with an anti-McyH antibody. Expression of McyH was found to be reduced in DeltamcyA and DeltamcyB mutants and completely absent in the DeltamcyH mutant. By virtue of its association with the mcy gene cluster and the bioinformatic and experimental data presented in this study, we predict that McyH functions as a microcystin exporter and is, in addition, intimately associated with the microcystin biosynthesis pathway.