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Chlorophylls are essential for light-harvesting and energy transduction in photosynthesis. Four chemically distinct varieties have been known for the past 60 years. Here we report isolation of a fifth, which we designate chlorophyll f. Its in vitro absorption (706 nanometers) and fluorescence (722 nanometers) maxima are red-shifted compared to all other chlorophylls from oxygenic phototrophs. On the basis of the optical, mass, and nuclear magnetic resonance spectra, we propose that chlorophyll f is [2-formyl]-chlorophyll a (C₅₅H₇₀O₆N₄Mg). This finding suggests that oxygenic photosynthesis can be extended further into the infrared region and may open associated bioenergy applications.

Saxitoxin is a potent neurotoxin that occurs in aquatic environments worldwide. Ingestion of vector species can lead to paralytic shellfish poisoning, a severe human illness that may lead to paralysis and death. In freshwaters, the toxin is produced by prokaryotic cyanobacteria; in marine waters, it is associated with eukaryotic dinoflagellates. However, several studies suggest that saxitoxin is not produced by dinoflagellates themselves, but by co-cultured bacteria. Here, we show that genes required for saxitoxin synthesis are encoded in the nuclear genomes of dinoflagellates. We sequenced >1.2×10(6) mRNA transcripts from the two saxitoxin-producing dinoflagellate strains Alexandrium fundyense CCMP1719 and A. minutum CCMP113 using high-throughput sequencing technology. In addition, we used in silico transcriptome analyses, RACE, qPCR and conventional PCR coupled with Sanger sequencing. These approaches successfully identified genes required for saxitoxin-synthesis in the two transcriptomes. We focused on sxtA, the unique starting gene of saxitoxin synthesis, and show that the dinoflagellate transcripts of sxtA have the same domain structure as the cyanobacterial sxtA genes. But, in contrast to the bacterial homologs, the dinoflagellate transcripts are monocistronic, have a higher GC content, occur in multiple copies, contain typical dinoflagellate spliced-leader sequences and eukaryotic polyA-tails. Further, we investigated 28 saxitoxin-producing and non-producing dinoflagellate strains from six different genera for the presence of genomic sxtA homologs. Our results show very good agreement between the presence of sxtA and saxitoxin-synthesis, except in three strains of A. tamarense, for which we amplified sxtA, but did not detect the toxin. Our work opens for possibilities to develop molecular tools to detect saxitoxin-producing dinoflagellates in the environment.

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a 'model' dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of 'cryptic' species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them.

The complexity of microbial communities, combined with technical biases in next-generation sequencing, pose a challenge to metagenomic analysis. Here, we develop a set of internal DNA standards, termed “sequins” (sequencing spike-ins), that together constitute a synthetic community of artificial microbial genomes. Sequins are added to environmental DNA samples prior to library preparation, and undergo concurrent sequencing with the accompanying sample. We validate the performance of sequins by comparison to mock microbial communities, and demonstrate their use in the analysis of real metagenome samples. We show how sequins can be used to measure fold change differences in the size and structure of accompanying microbial communities, and perform quantitative normalization between samples. We further illustrate how sequins can be used to benchmark and optimize new methods, including nanopore long-read sequencing technology. We provide metagenome sequins, along with associated data sets, protocols, and an accompanying software toolkit, as reference standards to aid in metagenomic studies. Complex microbial communities pose a challenge to metagenomic analysis. Here the authors develop ‘sequins’, internal DNA standards that represent a synthetic community of artificial genomes.

The association of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) with Crohn's disease is a controversial issue. M. paratuberculosis is detected by amplifying the IS900 gene, as microbial culture is unreliable from humans. We determined the presence of M. paratuberculosis in patients with Crohn's disease (CD) (n = 22), ulcerative colitis (UC) (n = 20), aphthous ulcers (n = 21) and controls (n = 42) using PCR assays validated on bovine tissue. Culture from human tissue was also performed. M. paratuberculosis prevalence in the CD and UC groups was compared to the prevalence in age and sex matched non-inflammatory bowel disease controls. Patients and controls were determined to be M. paratuberculosis positive if all three PCR assays were positive. A significant association was found between M. paratuberculosis and Crohn's disease (p = 0.02) that was not related to age, gender, place of birth, smoking or alcohol intake. No significant association was detected between M. paratuberculosis and UC or aphthous ulcers; however, one M. paratuberculosis isolate was successfully cultured from a patient with UC. We report the resistance of this isolate to ethambutol, rifampin, clofazamine and streptomycin. Interestingly this isolate could not only survive but could grow slowly at 5°C. We demonstrate a significant association between M. paratuberculosis and CD using multiple pre-validated PCR assays and that M. paratuberculosis can be isolated from patients with UC.

The recent determination of the genetic basis for the biosynthesis of the neurotoxin, saxitoxin, produced by cyanobacteria, has revealed a highly complex sequence of reactions, involving over 30 biosynthetic steps encoded by up to 26 genes clustered at one genomic locus, sxt. Insights into evolutionary–ecological processes have been found through the study of such secondary metabolites because they consist of a measurable phenotype with clear ecological consequences, synthesized by known genes in a small number of species. However, the processes involved in and timing of the divergence of prokaryotic secondary metabolites have been difficult to determine due to their antiquity and the possible frequency of horizontal gene transfer and homologous recombination. Through analyses of gene synteny, phylogenies of individual genes, and analyses of recombination and selection, we identified the evolutionary processes of this cluster in five species of cyanobacteria. Here, we provide evidence that the sxt cluster appears to have been largely vertically inherited and was therefore likely present early in the divergence of the Nostocales, at least 2,100 Ma, the earliest reliably dated appearance of a secondary metabolite. The sxt cluster has been extraordinarily conserved through stabilizing selection. Genes have been lost and rearranged, have undergone intra- and interspecific recombination, and have been subject to duplication followed by positive selection along the duplicated lineage, with likely consequences for the toxin analogues produced. Several hypotheses exist as to the ecophysiological role of saxitoxin: as a method of chemical defense, cellular nitrogen storage, DNA metabolism, or chemical signaling. The antiquity of this gene cluster indicates that potassium channels, not sodium channels, may have been the original targets of this compound. The extraordinary conservation of the machinery for saxitoxin synthesis, under radically changing environmental conditions, shows that it has continued to play an important adaptive role in some cyanobacteria.

Animals that modify their physical environment by foraging in the soil can have dramatic effects on ecosystem functions and processes. We compared bacterial and fungal communities in the foraging pits created by bilbies and burrowing bettongs with undisturbed surface soils dominated by biocrusts. Bacterial communities were characterized by Actinobacteria and Alphaproteobacteria, and fungal communities by Lecanoromycetes and Archaeosporomycetes. The composition of bacterial or fungal communities was not observed to vary between loamy or sandy soils. There were no differences in richness of either bacterial or fungal operational taxonomic units (OTUs) in the soil of young or old foraging pits, or undisturbed soils. Although the bacterial assemblage did not vary among the three microsites, the composition of fungi in undisturbed soils was significantly different from that in old or young foraging pits. Network analysis indicated that a greater number of correlations between bacterial OTUs occurred in undisturbed soils and old pits, whereas a greater number of correlations between fungal OTUs occurred in undisturbed soils. Our study suggests that digging by soil-disturbing animals is likely to create successional shifts in soil microbial and fungal communities, leading to functional shifts associated with the decomposition of organic matter and the fixation of nitrogen. Given the primacy of organic matter decomposition in arid and semi-arid environments, the loss of native soil-foraging animals is likely to impair the ability of these systems to maintain key ecosystem processes such as the mineralization of nitrogen and the breakdown of organic matter, and to recover from disturbance.

Summary Genome mining of Ascomycete sp. F53 (F53), a fungal endophyte of the traditional Chinese medicinal plant Taxus yunnanensis (Chinese yew), revealed 35 putative specialized metabolite biosynthesis gene clusters, one of which encodes a rarely seen tandem polyketide synthase pathway with close homology to azaphilone biosynthesis pathways. A novel compound, lijiquinone 1, was subsequently isolated from F53 and structurally and functionally characterized. The m/z 385 [M + H+]+ compound, comprised of a cyclohexenone side group attached to a core bicyclic ring, displayed cytotoxicity against human myeloma cells (IC50 = 129 μM), as well as antifungal activity against Candida albicans (IC50 = 79 μM) and Cryptococcus albidus (IC50 = 141 μM). Our results suggest that enzymes encoded on the lij gene cluster are responsible for the synthesis of 1 and that the medicinal properties of T. yunnanensis could be partially mediated by this novel azaphilone. This study highlights the utility of combining traditional knowledge with contemporary genomic approaches for the discovery of new bioactive compounds. Genome mining of Ascomycete sp. F53 (F53), a fungal endophyte of the traditional Chinese medicinal plant Taxus yunnanensis (Chinese yew), revealed 35 putative specialized metabolite biosynthesis gene clusters, one of which encodes a rarely seen tandem polyketide synthase pathway. We believe this is the pathway for the novel compound, lijiquinone, which was isolated from F53 and structurally and functionally characterised.

Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds.

Traditional Chinese medicine encompasses a rich empirical knowledge of the use of plants for the treatment of disease. In addition, the microorganisms associated with medicinal plants are also of interest as the producers of the compounds responsible for the observed plant bioactivity. The present study has pioneered the use of genetic screening to assess the potential of endophytes to synthesize bioactive compounds, as indicated by the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes. The total DNA extracts of 30 traditional Chinese herbs, were screened for functional genes involved in the biosynthesis of bioactive compounds. The four PCR screens were successful in targeting four bacterial PKS, six bacterial NRPS, ten fungal PKS and three fungal NRPS gene fragments. Analysis of the detected endophyte gene fragments afforded consideration of the possible bioactivity of the natural products produced by endophytes in medicinal herbs. This investigation describes a rapid method for the initial screening of medicinal herbs and has highlighted a subset of those plants that host endophytes with biosynthetic potential. These selected plants can be the focus of more comprehensive endophyte isolation and natural product studies.