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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.

The frequency of freshwater cyanobacterial blooms is at risk of increasing as a consequence of climate change and eutrophication of waterways. It is increasingly apparent that abiotic data are insufficient to explain variability within the cyanobacterial community, with biotic factors such as heterotrophic bacterioplankton, viruses and protists emerging as critical drivers. During the Australian summer of 2012-2013, a bloom that occurred in a shallow ephemeral lake over a 6-month period was comprised of 22 distinct cyanobacteria, including Microcystis, Dolichospermum, Oscillatoria and Sphaerospermopsis. Cyanobacterial cell densities, bacterial community composition and abiotic parameters were assessed over this period. Alpha-diversity indices and multivariate analysis were successful at differentiating three distinct bloom phases and the contribution of abiotic parameters to each. Network analysis, assessing correlations between biotic and abiotic variables, reproduced these phases and assessed the relative importance of both abiotic and biotic factors. Variables possessing elevated betweeness centrality included temperature, sodium and operational taxonomic units belonging to the phyla Verrucomicrobia, Planctomyces, Bacteroidetes and Actinobacteria. Species-specific associations between cyanobacteria and bacterioplankton, including the free-living Actinobacteria acI, Bacteroidetes, Betaproteobacteria and Verrucomicrobia, were also identified. We concluded that changes in the abundance and nature of freshwater cyanobacteria are associated with changes in the diversity and composition of lake bacterioplankton. Given this, an increase in the frequency of cyanobacteria blooms has the potential to alter nutrient cycling and contribute to long-term functional perturbation of freshwater systems.

Modern microbial mats are potential analogues of some of Earth's earliest ecosystems. Excellent examples can be found in Shark Bay, Australia, with mats of various morphologies. To further our understanding of the functional genetic potential of these complex microbial ecosystems, we conducted for the first time shotgun metagenomic analyses. We assembled metagenomic next-generation sequencing data to classify the taxonomic and metabolic potential across diverse morphologies of marine mats in Shark Bay. The microbial community across taxonomic classifications using protein-coding and small subunit rRNA genes directly extracted from the metagenomes suggests that three phyla Proteobacteria, Cyanobacteria and Bacteriodetes dominate all marine mats. However, the microbial community structure between Shark Bay and Highbourne Cay (Bahamas) marine systems appears to be distinct from each other. The metabolic potential (based on SEED subsystem classifications) of the Shark Bay and Highbourne Cay microbial communities were also distinct. Shark Bay metagenomes have a metabolic pathway profile consisting of both heterotrophic and photosynthetic pathways, whereas Highbourne Cay appears to be dominated almost exclusively by photosynthetic pathways. Alternative non-rubisco-based carbon metabolism including reductive TCA cycle and 3-hydroxypropionate/4-hydroxybutyrate pathways is highly represented in Shark Bay metagenomes while not represented in Highbourne Cay microbial mats or any other mat forming ecosystems investigated to date. Potentially novel aspects of nitrogen cycling were also observed, as well as putative heavy metal cycling (arsenic, mercury, copper and cadmium). Finally, archaea are highly represented in Shark Bay and may have critical roles in overall ecosystem function in these modern microbial mats.

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.

The biosynthesis of non-ribosomal peptide and polyketide natural products is facilitated by multimodular enzymes that contain domains responsible for the sequential condensation of amino and carboxylic subunits. These conserved domains provide molecular targets for the discovery of natural products from microbial metagenomes. This study demonstrates the application of tag-encoded FLX amplicon pyrosequencing (TEFAP) targeting non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes as a method for determining the identity and diversity of natural product biosynthesis genes. To validate this approach, we assessed the diversity of NRPS and PKS genes within the microbiomes of six Australian marine sponge species using both TEFAP and metagenomic whole-genome shotgun sequencing approaches. The TEFAP approach identified 100 novel ketosynthase (KS) domain sequences and 400 novel condensation domain sequences within the microbiomes of the six sponges. The diversity of KS domains within the microbiome of a single sponge species Scopalina sp. exceeded that of any previously surveyed marine sponge. Furthermore, this study represented the first to target the condensation domain from NRPS biosynthesis and resulted in the identification of a novel condensation domain lineage. This study highlights the untapped potential of Australian marine sponges for the isolation of novel bioactive natural products. Furthermore, this study demonstrates that TEFAP approaches can be applied to functional genes, involved in natural product biosynthesis, as a tool to aid natural product discovery. It is envisaged that this approach will be used across multiple environments, offering an insight into the biological processes that influence the production of secondary metabolites.

The nitrogen-fixing bacterium, Nostoc, is a commonly occurring cyanobacterium often found in symbiotic associations. We investigated the potential of cycad cyanobacterial endosymbionts to synthesize microcystin/nodularin. Endosymbiont DNA was screened for the aminotransferase domain of the toxin biosynthesis gene clusters. Five endosymbionts carrying the gene were screened for bioactivity. Extracts of two isolates inhibited protein phosphatase 2A and were further analyzed using electrospray ionization mass spectrometry (ESI-MS)/MS. Nostoc sp. 'Macrozamia riedlei 65.1' and Nostoc sp. 'Macrozamia serpentina 73.1' both contained nodularin. High performance liquid chromatography (HPLC) HESI-MS/MS analysis confirmed the presence of nodularin at 9.55±2.4 ng μg-1 chlorophyll a in Nostoc sp. 'Macrozamia riedlei 65.1' and 12.5±8.4 ng μg-1 Chl a in Nostoc sp. 'Macrozamia serpentina 73.1' extracts. Further scans indicated the presence of the rare isoform [L-Har(2)] nodularin, which contains L-homoarginine instead of L-arginine. Nodularin was also present at 1.34±0.74 ng ml(-1) (approximately 3 pmol per g plant ww) in the methanol root extracts of M. riedlei MZ65, while the presence of [L-Har(2)] nodularin in the roots of M. serpentina MZ73 was suggested by HPLC HESI-MS/MS analysis. The ndaA-B and ndaF genomic regions were sequenced to confirm the presence of the hybrid polyketide/non-ribosomal gene cluster. A seven amino-acid insertion into the NdaA-C1 domain of N. spumigena NSOR10 protein was observed in all endosymbiont-derived sequences, suggesting the transfer of the nda cluster from N. spumigena to terrestrial Nostoc species. This study demonstrates the synthesis of nodularin and [L-Har(2)] nodularin in a non-Nodularia species and the production of cyanobacterial hepatotoxin by a symbiont in planta.

The stromatolites at Shark Bay, Western Australia, are analogues of some of the oldest evidence of life on Earth. The aim of this study was to identify and spatially characterize the specific microbial communities associated with Shark Bay intertidal columnar stromatolites. Conventional culturing methods and construction of 16S rDNA clone libraries from community genomic DNA with both universal and specific PCR primers were employed. The estimated coverage, richness and diversity of stromatolite microbial populations were compared with earlier studies on these ecosystems. The estimated coverage for all clone libraries indicated that population coverage was comprehensive. Phylogenetic analyses of stromatolite and surrounding seawater sequences were performed in ARB with the Greengenes database of full-length non-chimaeric 16S rRNA genes. The communities identified exhibited extensive diversity. The most abundant sequences from the stromatolites were alpha- and gamma-proteobacteria (58%), whereas the cyanobacterial community was characterized by sequences related to the genera Euhalothece, Gloeocapsa, Gloeothece, Chroococcidiopsis, Dermocarpella, Acaryochloris, Geitlerinema and Schizothrix. All clones from the archaeal-specific clone libraries were related to the halophilic archaea; however, no archaeal sequence was identified from the surrounding seawater. Fluorescence in situ hybridization also revealed stromatolite surfaces to be dominated by unicellular cyanobacteria, in contrast to the sub-surface archaea and sulphate-reducing bacteria. This study is the first to compare the microbial composition of morphologically similar stromatolites over time and examine the spatial distribution of specific microorganismic groups in these intertidal structures and the surrounding seawater at Shark Bay. The results provide a platform for identifying the key microbial physiology groups and their potential roles in modern stromatolite morphogenesis and ecology.