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Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number of methodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance in microbiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced with MiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10 million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.

The human gut microbiome consists of at least 3 million non-redundant genes, 150 times that of the core human genome. Herein, we report the identification and characterisation of a novel stress tolerance gene from the human gut metagenome. The locus, assigned brpA, encodes a membrane protein with homology to a brp/blh-family β-carotene monooxygenase. Cloning and heterologous expression of brpA in Escherichia coli confers a significant salt tolerance phenotype. Furthermore, when cultured in the presence of exogenous β-carotene, cell pellets adopt a red/orange pigmentation indicating the incorporation of carotenoids in the cell membrane.

The recent detection and isolation of C. ureolyticus from patients with diarrhoeal illness and inflammatory bowel diseases warrants further investigation into its role as an emerging pathogen of the human gastrointestinal tract. Regarding the pathogenic mechanisms employed by this species we provide the first whole genome analysis of two C. ureolyticus isolates including the type strain. Comparative analysis, subtractive hybridisation and gene ontology searches against other Campylobacter species identifies the high degree of heterogenicity between C. ureolyticus isolates, in addition to the identification of 106 putative virulence associated factors, 52 of which are predicted to be secreted. Such factors encompass each of the known virulence tactics of pathogenic Campylobacter spp. including adhesion and colonisation (CadF, PEB1, IcmF and FlpA), invasion (ciaB and 16 virB-virD4 genes) and toxin production (S-layer RTX and ZOT). Herein, we provide the first virulence catalogue for C. ureolyticus, the components of which theoretically provide this emerging species with sufficient arsenal to establish pathology.

Functional environmental screening of metagenomic libraries is a powerful means to identify and assign function to novel genes and their encoded proteins without any prior sequence knowledge. In the current study we describe the identification and subsequent analysis of a salt-tolerant clone from a human gut metagenomic library. Following transposon mutagenesis we identified an unknown gene (stlA, for "salt tolerance locus A") with no current known homologues in the databases. Subsequent cloning and expression in Escherichia coli MKH13 revealed that stlA confers a salt tolerance phenotype in its surrogate host. Furthermore, a detailed in silico analysis was also conducted to gain additional information on the properties of the encoded StlA protein. The stlA gene is rare when searched against human metagenome datasets such as MetaHit and the Human Microbiome Project and represents a novel and unique salt tolerance determinant which appears to be found exclusively in the human gut environment.

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.