BackgroundLittle is known regarding the pool of mobile genetic elements associated with the human gut microbiome. In this study we employed the culture independent TRACA system to isolate novel plasmids from the human gut microbiota, and a comparative metagenomic analysis to investigate the distribution and relative abundance of functions encoded by these plasmids in the human gut microbiome.ResultsNovel plasmids were acquired from the human gut microbiome, and homologous nucleotide sequences with high identity (>90%) to two plasmids (pTRACA10 and pTRACA22) were identified in the multiple human gut microbiomes analysed here. However, no homologous nucleotide sequences to these plasmids were identified in the murine gut or environmental metagenomes. Functions encoded by the plasmids pTRACA10 and pTRACA22 were found to be more prevalent in the human gut microbiome when compared to microbial communities from other environments. Among the most prevalent functions identified was a putative RelBE toxin-antitoxin (TA) addiction module, and subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes. A broad phylogenetic distribution of RelE toxin genes was observed in gut associated bacterial species (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria), but no RelE homologues were identified in gut associated archaeal species. We also provide indirect evidence for the horizontal transfer of these genes between bacterial species belonging to disparate phylogenetic divisions, namely Gram negative Proteobacteria and Gram positive species from the Firmicutes division.ConclusionsThe application of a culture independent system to capture novel plasmids from the human gut mobile metagenome, coupled with subsequent comparative metagenomic analysis, highlighted the unexpected prevalence of plasmid encoded functions in the gut microbial ecosystem. In particular the increased relative abundance and broad phylogenetic distribution was identified for a putative RelBE toxin/antitoxin addiction module, a putative phosphohydrolase/phosphoesterase, and an ORF of unknown function. Our analysis also indicates that some plasmids or plasmid families are present in the gut microbiomes of geographically isolated human hosts with a broad global distribution (America, Japan and Europe), and are potentially unique to the human gut microbiome. Further investigation of the plasmid population associated with the human gut is likely to provide important insights into the development, functioning and evolution of the human gut microbiota.

Aims:  Functional screens using skimmed‐milk agar to obtain protease activity is a common approach. The aim of this study was to determine the efficacy of this screen to obtain protease activity from a metagenomic library. Methods and Results:  A distal gut metagenomic library was functionally screened using a skimmed‐milk agar. The functional screen provided 231 clones generating the characteristic clear halo indicative of protease production. Clone analysis revealed that they were not protease‐positive, but expressed glycosidic hydrolases and produced acid, which was responsible for the clear halos. Conclusions:  The current skimmed‐milk agar method to obtain proteases is not sufficiently robust to provide a definitive screen. Other‐ non‐protease activities will also give the same clear halo and these would be interpreted as protease positive clones without further analysis. Hence a more robust buffered medium or a specific protein should be used. Significance and Impact of the Study:  Functional screens are a powerful approach to obtaining enzymes from large metagenomic libraries and proteases are a particularly interesting target. The skimmed‐milk agar is not sufficiently robust to ensure that only proteases are isolated and in order to save time and money this study has shown that better designed media can aid in the process.

Lactic acid bacteria (LAB) were isolated from malted cereals by means of a high-throughput screening approach and investigated for antimicrobial activity against a range of beer-spoiling bacteria. Putative bacteriocin-producing strains were identified by 16S rRNA analysis and the inhibitory compounds were partially characterized. Following determination of the inhibitory spectra of the strains, an unspeciated Lactobacillus sp. UCC128, with inhibitory activity against a range of beer-spoiling strains was subjected to further characterization. A bacteriocin was purified from this strain and analyzed by mass spectrometry to determine the weight of the protein. The result indicated that the bacteriocin was highly similar to pediocin AcH /PA-1 from Pediococcus acidilactici. The bacteriocin-producers identified in this study have the potential to be used in the brewing industry to enhance the microbiological stability of beer in conjunction with hurdles already in place in the brewing process.