Exploiting the diverse microbial ecology of marine sponges

Marine sponges (phylum Porifera) are the oldest extant metazoan animals on earth and host large populations of symbiotic microbes: Bacteria, Archaea and unicellular Eukaryota. Those microbes play ecological functions which are essential to the health of the host including carbon, nitrogen and sulfur cycling as well as host defence through the production of bioactive secondary metabolites which protect against infection and predation. The diversity of sponge-associated microbes is remarkable with thousands of OTUs reported from individual sponge species. Amongst those populations are sponge-specific microbes which may be specific to sponges or specific to sponge species. While marine natural product discovery concerns many animal phyla, Porifera account for the largest proportion of novel compounds. Evidence suggests that many of these compounds are the products of symbiotic microbes. Descriptions of sponge-associated microbial community structures have been advanced by the development of next-generation sequencing technologies while the discovery and exploitation of sponge derived bioactive compounds has increased due to developments in sequence-based and function-based metagenomics. Here, we use pyrosequencing to describe the bacterial communities associated with two shallow, temperate water sponges (Raspailia ramosa and Stelligera stuposa) from Irish coastal waters and to describe the bacterial and archaeal communities of a single sponge species (Inflatella pellicula) from two different depths in deep waters in the Atlantic Ocean, including at a depth of 2900m, a depth far greater than that of any previous sequence-based sponge-microbe investigation. We identified diverse microbial communities in all sponges and the presence of sponge-specific taxa recruiting to previously described and novel spongespecific clusters. We also identified archaeal communities which dominated sponge-microbe communities. We demonstrate that sponge-associated microbial communities differ from seawater communities indicating host selection processes. We used sequence-based metagenomic techniques to identify genes of potential industrial and pharmacological interest in the metagenomes of various sponge species and functionbased metagenomic screening in an attempt to identify lipolytic and antibacterial activities from metagenomic clones from the metagenome of the marine sponge Stelletta normani. In addition we have cultured diverse bacterial species from sponge tissues, many of which display antimicrobial activities against clinically relevant bacterial and yeast test strains. Other isolates represent novel species in the genus Maribacter and require emendments to the description of that genus.

Functional genomics of motile commensal intestinal bacteria

Flagella confer upon bacteria the ability to move and are therefore organelles of significant bacteriological importance. The innate immune system has evolved to recognise flagellin, (the major protein component of the bacterial flagellar filament). Flagellate microbes can potentially stimulate the immune systems of mammals, and thus have significant immunomodulatory potential. The flagellum-biogenesis genotype and phenotype of Lactobacillus ruminis, an autochthonous intestinal commensal, was studied. The flagellum-biogenesis genotypes of motile enteric Eubacterium and Roseburia species were also investigated. Flagellin proteins were recovered from these commensal species, their amino-termini were sequenced and the proteins were found to be pro-inflammatory, as assessed by measurement of interleukin-8 (IL-8) secretion from human intestinal epithelial cell lines. For L. ruminis, this IL-8 secretion required signalling through Toll Like Receptor 5. A model for the regulation of flagellum-biogenesis in L. ruminis was inferred from transcriptomics data and bioinformatics analyses. Motility gene expression in this species may be under the control of a novel regulator, LRC_15730. Potential promoters for genes encoding flagellin proteins in the Eubacterium and Roseburia genomes analysed were inferred in silico. Relative abundances of the target Eubacterium and Roseburia species in the intestinal microbiota of 25 elderly individuals were determined. These species were found to be variably abundant in these individuals. Motility genes from these species were variably detected in the shotgun metagenome databases generated by the ELDERMET project. This suggested that a greater depth of sequencing, or improved evenness of sequencing, would be required to capture the full diversity of microbial functions for specific target or low abundance species in microbial communities by metagenomics. In summary, this thesis used a functional genomics approach to describe flagellum-mediated motility in selected Gram-positive commensal bacteria. The regulation of flagellum biosynthesis in these species, and the consequences of flagella expression from a host-interaction perspective were also considered.

Genetic analysis of bacteriophages from clinical and environmental samples

Bacteriophages, viruses infecting bacteria, are uniformly present in any location where there are high numbers of bacteria, both in the external environment and the human body. Knowledge of their diversity is limited by the difficulty to culture the host species and by the lack of the universal marker gene present in all viruses. Metagenomics is a powerful tool that can be used to analyse viral communities in their natural environments. The aim of this study was to investigate diverse populations of uncultured viruses from clinical (a sputum of patient with cystic fibrosis, CF) and environmental samples (a sludge from a dairy food wastewater treatment plant) containing rich bacterial populations using genetic and metagenomic analyses. Metagenomic sequencing of viruses obtained from these samples revealed that the majority of the metagenomic reads (97-99%) were novel when compared to the NCBI protein database using BLAST. A large proportion of assembled contigs were assignable as novel phages or uncharacterised prophages, the next largest assignable group being single-stranded eukaryotic virus genomes. Sputum from a cystic fibrosis patient contained DNA typical of phages of bacteria that are traditionally involved in CF lung infections and other bacteria that are part of the normal oral flora. The only eukaryotic virus detected in the CF sputum was Torque Teno virus (TTV). A substantial number of assigned sequences from dairy wastewater could be affiliated with phages of bacteria that are typically found in the soil and aquatic environments, including wastewater. Eukaryotic viral sequences were dominated by plant pathogens from the Geminiviridae and Nanoviridae families, and animal pathogens from the Circoviridae family. Antibiotic resistance genes were detected in both metagenomes suggesting phages could be a source for transmissible antimicrobial resistance. Overall, diversity of viruses in the CF sputum was low, with 89 distinct viral genotypes predicted, and higher (409 genotypes) in the wastewater. Function-based screening of a metagenomic library constructed from DNA extracted from dairy food wastewater viruses revealed candidate promoter sequences that have ability to drive expression of GFP in a promoter-trap vector in Escherichia coli. The majority of the cloned DNA sequences selected by the assay were related to ssDNA circular eukaryotic viruses and phages which formed a minority of the metagenome assembly, and many lacked any significant homology to known database sequences. Natural diversity of bacteriophages in wastewater samples was also examined by PCR amplification of the major capsid protein sequences, conserved within T4-type bacteriophages from Myoviridae family. Phylogenetic analysis of capsid sequences revealed that dairy wastewater contained mainly diverse and uncharacterized phages, while some showed a high level of similarity with phages from geographically distant environments.

Pro-inflammatory flagellin proteins of prevalent motile commensal bacteria are variably abundant in the intestinal microbiome of elderly humans

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute "cell motility" category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the beta- and gamma-proteobacteria. Many fliC genes were deduced to be under the control of sigma(28). The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13-4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (<= 1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.