School of Biological Sciences

Members of the bacterial genus Fibrobacter have long been considered important components of the anaerobic cellulolytic community in the herbivore gut, but their presence and activity in other environments is largely unknown. In this study, a specific polymerase chain reaction (PCR) primer set, targeting the 16S rRNA gene of Fibrobacter spp., was applied to community DNA from five landfill sites followed by temporal thermal gel electrophoresis (TTGE) analysis of cloned amplification products. Phylogenetic analysis of clone sequences indicated the presence of novel clusters closely related to the genus Fibrobacter. There are two named species, Fibrobacter succinogenes and F. intestinalis, and only two of the 58 sequenced clones were identified with them, and both were F. succinogenes. The clone sequences from landfill were recovered in five distinct clusters within the Fibrobacter lineage, and four of these were novel. Quantitative PCR (qPCR) assays of reverse-transcribed community RNA from landfill leachates and rumen fluid samples indicated that the abundance of Fibrobacter spp. relative to total bacteria varied from 0.2% to 40% in landfill, and 21% to 32% in the rumen, and these data demonstrate that fibrobacters can be a significant component of the microbial community in landfill ecosystems. This is the first evidence for Fibrobacter spp. outside the gut ecosystem, and as the only cultivated representatives of this group are actively cellulolytic, their diversity and abundance points to a possible role in cellulose hydrolysis in landfill, and perhaps other anaerobic environments also.

Polysaccharides are an important source of organic carbon in the marine environment and degradation of the insoluble and globally abundant cellulose is a major component of the marine carbon cycle. Although a number of species of cultured bacteria are known to degrade crystalline cellulose, little is known of the polysaccharide hydrolases expressed by cellulose-degrading microbial communities, particularly in the marine environment. Next generation 454 Pyrosequencing was applied to analyze the microbial community that colonizes and degrades insoluble polysaccharides in situ in the Irish Sea. The bioinformatics tool MG-RAST was used to examine the randomly sampled data for taxonomic markers and functional genes, and showed that the community was dominated by members of the Gammaproteobacteria and Bacteroidetes. Furthermore, the identification of 211 gene sequences matched to a custom-made database comprising the members of nine glycoside hydrolase families revealed an extensive repertoire of functional genes predicted to be involved in cellulose utilization. This demonstrates that the use of an in situ cellulose baiting method yielded a marine microbial metagenome considerably enriched in functional genes involved in polysaccharide degradation. The research reported here is the first designed to specifically address the bacterial communities that colonize and degrade cellulose in the marine environment and to evaluate the glycoside hydrolase (cellulase and chitinase) gene repertoire of that community, in the absence of the biases associated with PCR-based molecular techniques.

Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785) were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx) encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%). The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases.

The phylum Fibrobacteres currently comprises one formal genus, Fibrobacter, and two cultured species, Fibrobacter succinogenes and Fibrobacter intestinalis, that are recognised as major bacterial degraders of lignocellulosic material in the herbivore gut. Historically, members of the genus Fibrobacter were thought to only occupy mammalian intestinal tracts. However, recent 16S rRNA gene-targeted molecular approaches have demonstrated that novel centres of variation within the genus Fibrobacter are present in landfill sites and freshwater lakes, and their relative abundance suggests a potential role for fibrobacters in cellulose degradation beyond the herbivore gut. Furthermore, a novel subphylum within the Fibrobacteres has been detected in the gut of wood-feeding termites, and proteomic analyses have confirmed their involvement in cellulose hydrolysis. The genome sequence of F. succinogenes rumen strain S85 has recently suggested that within this group of organisms a “third” way of attacking the most abundant form of organic carbon in the biosphere, cellulose, has evolved. This observation not only has evolutionary significance, but the superior efficiency of anaerobic cellulose hydrolysis by Fibrobacter spp., in comparison to other cellulolytic rumen bacteria that typically utilise membrane-bound enzyme complexes (cellulosomes), may be explained by this novel cellulase system. There are few bacterial phyla with potential functional importance for which there is such a paucity of phenotypic and functional data. In this review, we highlight current knowledge of the Fibrobacteres phylum, its taxonomy, phylogeny, ecology and potential as a source of novel glycosyl hydrolases of biotechnological importance.

The relative abundance of micromonosporas in the bacterial communities inhabiting cellulose baits, the water columns and sediments of two freshwater lakes was determined by qPCR of reverse transcribed 16S rRNA. Micromonospora spp. were shown to be significant members of the active bacterial population colonising cellulosic substrates at the lake sediment, and their increased prevalence with depth was confirmed by enumeration of colony forming units.

Faecal contamination of estuarine and coastal waters can pose a risk to human health, particularly in areas used for shellfish production or recreation. Routine microbiological water quality testing highlights areas of faecal indicator bacteria (FIB) contamination within the water column, but fails to consider the abundance of FIB in sediments, which under certain hydrodynamic conditions can become resuspended. Sediments can enhance the survival of FIB in estuarine environments, but the influence of sediment composition on the ecology and abundance of FIB is poorly understood. To determine the relationship between sediment composition (grain size and organic matter) and the abundance of pathogen indicator bacteria (PIB), sediments were collected from four transverse transects of the Conwy estuary, UK. The abundance of culturable Escherichia coli, total coliforms, enterococci, Campylobacter, Salmonella and Vibrio spp. in sediments was determined in relation to sediment grain size, organic matter content, salinity, depth and temperature. Sediments that contained higher proportions of silt and/or clay and associated organic matter content showed significant positive correlations with the abundance of PIB. Furthermore, the abundance of each bacterial group was positively correlated with the presence of all other groups enumerated. Campylobacter spp. were not isolated from estuarine sediments. Comparisons of the number of culturable E. coli, coliform and Vibrio spp. in sediments and the water column revealed that their abundance was 281, 433 and 58-fold greater in sediments (colony forming units (CFU)/100 g) when compared with the water column (CFU/100 ml), respectively. These data provide important insights into sediment compositions that promote the abundance of PIB in estuarine environments, with important implications for the modelling and prediction of public health risk based on sediment resuspension and transport.

In insects, the homologue of the Down syndrome cell adhesion molecule (Dscam) is a unique case of a single-locus gene whose expression has extensive somatic diversification in both the nervous and immune systems. How this situation evolved is best understood through comparative studies. We describe structural, expression, and evolutionary aspects of a Dscam homolog in 2 species of the crustacean Daphnia. The Dscam of Daphnia generates up to 13,000 different transcripts by the alternative splicing of variable exons. This extends the taxonomic range of a highly diversified Dscam beyond the insects. Additionally, we have identified 4 alternative forms of the cytoplasmic tail that generate isoforms with or without inhibitory or activating immunoreceptor tyrosine-based motifs (ITIM and ITAM respectively), something not previously reported in insect's Dscam. In Daphnia, we detected exon usage variability in both the brain and hemocytes (the effector cells of immunity), suggesting that Dscam plays a role in the nervous and immune systems of crustaceans, as it does in insects. Phylogenetic analysis shows a high degree of amino acid conservation between Daphnia and insects except in the alternative exons, which diverge greatly between these taxa. Our analysis shows that the variable exons diverged before the split of the 2 Daphnia species and is in agreement with the nearest-neighbor model for the evolution of the alternative exons. The genealogy of the Dscam gene family from vertebrates and invertebrates confirmed that the highly diversified form of the gene evolved from a nondiversified form before the split of insects and crustaceans.

Yeast species have undergone extensive genome reorganization in their evolutionary history, including variations in chromosome number and large chromosomal rearrangements, such as translocations. To determine directly the contribution of chromosomal translocations to the whole organism's fitness, we devised a strategy to construct in Saccharomyces cerevisiae collinear ‘evolutionary mimics' of other species originally differing by the presence of reciprocal translocations in their genome. A modification of the Cre/loxP system was used to create in S. cerevisiae the translocations detected in the sibling species Saccharomyces mikatae IFO 1815 and 1816. Competition experiments under different physiological conditions showed that the translocated strains of S. cerevisiae consistently outcompeted the reference S. cerevisiae strain with no translocation, both in batch and chemostat culture, especially under glucose limitation. These results indicate that chromosomal translocations in Saccharomyces may have an adaptive significance, and lend support to a model of fixation by natural selection of reciprocal translocations in Saccharomyces species.

Two ecotypes of Nucella lapillus can occur allopatrically or sympatrically, according to different degrees of wave exposure and microhabitats, on rocky shores of NW Spain. We studied differences in shell size and shape in adults and families of shelled embryos captured in the wild, and molecular variation at 9 microsatellite loci and 4 mtDNA regions. Both adults and shelled embryos of the two ecotypes showed significant differences in size and shell-shape components (the most important being size of the shell aperture). Strong among-family variation suggests that shell-shape variation has an important genetic component, and the pattern of genetic differentiation was in agreement with traits being affected by divergent natural selection. No significant overall molecular differentiation was observed between the two ecotypes for mtDNA, although we found significant genetic structure at two microsatellite loci. Microsatellite differentiation between ecotypes at these two loci was larger in sympatry than in allopatry. These results and the comparison of tenacity suggest that the two forms are distinctly adapted to differences in shore level and degree of exposure to wave action.

Drosophila sechellia is a specialist species which feeds and breeds on a toxic plant, Morinda citrifolia. All other Drosophila species are killed by ripe fruits of Morinda. D. simulans was subjected to laboratory selection for survival in presence of octanoic acid, the toxic compound of M. citrifolia. After 20 generations of selection, selected lines showed an increased tolerance to octanoic acid, although the response was small compared to the interspecific differences. The genetic response to selection was assessed by studying the changes in allele frequencies at 28 microsatellite loci. Three loci, located in three distinct genomic regions, showed changes in allele frequencies significantly different from what is expected under drift alone in the selected lines. An oligogenic determination for tolerance to octanoic acid is in agreement with published results based on interspecific crosses.