Genomic diversity of Lactobacillus salivarius

Lactobacillus salivarius is unusual among the lactobacilli due to its multireplicon genome architecture. The circular megaplasmids harboured by L. salivarius strains encode strain-specific traits for intestinal survival and probiotic activity. L. salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. In terms of probiotic strain selection, it is important to have an understanding of the level of genomic diversity present in this species. Comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) were employed to assess the level of genomic diversity in L. salivarius. The wellcharacterised probiotic strains L. salivarius UCC118 was employed as a genetic reference strain. The group of test strains were chosen to reflect the range of habitats from which L. salivarius strains are frequently recovered, including human, animal, and environmental sources. Strains of L. salivarius were found to be genetically diverse when compared to the UCC118 genome. The most conserved strains were human GIT isolates, while the greatest level of divergence were identified in animal associated isolates. MLST produced a better separation of the test strains according to their isolation origins, than that produced by CGHbased strain clustering. The exopolysaccharide (EPS) associated genes of L. salivarius strains were found to be highly divergent. The EPS-producing phenotype was found to be carbonsource dependent and inversely related to a strain's ability to produce a biofilm. The genome of the porcine isolate L. salivarius JCM1046 was shown by sequencing to harbour four extrachromosomal replicons, a circular megaplasmid (pMP1046A), a putative chromid (pMP1046B), a linear megaplasmid (pLMP1046) and a smaller circular plasmid (pCTN1046) which contains an integrated Tn916-like element (Tn6224), which carries the tetracycline resistance gene tetM. pLMP1046 represents the first sequence of a linear plasmid in a Lactobacillus species. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable, and the identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications. In summary, this thesis used a comparative genomics approach to examine the level of genotypic diversity in L. salivarius, a species which contains probiotic strains. The genome sequence of strain JCM1046 provides additional insight into the spectrum of extrachromosomal replicons present in this species.

From field to fermentation: characterisation and application of non-dairy cultures in dairy foods

This thesis investigates the phenotypic and genotypic diversity of non-dairy L. lactis strains and their application to dairy fermentations. A bank of non-dairy lactococci were isolated from grass, vegetables and the bovine rumen. Subsequent analysis of these L. lactis strains revealed seven strains to possess cremoris genotypes which did not correlate with their observed phenotypes. Multi-locus sequence typing (MLST) and average nucleotide identity (ANI) highlighted the genetic diversity of lactis and cremoris subspecies. The application of these non-dairy lactococci to cheese production was also assessed. In milk, non-dairy strains formed diverse volatile profiles and selected strains were used as adjuncts in a mini Gouda-type cheese system. Sensory analysis showed non-dairy strains to be strongly associated with the development of off-flavours and bitterness. However, microfluidisation appeared to reduce bitterness. A novel bacteriophage, ɸL47, was isolated using the grass isolate L. lactis ssp. cremoris DPC6860 as a host. The phage, a member of the Siphoviridae, possessed a long tail fiber, previously unseen in dairy lactococcal phages. Genome sequencing revealed ɸL47 to be the largest sequenced lactococcal phage to date and owing to the high % similarity with ɸ949, a second member of the 949 group. Finally, to identify and characterise specific genes which may be important in niche adaptation and for applications to dairy fermentations, comparative genome sequence analysis was performed on L. lactis from corn (DPC6853), the bovine rumen (DPC6853) and grass (DPC6860). This study highlights the contribution of niche specialisation to the intra-species diversity of L. lactis and the adaptation of this organism to different environments. In summary this thesis describes the genetic diversity of L. lactis strains from outside the dairy environment and their potential application in dairy fermentations.

Managing diversity in practical ambient assisted living ecosystems

Though the motivation for developing Ambient Assisted Living (AAL) systems is incontestable, significant challenges exist in realizing the ambience that is essential to the success of such systems. By definition, an AAL system must be omnipresent, tracking occupant activities in the home and identifying those situations where assistance is needed or would be welcomed. Embedded sensors offer an attractive mechanism for realizing ambience as their form factor and harnessing of wireless technologies aid in their seamless integration into pre-existing environments. However, the heterogeneity of the end-user population, their disparate needs and the differing environments in which they inhabit, all pose particular problems regarding sensor integration and management

Antimicrobial activities and diversity of sponge derived microbes

In this study, marine sponges collected in Irish waters were analysed for their associated microbiota. Of the approximately 240 bacterial isolates obtained from two sponges several showed antimicrobial activity; among them members of genera which have rarely been shown to produce antimicrobial compounds. Differences observed from the sponge-derived groups of isolates in terms of bioactivity suggests that S. carnosus isolates may be a better source of antibacterial compounds, while Leucosolenia sp. isolates appear to be a better source of antifungal compounds. More than 60% of fungal isolates obtained from 12 sponge samples proved to be bioactive. One of the isolates, which was closely related to Fusarium oxysporum and showed activity against bacteria and fungi, was investigated for its secondary metabolite genes. At least 5 different NRPS genes, with a sequence similarity as low as 50 % to known genes, were identified highlighting the likelihood that this isolate may be capable of producing novel secondary metabolites. A Micromonospora sp. was isolated from a Haliclona simulans sample collected in Irish waters. The isolate inhibited the growth of Gram positive bacterial test strains in three different antimicrobial assays. Employing preparative layer chromatography the compound responsible for the bioactivity could be isolated. According to LC-MS andNMR data the bioactive compound could indeed be novel. Finally, two deep water sponges were shown to host a remarkably different bacterial and archaeal diversity by application of 454 Pyrosequencing. The L. diversichela –proteobacterial community was dominated by a single ƴ-proteobacterial bacterium whereas the S. normani sample hosted a largely sponge specific microbial community, even more diverse than has been previously reported for shallow water sponges. Organisms potentially involved in nitrification, sulphate reduction and secondary metabolite production were found to be spatially distributed in the sponge. Furthermore, a deep sea specific population was implied.

Sectoral impacts on the diversity of carabid beetles (Coleoptera: Carabidae) and their associated ecosystem service of biocontrol

This thesis aimed to provide an understanding of how human-induced changes in the economic sectors of agriculture and transport affect carabid diversity, potential carabidmediated biocontrol and predator-pest interactions. The research involved both observational and manipulative laboratory and field-based studies. Observational research consisted of two large-scale investigations of (1) the impact of Miscanthus and oilseed rape production (n=45) and (2) the impact of horticultural and ecological based landscaping of roadside verges (n=64). This research is the first record of carabid diversity, potential biocontrol and community assemblage with respect to bioenergy crop production and roadside landscaping in an Irish context and it is also an important addition to the limited knowledge of carabid populations in these ecosystems internationally. Manipulative work involved the examination of the role predator identity, diversity and biomass play in the suppression of pollen beetle larvae (an economically damaging insect pest of oilseed rape in Europe), using a novel experimental design called ‘simplex’. To complement this research, an additional field study on the impact of low and high oilseed rape pesticide management on carabid species richness and abundance, and crop yield, was also conducted. This research is a great contribution to the existing understanding of what constitutes the important components of predator biodiversity and expands the knowledge of the usefulness of carabid predators in the context of pollen beetle larvae control. In particular, the work shows that the abundance or biomass of beetles has an effect that is far larger than the effect of diversity on the capacity of beetles to consume prey. In turn, the field study showed that pesticide applications had little impact on yield, or carabid richness, but that carabid abundance/biomass declined drastically. The work provides compelling evidence that management practices erode the useful components of biodiversity that are essential for the delivery of biocontrol services.

Invertebrate diversity in Irish and British forests

Ireland and Britain were once covered in natural forest, but extensive anthropogenic deforestation reduced forest cover to less than 1% and 5 %, respectively, by the beginning of the 20th century. Large-scale afforestation has since increased the level of forest cover to 11% in Ireland and 12% in Britain, with the majority of planted forests comprising small monoculture plantations, many of which are of non - native conifer tree species. At present the forest cover of Ireland and Britain generally consists of small areas of remnant semi-natural woodland and pockets of these plantation forests within a predominantly agricultural landscape. Invertebrates comprise a large proportion of the biodiversity found within forested habitats. In particular, spiders and carabid beetles play an important role in food webs as both predators and prey and respond to small-scale changes in habitat structure, meaning they are particularly sensitive to forest management. Hoverflies play an important role in control and pollination and have been successfully used as indicators of habitat disturbance and quality. This research addressed a number of topics pertinent to the forest types present in the contemporary Irish and British landscapes and aimed to investigate the invertebrate diversity of these forests. Spiders and carabid beetles were sampled using pitfall trapping and hoverflies were sampled using Malaise net trapping. Topics included the impacts of afforestation, the importance of open space, the choice of tree species, and the use of indicators for biodiversity assessment, as well as rare native woodlands and the effect of grazing on invertebrate diversity. The results are discussed and evidence-based recommendations are made for forest policy and management to protect and enhance invertebrate biodiversity in order to promote sustainable forest management in Ireland and Britain.

Analysing energy detector diversity receivers for spectrum sensing

The analysis of energy detector systems is a well studied topic in the literature: numerous models have been derived describing the behaviour of single and multiple antenna architectures operating in a variety of radio environments. However, in many cases of interest, these models are not in a closed form and so their evaluation requires the use of numerical methods. In general, these are computationally expensive, which can cause difficulties in certain scenarios, such as in the optimisation of device parameters on low cost hardware. The problem becomes acute in situations where the signal to noise ratio is small and reliable detection is to be ensured or where the number of samples of the received signal is large. Furthermore, due to the analytic complexity of the models, further insight into the behaviour of various system parameters of interest is not readily apparent. In this thesis, an approximation based approach is taken towards the analysis of such systems. By focusing on the situations where exact analyses become complicated, and making a small number of astute simplifications to the underlying mathematical models, it is possible to derive novel, accurate and compact descriptions of system behaviour. Approximations are derived for the analysis of energy detectors with single and multiple antennae operating on additive white Gaussian noise (AWGN) and independent and identically distributed Rayleigh, Nakagami-m and Rice channels; in the multiple antenna case, approximations are derived for systems with maximal ratio combiner (MRC), equal gain combiner (EGC) and square law combiner (SLC) diversity. In each case, error bounds are derived describing the maximum error resulting from the use of the approximations. In addition, it is demonstrated that the derived approximations require fewer computations of simple functions than any of the exact models available in the literature. Consequently, the regions of applicability of the approximations directly complement the regions of applicability of the available exact models. Further novel approximations for other system parameters of interest, such as sample complexity, minimum detectable signal to noise ratio and diversity gain, are also derived. In the course of the analysis, a novel theorem describing the convergence of the chi square, noncentral chi square and gamma distributions towards the normal distribution is derived. The theorem describes a tight upper bound on the error resulting from the application of the central limit theorem to random variables of the aforementioned distributions and gives a much better description of the resulting error than existing Berry-Esseen type bounds. A second novel theorem, providing an upper bound on the maximum error resulting from the use of the central limit theorem to approximate the noncentral chi square distribution where the noncentrality parameter is a multiple of the number of degrees of freedom, is also derived.