Bacterial Community Structure in the Hindgut of Wild and Captive Dugongs (Dugong dugon)

Dugongs (Dugong dugon) are marine mammals that obtain nutrients through hindgut fermentation of seagrass, however, the microbes responsible have not been identified. This study used denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing to profile hindgut bacterial communities in wild dugongs. Faecal samples obtained from 32 wild dugongs representing four size/maturity classes, and two captive dugongs fed on cos lettuce were screened using DGGE. Partial 16S rRNA gene profiles of hindgut bacteria from wild dugong calves and juveniles were grouped together and were different to those in subadults and adults. Marked differences between hindgut bacterial communities of wild and captive dugongs were also observed, except for a single captive whose profile resembled wild adults following an unsuccessful reintroduction to the wild. Pyrosequencing of hindgut communities in two wild dugongs confirmed the stability of bacterial populations, and Firmicutes (average 75.6% of Operational Taxonomic Units [OTUs]) and Bacteroidetes (19.9% of OTUs) dominated. Dominant genera were Roseburia, Clostridium, and Bacteroides. Hindgut microbial composition and diversity in wild dugongs is affected by ontogeny and probably diet. In captive dugongs, the absence of the dominant bacterial DNA bands identified in wild dugongs is probably dependent upon prevailing diet and other captive conditions such as the use of antibiotics. This study represents a first step in the characterisation of a novel microbial ecosystem-the marine hindgut of Sirenia.

Variation in the hindgut microbial communities of the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, Florida

The Florida manatee, Trichechus manatus latirostris, is a hindgut-fermenting herbivore. In winter, manatees migrate to warm water overwintering sites where they undergo dietary shifts and may suffer from cold-induced stress. Given these seasonally induced changes in diet, the present study aimed to examine variation in the hindgut bacterial communities of wild manatees overwintering at Crystal River, west Florida. Faeces were sampled from 36 manatees of known sex and body size in early winter when manatees were newly arrived and then in mid-winter and late winter when diet had probably changed and environmental stress may have increased. Concentrations of faecal cortisol metabolite, an indicator of a stress response, were measured by enzyme immunoassay. Using 454-pyrosequencing, 2027 bacterial operational taxonomic units were identified in manatee faeces following amplicon pyrosequencing of the 16S rRNA gene V3/V4 region. Classified sequences were assigned to eight previously described bacterial phyla; only 0.36% of sequences could not be classified to phylum level. Five core phyla were identified in all samples. The majority (96.8%) of sequences were classified as Firmicutes (77.3 ± 11.1% of total sequences) or Bacteroidetes (19.5 ± 10.6%). Alpha-diversity measures trended towards higher diversity of hindgut microbiota in manatees in mid-winter compared to early and late winter. Beta-diversity measures, analysed through permanova, also indicated significant differences in bacterial communities based on the season.

Fungal community structure in disease suppressive soils assessed by 28S LSU gene sequencing

Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils 'suppressive' or 'non-suppressive' for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria , Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression. © 2014 Penton et al.

Programming rumen bacterial communities in newborn Merino lambs

Establishment of the rumen microbiome can be affected by both early-life dietary measures and rumen microbial inoculation. This study used a 2 × 3 factorial design to evaluate the effects of inclusion of dietary fat type and the effects of rumen inoculum from different sources on ruminal bacterial communities present in early stages of the lambs’ life. Two different diets were fed ad libitum to 36 pregnant ewes (and their lambs) from 1 month pre-lambing until weaning. Diets consisted of chaffed lucerne and cereal hay and 4% molasses, with either 4% distilled coconut oil (CO) provided as a source of rumen-active fat or 4% Megalac® provided as a source of rumen-protected fat (PF). One of three inoculums was introduced orally to all lambs, being either (1) rumen fluid from donor ewes fed the PF diet; (2) rumen fluid from donor ewes fed CO; or (3) a control treatment of MilliQ-water. After weaning at 3 months of age, each of the six lamb treatment groups were grazed in spatially separated paddocks. Rumen bacterial populations of ewes and lambs were characterised using 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Species richness and biodiversity of the bacterial communities were found to be affected by the diet in ewes and lambs and by inoculation treatment of the lambs. Principal coordinate analysis and analysis of similarity (ANOSIM) showed between diet differences in bacterial community groups existed in ewes and differential bacterial clusters occurred in lambs due to both diet and neonatal inoculation. Diet and rumen inoculation acted together to clearly differentiate the bacterial communities through to weaning, however the microbiome effects of these initial early life interventions diminished with time so that rumen bacterial communities showed greater similarity 2 months after weaning. These results demonstrate that ruminal bacterial communities of newborn lambs can be altered by modifying the diet of their mothers. Moreover, the rumen microbiome of lambs can be changed by diet while they are suckling or by inoculating their rumen, and resulting changes in the rumen bacterial microbiome can persist beyond weaning.

Production attributes of Merino sheep genetically divergent for wool growth are reflected in differing rumen microbiotas

Divergent genetic selection for wool growth as a single trait has led to major changes in sheep physiology and metabolism, including variations in rumen microbial protein production and uptake of α-amino nitrogen in portal blood. This study was conducted to determine if sheep with different genetic merit for wool growth exhibit distinct rumen bacterial diversity. Eighteen Merino wethers were separated into groups of contrasting genetic merit for clean fleece weight (CFW; low: WG− and high: WG+) and fed a blend of oaten and lucerne chaff diet at two levels of intake (LOI; 1 or 1.5 times maintenance energy requirements) for two seven-week periods in a crossover design. Bacterial diversity in rumen fluid collected by esophageal intubation was characterized using 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Bacterial diversity estimated by Phylogenetic distance, Chao1 and observed species did not differ significantly with CFW or LOI; however, the Shannon diversity index differed (P=0.04) between WG+ (7.67) and WG− sheep (8.02). WG+ animals had a higher (P=0.03) proportion of Bacteroidetes (71.9% vs 66.5%) and a lower (P=0.04) proportion of Firmicutes (26.6% vs 31.6%) than WG− animals. Twenty-four specific operational taxonomic units (OTUs), belonging to the Firmicutes and Bacteroidetes phyla, were shared among all the samples, whereas specific OTUs varied significantly in presence/abundance (P<0.05) between wool genotypes and 50 varied (P<0.05) with LOI. It appears that genetic selection for fleece weight is associated with differences in rumen bacterial diversity that persist across different feeding levels. Moderate correlations between seven continuous traits, such as methane production or microbial protein production, and the presence and abundance of 17 OTUs were found, indicating scope for targeted modification of the microbiome to improve the energetic efficiency of rumen microbial synthesis and reduce the greenhouse gas footprint of ruminants.

Microbial communities in Pb contaminated boreal forest soil

Lead contamination in the environment is of particular concern, as it is a known toxin. Until recently, however, much less attention has been given to the local contamination caused by activities at shooting ranges compared to large-scale industrial contamination. In Finland, more than 500 tons of Pb is produced each year for shotgun ammunition. The contaminant threatens various organisms, ground water and the health of human populations. However, the forest at shooting ranges usually shows no visible sign of stress compared to nearby clean environments. The aboveground biota normally reflects the belowground ecosystem. Thus, the soil microbial communities appear to bear strong resistance to contamination, despite the influence of lead. The studies forming this thesis investigated a shooting range site at Hälvälä in Southern Finland, which is heavily contaminated by lead pellets. Previously it was experimentally shown that the growth of grasses and degradation of litter are retarded. Measurements of acute toxicity of the contaminated soil or soil extracts gave conflicting results, as enchytraeid worms used as toxicity reporters were strongly affected, while reporter bacteria showed no or very minor decreases in viability. Measurements using sensitive inducible luminescent reporter bacteria suggested that the bioavailability of lead in the soil is indeed low, and this notion was supported by the very low water extractability of the lead. Nevertheless, the frequency of lead-resistant cultivable bacteria was elevated based on the isolation of cultivable strains. The bacterial and fungal diversity in heavily lead contaminated shooting sectors were compared with those of pristine sections of the shooting range area. The bacterial 16S rRNA gene and fungal ITS rRNA gene were amplified, cloned and sequenced using total DNA extracted from the soil humus layer as the template. Altogether, 917 sequenced bacterial clones and 649 sequenced fungal clones revealed a high soil microbial diversity. No effect of lead contamination was found on bacterial richness or diversity, while fungal richness and diversity significantly differed between lead contaminated and clean control areas. However, even in the case of fungi, genera that were deemed sensitive were not totally absent from the contaminated area: only their relative frequency was significantly reduced. Some operational taxonomic units (OTUs) assigned to Basidiomycota were clearly affected, and were much rarer in the lead contaminated areas. The studies of this thesis surveyed EcM sporocarps, analyzed morphotyped EcM root tips by direct sequencing, and 454-pyrosequenced fungal communities in in-growth bags. A total of 32 EcM fungi that formed conspicuous sporocarps, 27 EcM fungal OTUs from 294 root tips, and 116 EcM fungal OTUs from a total of 8 194 ITS2 454 sequences were recorded. The ordination analyses by non-parametric multidimensional scaling (NMS) indicated that Pb enrichment induced a shift in the EcM community composition. This was visible as indicative trends in the sporocarp and root tip datasets, but explicitly clear in the communities observed in the in-growth bags. The compositional shift in the EcM community was mainly attributable to an increase in the frequencies of OTUs assigned to the genus Thelephora, and to a decrease in the OTUs assigned to Pseudotomentella, Suillus and Tylospora in Pb-contaminated areas when compared to the control. The enrichment of Thelephora in contaminated areas was also observed when examining the total fungal communities in soil using DNA cloning and sequencing technology. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem remain undetermined. The results indicate that at the Hälvälä shooting range, lead influences the fungal communities but not the bacterial communities. The forest ecosystem shows apparent functional redundancy, since no significant effects were seen on forest trees. Recently, by means of 454 pyrosequencing , the amount of sequences in a single analysis run can be up to one million. It has been applied in microbial ecology studies to characterize microbial communities. The handling of sequence data with traditional programs is becoming difficult and exceedingly time consuming, and novel tools are needed to handle the vast amounts of data being generated. The field of microbial ecology has recently benefited from the availability of a number of tools for describing and comparing microbial communities using robust statistical methods. However, although these programs provide methods for rapid calculation, it has become necessary to make them more amenable to larger datasets and numbers of samples from pyrosequencing. As part of this thesis, a new program was developed, MuSSA (Multi-Sample Sequence Analyser), to handle sequence data from novel high-throughput sequencing approaches in microbial community analyses. The greatest advantage of the program is that large volumes of sequence data can be manipulated, and general OTU series with a frequency value can be calculated among a large number of samples.

Methylenetetrahydrofolate reductase CpG islands: Epigenotyping

Background Determination of the differential DNA methylation patterns of methylenetetrahydrofolate reductase (MTHFR) that are associated with differential MTHFR activity is important to understand the pathogenesis of ischemic stroke. However, to date, no data are available on the differential DNA methylation profiles of Kelantanese Malays. Therefore, we developed a rapid and efficient serial pyrosequencing assay to determine differential DNA methylation profiles of MTHFR, which help to further our understanding of the pathogenesis of ischemic stroke. The developed assay also served as the validation platform for our previous computational epigenetic research on MTHFR. Methods Polymerase chain reaction primers were designed and validated to specifically amplify the cytosine that is followed by guanine residues (CpGs) A and B regions. Prior epigenotyping on 110 Kelantanese Malays, the serial pyrosequencing assays for the CpGs A and B regions were validated using five validation controls. The mean values of the DNA methylation profiles of CpGs A and B were calculated. Results The mean DNA methylation levels for CpGs A and B were 0.984 ± 0.582 and 2.456 ± 1.406, respectively. The CpGs 8 and 20 showed the highest (5.581 ± 4.497) and the lowest (0.414 ± 2.814) levels of DNA methylation at a single-base resolution. Conclusion We have successfully developed and validated a pyrosequencing assay that is fast and can yield high-quality pyrograms for DNA methylation analysis and is therefore applicable to high throughput study. Using this newly developed pyrosequencing assay, the MTHFR DNA methylation profiles of 110 Kelantanese Malays were successfully determined. It also validated our computational epigenetic research on MTHFR.

Protocol for assessing maternal, environmental and epigenetic risk factors for dental caries in children

Background Expenditure on dental and oral health services in Australia is $3.4 billion AUD annually. This is the sixth highest health cost and accounts for 7 % of total national health expenditure. Approximately 49 % of Australian children aged 6 years have caries experience in their deciduous teeth and this is rising. The aetiology of dental caries involves a complex interplay of individual, behavioural, social, economic, political and environmental conditions, and there is increasing interest in genetic predisposition and epigenetic modification. Methods The Oral Health Sub-study; a cross sectional study of a birth cohort began in November 2012 by examining mothers and their children who were six years old by the time of initiation of the study, which is ongoing. Data from detailed questionnaires of families from birth onwards and data on mothers’ knowledge, attitudes and practices towards oral health collected at the time of clinical examination are used. Subjects’ height, weight and mid-waist circumference are taken and Body Mass Index (BMI) computed, using an electronic Bio-Impedance balance. Dental caries experience is scored using the International Caries Detection and Assessment System (ICDAS). Saliva is collected for physiological measures. Salivary Deoxyribose Nucleic Acid (DNA) is extracted for genetic studies including epigenetics using the SeqCap Epi Enrichment Kit. Targets of interest are being confirmed by pyrosequencing to identify potential epigenetic markers of caries risk. Discussion This study will examine a wide range of potential determinants for childhood dental caries and evaluate inter-relationships amongst them. The findings will provide an evidence base to plan and implement improved preventive strategies.

The Biocontrol Agent Phlebiopsis gigantea: Efficacy and Impacts on the Stump Bacterial Biota and Conifer tree Defences

Phlebiopsis gigantea has been for a long time known as a strong competitor against Heterobasidion annosum and intensively applied as a biological control agent on stump surfaces of Picea abies in Fennoscandia. However, the mechanism underlying its antagonistic activity is still unknown. A primary concern is the possible impact of P. gigantea treatment on resident non-target microbial biota of conifer stumps. Additional risk factor is the potential of P. gigantea to acquire a necrotrophic habit through adaptation to living wood tissues. This study focused on the differential screening of several P. gigantea isolates from diverse geographical sources as well as the use of breeding approach to enhance the biocontrol efficacy against H. annosum infection. The results showed a significant positive correlation between growth rate in wood and high biocontrol efficacy. Furthermore, with aid of breeding approach, several progeny strains were obtained that had better growth rate and control efficacy than parental isolates. To address the issue of the potential of P. gigantea to acquire necrotrophic capability, a combination of histochemical, molecular and transcript profiling (454 sequencing) were used to investigate the interactions between these two fungi and ten year old P. sylvestris seedlings. The results revealed that both P. gigantea and H. annosum provoked strong necrotic lesions, but after prolonged incubation, P. gigantea lesions shrank and ceased to expand further. Tree seedlings pre-treated with P. gigantea further restricted H. annosum-induced necrosis and had elevated transcript levels of genes important for lignification, cell death regulation and jasmonic acid signalling. These suggest that induced localized resistance is a contributory factor for the biocontrol efficacy of P.gigantea, and it has a comparatively limited necrotrophic capability than H. annosum. Finally, to investigate the potential impact of P. gigantea on the stump bacterial biota, 16S rDNA isolated from tissue samples from stumps of P. abies after 1-, 6- and 13-year post treatment was sequenced using bar-coded 454 Titanium pyrosequencing. Proteobacteria were found to be the most abundant at the initial stages of stump decay but were selectively replaced by Acidobacteria at advanced stages of the decay. Moreover, P. gigantea treatment significantly decreased the bacterial richness at initial decay stage in the stumps. Over time, the bacterial community in the stumps gradually recovered and the negative effects of P. gigantea was attenuated.

Características biogeoquímicas da interação atmosfera criosfera na Antártica ocidental

O manto polar antártico retêm informação paleoclimatologica por entres suas camadas de neve e gelo. O gelo antártico tem revelado a base de dados paleoclimática de maior resolução para os últimos 800 mil anos. Os padrões de transporte atmosférico refletem a composição e a fonte do particulado encontrado na neve e no gelo do continente Antártico. Estando relacionado a processos climáticos, as características desse transporte alteram em quantidade e qualidade as espécies químicas que se depositam sobre o manto de gelo. Dessa forma, o estudo dos depósitos de particulado ao longo das camadas de neve/gelo na Antártica pode sugerir mudanças nos padrões de transporte atmosférico. Atualmente a comunidade científica discute as diferenças de padrões climáticos entre o leste e o oeste antártico. Enquanto de forma geral observa-se instabilidade no setor oeste, o clima da antártica oriental demonstra relativa estabilidade climática. Neste estudo, analisamos dois testemunhos de gelo recente de duas regiões com características climáticas diferentes do continente Antártico. No Platô Detroit situado na Península Antártica (6410′S/0600′O), analisamos a variabilidade de Black Carbon (BC) ao longo de 20 metros de neve. O BC encontrado na Península Antártica apresentou baixas concentrações comparáveis as encontradas no gelo do Artico período pré-industrial. Nossos resultados sugerem que sua variabiliade corresponde à sazonalidade dos períodos de queimada nos continentes do Hemisfério Sul. No interior do continente Antártico, analisamos o particulado em geral por um processo de microanálise ao longo de um testemunho de 40 metros extraído em Mont Johns (79o55′S/09423′O). Encontramos uma tendência negativa na deposição de poeira mineral (AlSi) entre 1967 e 2007. Nossos resultados sugerem que esta tendência seja resultado de um crescente isolamento atmosférico da região central do continente antártico pelo aumento da intensidade dos ventos ao redor da Antártica. Este aumento na intensidade dos ventos reflete por sua vez o resfriamento da alta atmosfera no centro antártico causado pela depleção da camada de ozônio na região. Adicionalmente, amostras de diferentes microambientes de Patriot Hills (8018′S/08121′O) foram coletadas de maneira asséptica para análise microbiológica. As amostras foram cultivadas em meio R2 e paralelamente o DNA total extraído foi sequenciado pela técnica de pirosequenciamento. Os resultados preliminares desta analise mostram grande riqueza de espécies dos mais variados grupos. Os resultados deste trabalho caracterizam três diferentes parâmetros relacionados a deposição atmosférica em duas áreas pouco exploradas e de grande interesse científico do continente antártico.

Diversidade genética em um grupo de anuros em miniatura endêmico da Mata Atlântica brasileira (Euparkerella Griffiths 1959).

A Mata Atlântica (MA) está entre as regiões com maior biodiversidade e mais ameaçadas do planeta. Esforços em diversas áreas do conhecimento têm sido feitos para que se tenha uma estimativa mais refinada da diversidade existente e sua organização ao longo do bioma. O crescente número de estudos que buscam reconstituir a história da diversificação da MA apontam para um cenário espacial e temporal complexo, havendo ainda uma lacuna no conhecimento dos processos em pequena escala. Vertebrados em miniatura têm se mostrado uma boa ferramenta para estudos de processos evolutivos em pequena escala. Assim, o gênero Euparkerella, endêmico de uma pequena região da MA dos Estados do Rio de Janeiro (RJ) e Espírito Santo (ES), foi escolhido como modelo para este estudo. No primeiro capítulo buscou-se descrever a diversidade existente dentro do gênero a partir de uma filogenia molecular. Para isso, utilizaram-se métodos bayesianos para gerar genealogias de genes e de espécies a partir de um fragmento de gene mitocondrial e quatro fragmentos de genes nucleares. Os resultados obtidos apontaram para uma grande diversidade críptica no gênero. Foram identificadas seis unidades evolutivas significativamente divergentes para o RJ: duas em Euparkerella cochranae, três em Euparkerella brasiliensis, e Euparkerella sp.. A espécie mais basal recuperada foi Euparkerella robusta, do ES, e estimou-se o início da diversificação do gênero para o final do Mioceno. O segundo capítulo descreve onze marcadores de microssatélites desenvolvidos para Euparkerella brasiliensis através do método de pirosequenciamento de nova geração 454. No terceiro capítulo estudou-se apenas uma unidade evolutiva, Euparkerella brasiliensis da área dos Três Picos/ RJ. A partir de marcadores de evolução rápida (microssatélites) e lenta (sequências de DNA) buscou-se compreender a estrutura e a dinâmica populacional desta unidade evolutiva em uma área bastante pequena (aprox. 20 km) sob influência de um gradiente ambiental altitudinal (40 m 1000 m). Foram identificadas, a partir dos microssatélites, duas subpopulações geneticamente distintas nas bordas do gradiente. O fluxo gênico se deu predominantemente das bordas para a zona de contato, onde foi observado o maior efetivo populacional. Tais resultados indicam que pequenas variações ambientais podem atuar no isolamento populacional em Euparkerella e corroboram o padrão de formas microendêmicas identificadas na filogenia. Futuros estudos devem ser feitos no sentido de buscar caracterizar morfologicamente as unidades evolutivas aqui identificadas; preencher as lacunas amostrais, especialmente no ES; e descrever os processos que atuam em pequena escala nas zonas de contato entre as unidades evolutivas e fatores limitantes a distribuição das mesmas.

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.

Biodiscovery of natural products from microbes associated with Irish coastal sponges

Marine sponges have been an abundant source of new metabolites in recent years. The symbiotic association between the bacteria and the sponge has enabled scientists to access the bacterial diversity present within the bacterial/sponge ecosystem. This study has focussed on accessing the bacterial diversity in two Irish coastal marine sponges, namely Amphilectus fucorum and Eurypon major. A novel species from the genus Aquimarina has been isolated from the sponge Amphilectus fucorum. The study has also resulted in the identification of an α–Proteobacteria, Pseudovibrio sp. as a potential producer of antibiotics. Thus a targeted based approach to specifically cultivate Pseudovibrio sp. may prove useful for the development of new metabolites from this particular genus. Bacterial isolates from the marine sponge Haliclona simulans were screened for anti–fungal activity and one isolate namely Streptomyces sp. SM8 displayed activity against all five fungal strains tested. The strain was also tested for anti–bacterial activity and it showed activity against both against B. subtilis and P. aeruginosa. Hence a combinatorial approach involving both biochemical and genomic approaches were employed in an attempt to identify the bioactive compounds with these activities which were being produced by this strain. Culture broths from Streptomyces sp. SM8 were extracted and purified by various techniques such as reverse–phase HPLC, MPLC and ash chromatography. Anti–bacterial activity was observed in a fraction which contained a hydroxylated saturated fatty acid and also another compound with a m/z 227 but further structural elucidation of these compounds proved unsuccessful. The anti–fungal fractions from SM8 were shown to contain antimycin–like compounds, with some of these compounds having different retention times from that of an antimycin standard. A high–throughput assay was developed to screen for novel calcineurin inhibitors using yeast as a model system and three putative bacterial extracts were found to be positive using this screen. One of these extracts from SM8 was subsequently analysed using NMR and the calcineurin inhibition activity was con rmed to belong to a butenolide type compound. A H. simulans metagenomic library was also screened using the novel calcineurin inhibitor high–throughput assay system and eight clones displaying putative calcineurin inhibitory activity were detected. The clone which displayed the best inhibitory activity was subsequently sequenced and following the use of other genetic based approaches it became clear that the inhibition was being caused by a hypothetical protein with similarity to a hypothetical Na+/Ca2+ exchanger protein. The Streptomyces sp. SM8 genome was sequenced from a fragment library using Roche 454 pyrosequencing technology to identify potential secondary metabolism clusters. The draft genome was annotated by IMG/ER using the Prodigal pipeline. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMPN00000000. The genome contains genes which appear to encode for several polyketide synthases (PKS), non–ribosomal peptide synthetases (NRPS), terpene and siderophore biosynthesis and ribosomal peptides. Transcriptional analyses led to the identification of three hybrid clusters of which one is predicted to be involved in the synthesis of antimycin, while the functions of the others are as yet unknown. Two NRPS clusters were also identified, of which one may be involved in gramicidin biosynthesis and the function of the other is unknown. A Streptomyces sp. SM8 NRPS antC gene knockout was constructed and extracts from the strain were shown to possess a mild anti–fungal activity when compared to the SM8 wild–type. Subsequent LCMS analysis of antC mutant extracts confirmed the absence of the antimycin in the extract proving that the observed anti–fungal activity may involve metabolite(s) other than antimycin. Anti–bacterial activity in the antC gene knockout strain against P. aeruginosa was reduced when compared to the SM8 wild–type indicating that antimycin may be contributing to the observed anti–bacterial activity in addition to the metabolite(s) already identified during the chemical analyses. This is the first report of antimycins exhibiting anti–bacterial activity against P. aeruginosa. One of the hybrid clusters potentially involved in secondary metabolism in SM8 that displayed high and consistent levels of gene–expression in RNA studies was analysed in an attempt to identify the metabolite being produced by the pathway. A number of unusual features were observed following bioinformatics analysis of the gene sequence of the cluster, including a formylation domain within the NRPS cluster which may add a formyl group to the growing chain. Another unusual feature is the lack of AT domains on two of the PKS modules. Other unusual features observed in this cluster is the lack of a KR domain in module 3 of the cluster and an aminotransferase domain in module 4 for which no clear role has been hypothesised.

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.

Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms