Browsing by Author "Mahoney-Kurpe, Sam"

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    Identification and characterisation of rumen bacteria with prominent roles in the ruminal metabolism of forages : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Microbiology and Genetics) at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Mahoney-Kurpe, Sam
    This thesis documents the characterisation of two groups of rumen bacteria that are both prominent in forage-fed ruminants, with the aim to better understand their roles in ruminal metabolism. The first group, referred to as the R-7 group, has in recent years been shown to be one of the most abundant rumen bacterial groups, though the few isolated representative strains available were uncharacterised. Two strains of the group included in the Hungate1000 culture collection, R-7 and WTE2008, were selected for characterisation. To facilitate phylogenetic analyses of this group, the complete genomes of an additional three previously isolated R-7 group strains were sequenced. Genomic, phylogenetic and phenotypic characterisation of R-7 and WTE2008 demonstrated that despite their 16S rRNA gene sequences sharing 98.6-99.0% nucleotide identity, their genome-wide average nucleotide identity of 84% assigned them as separate species of a novel genus and family of the proposed order ‘Christensenellales’ using the Genome Taxonomy Database. Phenotypic characterisation showed that the strains were identical in morphology, and both possessed the ability to degrade plant cell wall polysaccharides xylan and pectin, but not cellulose. Acetate, ethanol, hydrogen and lactate were produced by both strains, though R-7 produced greater amounts of hydrogen than WTE2008, which instead produced more lactate. Based on these analyses, it is proposed that R-7 and WTE2008 belong to separate species (Aristaeella gen. nov. hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively) of a newly proposed family (Aristaeellaceae fam. nov.). The second bacterial group of interest, due to their dominant role in ruminal propionate production, was the Prevotella 1 group, following analyses of metatranscriptome datasets of rumen microbial communities of lucerne-fed sheep for dominant community members that express propionate pathway genes from succinate. Screening of 14 strains spanning the diversity of Prevotella 1 found that all except one P. brevis strain produced propionate in a cobalamin (vitamin B12)-dependent manner. To better understand the pathway and regulation of propionate production from succinate, a comparative multi-omics approach was used to test the hypothesis that propionate production is regulated by a cobalamin-binding riboswitch. Scanning of a completed genome assembly of Prevotella ruminicola KHP1 identified four ‘cobalamin’ family riboswitches. However, the riboswitches were not in close proximity to genes putatively involved in converting succinate to propionate, nor were these genes arranged in a single operon. Comparative genomics of the 14 screened strains found that all strains possessed all homologues of candidate propionate pathway genes identified in the KHP1 genome. However, the 13 propionate-producing strains possessed a putative transporter and three subunits encoding a putative methylmalonyl-CoA decarboxylase upstream but antisense to two genes encoding methylmalonyl-CoA mutase subunits, whereas the non-producing strain did not. Comparative transcriptomics and proteomics of KHP1 cultures in the presence and absence of cobalamin demonstrated that some gene candidates were upregulated by cobalamin at the transcriptome level, including co-located genes annotated as phosphate butyryltransferase and butyrate kinase, despite the strain not producing butyrate, suggesting that propionate production may occur via propionyl phosphate. However, only both subunits of methylmalonyl-CoA mutase showed greater transcript and protein abundances in the presence of cobalamin. These results show that while some propionate pathway candidate genes were differentially expressed between cobalamin treatments, they did not appear to be under direct control of a cobalamin-binding riboswitch. This study has contributed to our understanding of the roles of both Aristaeellaceae fam. nov. and Prevotella 1 in ruminal metabolism.
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    Metabarcoding of the rhizosphere microbiome of perennial ryegrass in response to Epichloë festucae var. lolii infection : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2017) Mahoney-Kurpe, Sam
    Epichloë endophytes inhabit the intercellular spaces of cool-season pasture grasses, and can confer upon their hosts agriculturally desirable benefits such as heightened resistance to biotic and abiotic stresses. The mechanisms underlying many of these benefits are not well understood. Previously observed Epichloë-associated impacts towards the rhizosphere microbiome of their hosts could be a contributing factor, however the overall extent to which specific taxa in the rhizosphere microbiome of perennial ryegrass are affected by Epichloë festucae var. lolii infection remains to be elucidated. To assess this, two independent experiments were carried out in which clonal perennial ryegrass (NuiD) plants inoculated or uninoculated with E. festucae var. lolii (Lp19) originating from sterile tissue culture were grown in soil collected from a natural ryegrass pasture. After approximately two months of growth under controlled conditions in a growth cabinet, their prokaryotic and fungal rhizosphere microbiomes were compared using high-throughput metabarcoding. For prokaryotes, endophyte infection had no significant impact on species richness or evenness of the rhizosphere microbiome of their hosts in either experiment. A very minor but significant shift in overall community composition was shown in the first experiment but not the second. At the level of phyla, aside from a minor 1.1% increase in the relative abundances of Bacteroidetes in the rhizosphere of infected compared with uninfected plants in the first experiment but not the second, there were no other significantly differentially abundant prokaryotic phyla due to endophyte infection. At the genus level rhizospheres of infected and uninfected plants showed a high degree of similarity in both experiments, with little variability between replicates within treatments. At the level of operational taxonomic units (OTUs), in the first experiment there was only one significantly differentially abundant OTU in the rhizosphere depending on endophyte infection, and nine in the second. However, all of which had relatively low abundances (<0.3%), and none were consistently significantly differentially abundant in both experiments. For fungi, there were no significant impacts of endophyte infection on species richness or evenness of the rhizosphere in either experiment, nor were there any significant endophyte-associated shifts detected in overall rhizosphere community composition. Taxonomic analyses found that in both experiments endophyte infected plants had decreased abundances of a single abundant OTU compared with uninfected plants, which was found to be significant across both experiments (P= 0.026). The OTU sequence mapped with moderate (76-90%) homology to a number of reference sequences assigned as belonging to the class Sordariomycetes. Given previously observed endophyte-associated effects on arbuscular mycorrhizal (AM) fungi, reads assigned as belonging to AM were filtered and analysed separately. This showed that there were no significant effects of endophyte infection towards AM diversity nor overall community composition in both experiments, although there was an endophyte-associated increase in the abundance of the AM family Acaulosporaceae in the first experiment but not the second. Thus, aside from an endophyte-associated antagonism towards an abundant OTU in the rhizosphere likely of the class Sordariomycetes, E. festucae var. lolii had an otherwise minor impact on the prokaryotic and fungal rhizosphere microbiome of their perennial ryegrass hosts. The minor magnitude of endophyte-associated effects was further emphasized by analyses consistently showing that both prokaryotic and fungal rhizosphere community composition differed to a greater extent between plants of each experiment irrespective of endophyte infection than between plants of differing endophyte status within each experiment- at least in this cultivar-endophyte strain interaction under the conditions of this study.