Comparative genome analysis of human bifidobacteria with commercial potential

This thesis describes two newly sequenced B. longum subsp. longum genomes and subsequent comparative analysis with publicly available B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis genomes (Chapter 2). The acquired data revealed a closed pan-genome for this bifidobacterial species and furthermore facilitated the definition of the B. longum core genome. The comparative analysis also highlights differences in the potential metabolic abilities of all three sub-species. Interestingly, phylogenetic analysis of the B. longum core genome indicated the existence of a novel B. longum subspecies. Characterisation of restriction-modification systems from two B. longum subsp. longum strains is described in Chapter 3. These defence mechanisms limit the uptake of genetic material, which was successfully demonstrated for some of the identified systems. When these systems were by-passed by methylation of DNA prior to the transformation procedure, the resulting transformation efficiency of both B. longum subsp. longum strains was increased to a level that allowed for the generation of mutants via homologous recombination. Arabinoxylan metabolism by B. longum subsp. longum NCIMB 8809 was investigated in Chapter 4 of this thesis. Transcriptome analysis allowed the identification of a number of genes involved in the degradation, uptake and utilisation of arabinoxylan. Biochemical analysis revealed that three of the identified genes encode arabinofuranosidase activity. Phenotypic assessment of a number of insertion mutants in genes identified by the transcriptome analysis revealed the essential role of two of these enzymes in arabinoxylan metabolism, and a third enzyme in the metabolism of debranched arabinan. Furthermore, this investigation revealed that B. longum subsp. longum NCIMB 8809 does not completely degrade arabinoxylan, but utilises the arabinose substitutions only, while leaving the xylan backbone untouched.Finally, Chapter 5 outlines that B. longum subsp. longum NCIMB 8809 is capable of removing ferulic and p-coumaric acid substitutions that originate from arabinoxylan. Analysis of the genome sequence led to the identification of a candidate gene for this activity, which was subsequently cloned and expressed in E. coli. Biochemical analysis revealed that the enzyme, designated here as FaeA, is indeed capable of releasing both ferulic and p-coumaric acid from arabinoxylan. Furthermore, it is shown that a derivative of B. longum subsp. longum NCIMB 8809 carrying an insertion mutation in faeA had lost the ability to release ferulic and p-coumaric acid from arabinoxylan, and that growth of this mutant strain is negatively affected when cultivated on growth-limiting levels of arabinoxylan.

Antibiotic resistance in the gut microbiota

Antibiotic resistance is an increasing threat to our ability to treat infectious diseases. Thus, understanding the effects of antibiotics on the gut microbiota, as well as the potential for such populations to act as a reservoir for resistance genes, is imperative. This thesis set out to investigate the gut microbiota of antibiotic treated infants compared to untreated controls using high-throughput DNA sequencing. The results demonstrated the significant effects of antibiotic treatment, resulting in increased proportions of Proteobacteria and decreased proportions of Bifidobacterium. The species diversity of bifidobacteria was also reduced. This thesis also highlights the ability of the human gut microbiota to act as an antibiotic resistance reservoir. Using metagenomic DNA extracted from faecal samples from adult males, PCR was employed to demonstrate the prevalence and diversity of aminoglycoside and β-lactam resistance genes in the adult gut microbiota and highlighted the merits of the approach adopted. Using infant faecal samples, we constructed and screened a second fosmid metagenomic bank for the same families of resistance genes and demonstrated that the infant gut microbiota is also a reservoir for resistance genes. Using in silico analysis we highlighted the existence of putative aminoglycoside and β-lactam resistance determinants within the genomes of Bifidobacterium species. In the case of the β- lactamases, these appear to be mis-annotated. However, through homologous recombination-mediated insertional inactivation, we have demonstrated that the putative aminoglycoside resistance proteins do contribute to resistance. In additional studies, we investigated the effects of short bowel syndrome on infant gut microbiota, the immune system and bile acid metabolism. We also sequenced the microbiota of the human vermiform appendix, highlighting its complexity. Finally, this thesis demonstrated the strain specific nature of 2 different probiotic CLA-producing Bifidobacterium breve on the murine gut microbiota.