Characterization of genomic diversity in cpn60 defined Enterococcus ecotypes

The astounding complexity of microbial communities limits the ability to study the role of genomic diversity in shaping the community composition at the species level. With the advancement and increased affordability of high-throughput sequencing methods, it is increasingly recognized that genomic diversity at the sub-species level plays an important role in selection during microbial community succession. Recent studies using the cpn60 universal target (UT) have shown that it is a high-resolution tool that provides superior resolution in comparison to 16S rRNA based tools and can predict genome relatedness. However, studies to characterize the nature and degree of genome content differences predicted by cpn60 UT sequence relationships have not been conducted. In this work, we focused on the Enterococcus community obtained from feces of healthy pigs. Enterococci are both accessible with selective culture, and interesting since the genus includes members that are significant human pathogens and others that are used as probiotics. Previous work has shown that cpn60 UT sequences can resolve pig fecal Enterococcus faecalis and E. hirae into phylogenetically and phenotypically distinct ecotypes. The utility of cpn60 UT sequences for resolution of Enterococcus species was first established in the year 2000, and this demonstration included 17 Enterococcus species. We have expanded the analysis to include all currently recognized Enterococcus species and confirmed that cpn60 UT sequences provide higher resolution than 16S rRNA sequences for identification of Enterococcus species. In addition, we showed that cpn60 UT sequences could resolve sub-groups in E. faecium consistent with results obtained from comparison of whole genome sequences. GTG rep PCR based clusters for E. faecalis and E. hirae isolates were generally consistent with the cpn60 defined Enterococcus ecotypes observed in our previous study, suggesting that cpn60 UT sequences predict overall genomic relationships. Results from analysis of CRISPR sequences provided insights into the extensive networking and transfer of genetic material that takes place within the intestinal Enterococcus community. We conducted whole genome sequencing of representative isolates to characterize further the genomic diversity in cpn60 defined E. hirae ecotypes to determine the nature and degree of genome content differences predicted by cpn60 UT sequences. Differences in phosphotransferase systems, amino acid metabolism pathways for glutamine, proline and selenocystiene, potassium-transporting ATPases, copper homeostasis systems and putative prophage associated sequences, CRISPRs and antibiotic resistance genes were observed. Results from in vitro growth competition assays showed that isolates from E. hirae-1 and E. hirae-2 were able to out-compete isolates from the E. hirae-3 ecotype, consistent with the relatively low abundance of E. hirae-3 relative to E. hirae-1 and E. hirae-2 previously observed in the pig fecal microbiome, and with observed gene content differences between the ecotypes. Results presented in this thesis provide a genomic basis for the definition of ecotypes within E. hirae and confirm the utility of the cpn60 UT sequence for high resolution profiling of complex microbial communities