A community genomics investigation of microorganisms involved in acid mine generation

Microorganisms are essential for the functioning of all ecosystems. However, our understanding of the natural microbial communities is limited by cultivation bottlenecks and the resolution of conventional molecular approaches. Microbial genome sequencing has begun to enable us to determine the genetic potential and genome structure of microorganisms in isolation but fails to provide the information necessary to fully understand the dynamics, diversity and functions of the community from which they were derived. The focus of this dissertation is a comprehensive genomic characterization of a low diversity microbial biofilm found growing in acid mine drainage (AMD) and details the community metabolic potential and interactions, structure and degree of variability within populations, and the potential of genome-directed cultivation methods.; All AMD microbial communities investigated to date comprise only a handful of phylogenetically distinct organisms, typically iron-oxidizing members of the Nitrospira and Thermoplasma lineages. An AMD community was selected for genomic characterization using fluorescence in situ hybridization, and 16S rRNA clone libraries confirmed the low species-level diversity and suggested that the extent of microheterogeneity within each of the populations was limited. The community selected contained ∼75% Leptospirillum group II (closely related to Leptospirillum ferriphilum), ∼10% Leptospirillum group III, and ∼10% archaea which fell into three distinct groups within the Thermoplasmatales order (Ferroplasma sp., for which genome data is already available, "A-plasma" and "E-plasma").; Random shotgun sequence was used to reconstruct near complete genomes of Leptospirillum group II and Ferroplasma type II and to partially reconstruct three other genomes. Analysis of the genomes of each of these microorganisms revealed the metabolic potential of these organisms including pathways used for carbon and nitrogen fixation and provided insights into survival strategies in an extreme environment.; Using the inherent resolution of shotgun sequencing, we were able to determine that single nucleotide polymorphisms are the predominant of form of heterogeneity at the strain level in each population. The Leptospirillum group II genome had remarkably few polymorphisms, implying strong recent environmental selection. However, the cluster regularly interspaced short palindrome repeats (CRISPR) regions have undergone extremely rapid evolution, creating individual-level variation in the dominant members of at least one Leptospirillum group II population. In contrast, the Ferroplasma type II composite genome was constructed from mosaic strain genomes derived primarily from three 'parental' types via homologous recombination. Detailed analysis of inter- and intra-species recombination frequency for the Ferroplasma populations, revealed rampant recombination within populations which declined exponentially with sequence divergence between populations.; Nitrogen fixation is an essential function in the AMD environment because there are no external inputs of fixed nitrogen. A single nif operon was identified in the genomic data suggesting only one member of the biofilm community performs this function. The operon was found to belong to a relatively minor component (∼10%) of the biofilm community, Leptospirillum group III, suggesting that this organism is a keystone species. A directed isolation strategy was pursued based the nitrogen fixing and iron oxidizing capabilities of this organism resulting in the first pure culture of Leptospirillum group III, Leptospirillum ferrodiazotrophum.