Culturing isolated microorganisms can be challenging, not only because usually the detailed environmental conditions where organisms grow optimally are not known, but also because many of them need to grow in the presence of other organisms. High-throughput sequencing and other `omics' technologies provide important approaches for the study of microorganisms in their natural environments. Specifically metagenomics methods enable culture-independent surveys of organisms and functions in microbial consortia, and can yield near-complete genomes of the most abundant community members, and partial genomes of lower abundance organisms. When coupled to community proteomic and/or transcriptomic analyses it is possible to predict what functions are being expressed within the community. Therefore, `omics' technologies provide a means for the study of community physiology and ecology in natural systems.
Acid mine drainage (AMD) is a mining-related problem caused by sulfide mineral dissolution coupled to microbial iron oxidation, which leads to acidification and metal contamination of the environment. The Richmond Mine AMD community is currently the best-studied AMD system. Bacteria of the genus Leptospirillum, of the Nitrospira phylum, generally dominate Richmond Mine AMD microbial communities. Current studies show that Leptospirillum rubarum (group II) tends to dominate early-formed biofilms, and Leptospirillum group II 5way CG (a genotype related to L. rubarum) or L. ferrodiazotrophum (group III) increase in abundance as environmental conditions change.
In chapter 1, community genomics was used to reconstruct the near-complete genome of Leptospirillum ferrodiazotrophum, and report the genome annotation and metabolic reconstruction of L. ferrodiazotrophum, Leptospirillum rubarum and an extrachromosomal plasmid associated to these bacteria. In addition, proteomic analyses were used to evaluate protein expression patterns in three AMD biofilms. Results indicate that, despite sharing only 92% identity at the 16S rRNA level, L. rubarum and L. ferrodiazotrophum share more than half of their genes. Both bacteria are motile, acidophilic iron-oxidizers, as evidenced by the presence of cytochrome Cyt572 and an electron transport chain. They are chemoautotrophs, using a reverse tricarboxilic acid (TCA) cycle for carbon fixation. Their metabolic potential indicates that L. rubarum and L. ferrodiazotrophum are capable of amino acid and vitamin biosynthesis, fatty acid biosynthesis, flagella biosynthesis, synthesis of polymers such as cellulose, and the synthesis of compatible solutes for osmotic tolerance. Only L. ferrodiazotrophum is capable of nitrogen fixation, although proteins were not detected by proteomics in the analyzed biofilms. Proteomic analyses indicate that core metabolic proteins are similarly expressed in both bacteria, however high expression of many hypothetical proteins unique to each Leptospirillum might contribute to their differentiation within the biofilms.
In chapter 2, the partial genome reconstruction of a new Leptospirillum bacterium, which is closely related to L. ferrodiazotrophum, is reported. The bacterium represents ~ 3% of the sequenced community, and comparison of its 16S rRNA gene sequence with those of other Leptospirilli identifies it as a new group within the Leptospirillum clade: Leptospirillum group IV UBA BS. The bacterium grows in unusually thick, Archaeal-dominated biofilms where other Leptospirillum spp. are found at very low abundance. It shares 98% 16S rRNA sequence identity and 70% amino acid identity between orthologs with L. ferrodiazotrophum. Its metabolic potential indicates that it too is a motile, iron oxidizing chemoautotroph capable of nitrogen fixation, although nitrogen fixation expression was not observed. Leptospirillum group IV UBA BS is distinguished from the other Leptospirilli in that it contains a unique multicopper oxidase likely involved in iron oxidation, and the presence of two clusters of hydrogenase genes. The cytoplasmic hydrogenase is likely used to take up H2 during nitrogen fixation, while the membrane-bound hydrogenase might be involved in anaerobic H2 oxidation for energy generation. Community transcriptomic and proteomic analyses confirm expression of the multicopper oxidase, as well as the expression of many hypothetical proteins and core metabolic genes. Transcription of hydrogenases in the only biofilm in which the nitrogen fixation operon in L. ferrodiazotrophum is transcribed points to potential cooperative interactions between the two bacteria.
AMD has long been considered a simple, low-diversity ecosystem. In chapter 3, a new view of the diversity of organisms in AMD was obtained by deep sequencing of the small subunit (SSU) rRNA from 13 biofilm communities. A total of 159 taxa, including Archaea, Bacteria, and Eukaryotes, were identified. Leptospirillum spp. dominate the samples, and members of diverse phyla, such as Actinobacteria, Acidobacteria, Firmicutes, Alpha-, Beta-, Gamma-, and Delta-Proteobacteria, Chloroflexi, and Deferribacter were present at low abundance. Interestingly, members related to Magnetobacterium spp. of the Nitrospira phylum were detected. These bacteria have not been reported present in AMD environments, and they have not been identified in community genomics datasets. However, the presence of magnetosome-like structures observed by cryo-TEM in some AMD biofilms supports the transcriptomics results. The findings indicate that it is dominance by a few taxa, and not lack of complexity of the system that has made AMD environments model systems for the study of microbial physiology and ecology.
In Chapter 4, non-ribosomal transcriptomic reads were mapped to several genomes of AMD organisms, including Archaea, Bacteria, and viruses, in order to evaluate the expression profiles of genes and non-coding regions (ncRNAs) in biofilms at increasing stages of development. More than 95% of the genes in the most abundant Leptospirillum group II and group III bacteria were detected by at least one transcriptomic read, indicating that the whole genome is transcribed at some level. More than half of the genes in the Archaea G-plasma and Ferroplasma Type II, and a virus associated to Leptospirillum were also detected. Transposases, cytochromes, and ncRNAs were among the most highly expressed genes in all samples. Gene expression profiles indicate that Leptospirillum group II 5way CG and L. ferrodiazotrophum prefer growth at higher pH and lower temperature, conditions generally present in bioreactors, while the opposite is true for L. rubarum and G-plasma, who prefer conditions found in early to mid-developmental stage environmental biofilms. High levels of expression were observed for a novel ectoine riboswitch predicted in the Leptospirillum group II genome, as well as for other non-coding RNAs. Results provide new insight into understanding functioning and adaptation of acidic ecosystems.