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The bacterial phylum, Bacteroidetes, is a remarkably diverse and ubiquitous lineage whose members are profoundly integral to the ecological niches they occupy such as the mammalian gut, the rhizosphere, and the ocean microbiome. They also possess a wealth of notable features that distinguishes them from other groups of bacteria. Despite their apparent ecological importance and the intriguing characteristics that they possess, they remain a poorly studied group. This deficiency in characterization is readily evident with regards to translation initiation in the Bacteroidetes, which is contrary to our common understanding of initiation in more well-studied bacteria. Among prokaryotic genomes, many protein coding genes have a Shine Dalgarno (SD) motif proximal to the start codon which serves to recruit ribosomes to that site. Translation initiation is often facilitated by pairing of the SD sequence to its motific counterpart, the anti-SD (ASD), on the 16S rRNA of the ribosome. This SD-ASD interaction was thought to be the predominant mechanism of translation initiation among bacteria and in many cases indispensable. Yet, SD sequences are greatly underrepresented in the genomes of Bacteroidetes, suggesting that the SD-ASD mechanism has become largely irrelevant. In the absence of SD sequences, alternative translational determinants must substitute and be more prominent in the Bacteroidetes. In this study, we identify translational determinants in the Bacteroidetes that supplant the SD motif. We observe similar alternative determinants in other organisms such as Escherichia coli that do use the SD sequences, but less so in organisms such as B. subtilis which relies heavily on SD sequences. However, what remains uncertain is why the Bacteroidetes lineage has largely dispensed with the SD sequence.Another intriguing aspect of the Bacteroidetes is that their ribosomes virtually retain the complete ASD sequence. The increasing irrelevance of the SD sequence should lessen the imposition to keep the ASD sequence intact within this lineage, yet degeneration of the ASD has not occurred in most Bacteroidetes. Conservation implies functionality and so the ASD in the Bacteroidetes must be obliged to participate in some other capacity. In this study we uncover an alternative role of the ASD by demonstrating that subunits carrying ASD substitutions in Flavobacterium johnsoniae retain substantial activity but are impaired in entering translation. The exclusion of SD sequences in F. johnsoniae suggests ASD mutations cause defects in 30S assembly or in general initiation, revealing an alternative role of the ASD sequence in the Bacteroidetes. Perhaps a more interesting aspect of the ASD sequence in the Bacteroidetes is that although this lineage bothers to maintain the integrity of the ASD sequence, they also opt to keep it in a quiescent state. Indeed, the occlusion of the ASD appears to be conserved across the phylum. Ribosomes from the Bacteroidetes seem unable to recognize typical SD sequences. No doubt the occlusion of the ASD sequence and the loss of the SD sequence in the Bacteroidetes are evolutionarily intertwined, although it remains unclear which process preceded and induced the other and a motivation for these events occurring at all is so far without explanation. In this study, we provide a basis for the inactivity of the ASD in the Bacteroidetes ribosomes. We solved the structure of the ribosome from F. johnsoniae and observe that the ASD is sequestered by three neighboring 30S proteins, bS6, bS18, and bS21. Uniquely conserved residues from these proteins that interact with the 3′ tail of the 16S may explain why the ASD is uniquely sequestered in the Bacteroidetes. In F. johnsoniae, the gene encoding bS21 (rpsU) has an extraordinarily strong SD sequence while virtually all other genes do not contain an SD. In fact, strong SD sequences are frequently found in the rpsU gene as well in the bS18 coding gene (rpsR) for many Bacteroidetes. We infer that these SD sequence function in an autoregulatory mechanism to control translation of bS21 and bS18 in the Bacteroidetes. Bioinformatic evidence reveals that the rpsU gene in organisms from many other phyla also has a uniquely extensive SD sequence indicating that bS21 autoregulation can be more widespread. In this study, we provide experimental data to support our model for bS21 autoregulation in F. johnsoniae. Our data suggest that ribosomes lacking bS21 exhibit altered specificity in initiation in a SD-dependent manner with gain of function. These data together with several other observations of the bS21 protein implicate bS21 in being a component of the ribosome whose presence or absence can impart specialized function.

Organic carbon in anoxic ecosystems flows in a cascade from complex plant material to more labile sugars, and ultimately to short-chain fatty acids (SCFA) and gasses like carbon dioxide and methane. Microbial communities, groups of microorganisms that interact with one another, facilitate this process. Microbial anaerobic carbon degradation is exemplified in ruminants. These animals harness energy from plant material using the power of interacting microorganisms, which break down plant carbon into SCFA under largely anoxic conditions in the rumen. Because microbial SCFA can provide up to 80% of the animal’s energy, understanding microbial carbon degradation mechanisms in the rumen is important for many agricultural industries including the production of meat, milk, leather, and wool. Beyond domesticated ruminants, there are over 75 million wild ruminants that are fundamental members in ecosystems from Alaska to Australia. Furthermore, the microbial enzymes that break down plant material in the rumen have industrial applications for modifying enzymatic cocktails in biofuel production.The research presented here uses cultivation-independent and laboratory approaches to assign carbon degradation capabilities to specific members of the microbial community in the moose rumen. Moose, animals that naturally forage on woody biomass, were selected to provide access to natural rumen microbial communities that are especially adapted to a high lignocellulose diet. We sampled rumen fluid from moose in the spring, summer, and winter, along a seasonal gradient in lignocellulose. Rumen fluid was sampled via the rumen cannula, offering access into the active microbial interactions mediating complex carbon degradation. From these rumen fluid samples, we performed high-throughput shotgun metagenomics and metaproteomics, coupled to multiple methods for metabolite quantification (1H NMR, sequential fiber analyses, and carbohydrate microarray polymer profiling (CoMPP)). We binned hundreds of genomes, resulting in 77 unique (~>80% complete) genomes. A majority of these genomes (71%) belong to novel genera, families, and orders. Five of these genomes belong to an uncultivated, Bacteroidetes family, the BS11, which represent the first ever genomic representatives from this family. A newly resolved genus in this family was the most enriched member on a high lignocellulose diet and was found to ferment hemicellulose sugars.To uncover interactions between these microorganisms and determine their functional role, we mapped metaproteomics data to the unique genome data. This revealed most of the carbon degradation enzymes were encoded within polysaccharide utilization loci from uncultivated Bacteroidetes genomes. We then characterized the carbon metabolite chemistry focusing primarily on carbohydrate polymers and sugars of using CoMPP and 1H NMR. Linking our proteomes to metabolomics, we discovered that proteins from only seven Bacteroidetes genomes were processing all plant polymers detected, suggesting that a few generalist microorganisms are responsible for most of the carbon degradation in the rumen. One of these highly active Bacteroidetes genomes contained protospacer linkages to viral genomes, indicating that immunity against viral predation that may be required for some organisms to sustain carbon degradation in the rumen.Finally, winter rumen fluid with elevated condensed tannins was used to enrich for tannin-degrading microorganisms. From these reactors, we isolated a Streptococcus sp. that can degrade Sorghum condensed tannins (CT) in the presence of glucose. Label-free proteomics was performed to evaluate the dynamic proteome when the isolate was grown in the presence or absence of CT. CT lead to the enrichment of many proteins annotated as tannase enzyme, transcriptional regulators for phenolic metabolism, putative enzymes involved in phenolic metabolism, stress response proteins, and proteins originating from prophage. Cumulatively, this dissertation research examines the rumen on multiple scales, to identify microbial community and viral interactions, microorganism physiology, and the putative enzymes that facilitate how carbon flows through the rumen.

Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate between ruminant and human fecal pollution. These assays are rapid and relatively inexpensive but have been used in a limited number of environmental applications. In this study, we evaluated the efficacy of human- and ruminant-specific 16S rDNA Bacteroidetes assays in determining the primary sources of fecal pollution in Plum Creek (NE) watershed. The sensitivity and specificity, as well as the temporal and spatial application of these assays were challenged against feces from different animals, water, and sediment samples from this watershed. Phylogenetic analyses of 981 fecal and environmental 16S rDNA clones were also performed to study the diversity of Bacteroidetes in this watershed. On average, the host specific assays indicated that ruminant feces were present in more than one-third of the water samples and in all sampling seasons, with increasing frequency in downstream sites along Plum Creek. The human-targeted assay indicated that only 5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (i.e., 24%) were detected in sediment samples. Phylogenetic analysis of Bacteroidetes 16S rDNA sequences derived from fecal and environmental samples demonstrated the presence of a high level of sequence diversity, with nearly half of the recovered sequences showing less than 97% identity to sequences found in publicly available databases. Approximately, 60% of all clones clustered with yet to be cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. The preponderance of clones affiliated with uncultured Bacteroidetes strongly suggests that the bacterial diversity of this group is not sufficiently represented by the culturable members for which sequence data are available. The sequencing data also indicated that other sources, including humans, sediments, and wildlife may be significantly contributing to the molecular diversity of fecal Bacteroidetes in this watershed. However, since several clusters contained sequences from multiple sources, future studies must take into consideration the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers.