The reverse flow of genetic information can occur when a special DNA polymerase called Reverse Transcriptase (RT) copies the genetic information in an RNA molecule back into a complementary DNA. One type of RT encoding gene found in bacteria is called a retron element. Recent bacterial genome sequencing projects have revealed many examples of retron RT genes. This gene assignment is based on comparison with a few known retron RT proteins. However, RT proteins are highly diverse in their amino acid sequences, and thus the assigned identity of these RT proteins as retrons in genome databases is questionable. One way to prove that these postulated RTs are indeed from retron elements is to see if they can produce msDNA. Retron RTs are known to synthesize a structurally unique satellite DNA called msDNA in the bacterial cells that contain them. Based on GenBank database matches to a known protein, 7 proteins designated as retron RTs were tested for their ability to synthesize msDNA. Five of these retron RTs did show evidence of producing msDNA and are from very different bacterial hosts. The other 2 RT proteins did not show any evidence that they produce msDNA.

<p>The inheritance of mitochondria and their genomes is predominantly uniparental in plants and animals. However, in fungi, the patterns of inheritance can become complex. Interestingly, in <em>Cryptococcus neoformans</em>, the inheritance pattern is predominantly uniparental. Similar to <em>C. neoformans</em>, closely related <em>Cryptococcus gattii </em>is also a pathogenic fungus, except that it is capable of infecting healthy individuals. In this research, PCR-RFLP was employed on progeny of 14 crosses in order to determine if the mtDNA inheritance pattern is the same in <em>Cryptococcus gattii</em> as was found in <em>C. neoformans.</em> Additionally, the effect of genetic distance between mating partners on mitochondrial inheritance was investigated by using a range of strains across three of the four distinct lineages in <em>C. gattii</em>. My results identified highly variable patterns of inheritance among strains and strain combinations, both within and between the analyzed lineages in <em>C. gattii</em>.</p> / Master of Science (MSc)

<p>Small molecule metabolites have important and diverse roles in every major cellular function. To study the activities of metabolites and the biological processes in which they are involved, it is important to be able to detect their levels within cells. Technologies that measure the concentrations of small molecules within the context of living, growing cells are highly advantageous but are challenging to produce. In this thesis, a novel class of intracellular small molecule sensors is produced, characterized and applied to address novel and relevant research questions. These sensors detect a specific target molecule within bacterial cells using RNA regulatory elements known as riboswitches and one of many possible reporter proteins. In addition to a project that yielded new methodology to create custom riboswitches, two projects that assess the capabilities of sensors that detect an active form of vitamin B12 are described. These projects present an abundance of data that provide novel insights into the transport and metabolism of vitamin B12 in <em>E. coli</em> cells. Overall, the results presented indicate that riboswitch-based sensors represent valuable and unique tools for the study of microbial biology. The thesis is concluded with a discussion that describes design strategies and several exciting potential applications for future riboswitch sensors.</p> / Doctor of Philosophy (PhD)

Porphyromonas gingivalis, a gram-negative anaerobic, pathogenic bacterium is a major etiological agent in the initiation and progression of periodontal disease. Due to the ever-changing environment of the oral cavity, inhabitants like Porphyromonas gingivalis must possess the ability to adapt to changes in environmental conditions like pH, temperature, oxygen tension, and metal concentration. P. gingivalis should therefore have an efficient regulatory system in order to adapt and survive in the oral cavity. This response adaptation occurs at the transcriptional level, which involves alternative sigma factors. Extracytoplasmic function sigma (ECF-s) factors are the largest group of alternative sigma factors that play a role in the bacterial response to environmental stress conditions. Here we analyze the s-70 factor gene, PG0214, an extracytoplasmic function sigma factor encoded in the P. gingivalis genome, and examine its role in the bacterial response to environmental stress and virulence. Our findings indicate that the PG0214 gene is important in regulating major functional gene groups and pathways in the P. gingivalis genome. Strains deficient in the PG0214 gene were analyzed and shown to have decreased protease activity, as well as reduced survivability and invasion rates in eukaryotic host cells when compared against wild-type W83 and ATCC 33277 strains. Collectively our studies demonstrate that the PG0214 gene is a positive regulator of gene expression for the survival and virulence of P. gingivalis in the presence of oxidative- and iron-stress, although further study is needed to fully characterize the gene and determine its specific function.

The emergence of genomics as a discrete field of biology has changed humanity’s understanding of our relationship with bacteria. Sequencing the genome of each newly-discovered bacterial species can reveal novel gene sequences, though the genome may contain genes coding for hundreds or thousands of proteins of unknown function (PUFs). In some cases, these coding sequences appear to be conserved across nearly all bacteria. Exploring the functional roles of these cases ideally requires an integrative, cross-species approach involving not only gene sequences but knowledge of interactions among their products. Protein interactions, studied at genome scale, extend genomics into the field of interactomics. I have employed novel computational methods to provide context for bacterial PUFs and to leverage the rich genomic, proteomic, and interactomic data available for hundreds of bacterial species. The methods employed in this study began with sets of protein complexes. I initially hypothesized that, if protein interactions reveal protein functions and interactions are frequently conserved through protein complexes, then conserved protein functions should be revealed through the extent of conservation of protein complexes and their components. The subsequent analyses revealed how partial protein complex conservation may, unexpectedly, be the rule rather than the exception. Next, I expanded the analysis by combining sets of thousands of experimental protein-protein interactions. Progressing beyond the scope of protein complexes into interactions across full proteomes revealed novel evolutionary consistencies across bacteria but also exposed deficiencies among interactomics-based approaches. I have concluded this study with an expansion beyond bacterial protein interactions and into those involving bacteriophage-encoded proteins. This work concerns emergent evolutionary properties among bacterial proteins. It is primarily intended to serve as a resource for microbiologists but is relevant to any research into evolutionary biology. As microbiomes and their occupants become increasingly critical to human health, similar approaches may become increasingly necessary.

Rhodococcus is a soil bacterium, member of the Actinobacteria, and a close relative of the prolific small molecule producer Streptomyces. Recent interest in Rhodococcus as an under investigated source of possible bioactive secondary metabolites is sparked by the discovery of many polyketide synthase and non-ribosomal peptide synthetase genes of unknown function from sequenced Rhodococcus genomes. Rhodococcus species strain MTM3W5.2 was recently shown to produce a strong inhibitory compound with activity against most strains of Rhodococcus and closely related genera. A goal of this investigation is to discover the gene(s) required to synthesize this inhibitory molecule. The engineered Rhodococcus transposon, pTNR, was used to generate random insertional mutations in the genome of MTM3W5.2. The transposon insertion sites for 8 non-producing mutants were cloned and sequenced. Genes that encode polyketide synthases usually form parts of large biosynthetic gene clusters responsible for the production of small polyketide molecules.

Variovorax paradoxus EPS is a gram-negative rod isolated from the sunflower rhizosphere at CSUSB. Preliminary research has shown that Variovorax paradoxus EPS has anti-staphylococcal activity in liquid and solid co-culture. Anti-staphylococcal activity of Wild type and V. paradoxus EPS 𝚫4519 on 0.5% YE agar with embedded S. aureus AH1710 supports the idea that a soluble molecule is responsible for this activity, as the agar acted as a physical barrier between V. paradoxus EPS and S. aureus colonies. Preliminary genetic analysis of V. paradoxus EPS identified three loci that suitable candidates for the synthesis of a potential anti-staphylococcal small molecule. Preliminary data failed to detect expression at two of the three identified loci and a strain with a mutation at the third locus continues to produce anti-staphylococcal activity. We hypothesize that the microbial agent is expressed at a different locus or loci that have not yet been identified. These gene products are responsible for the synthesis of the microbial agent and are controlled by exposure to Staphylococcus aureus. Optimal growth conditions were identified for V. paradoxus EPS and S. aureus to demonstrate the formation of a zone of inhibition on Tryptic Soy Agar. The use of a V. paradoxus EPS Δ 4519 transposon library at optimal growth conditions allowed us candidate mutants with altered antimicrobial activity phenotypes. We identified 28 insertion sites that resulted in altered antimicrobial activities, which will allow us to identify the genes involved in this biosynthetic pathway.

The vaginal microbiome plays an important role in reproductive health and pregnancy. It has coevolved with humans and has direct effects on reproductive success, rendering selective pressure more pronounced at this site. Herein, we probe coevolution of the vaginal microbiome using a systems-level approach. In Chapter 2, we examine the evolutionary trajectory of two vaginal Veillonellaceae phylotypes evolved from an ancestral gastrointestinal lineage to inhabit the vaginal niche. We present evidence of their divergence and subniche specification and describe their differential associations with vaginal infection and pregnancy. In Chapter 3, we identify ten bacterial taxa, predicted to contribute to the underlying pathology of the sexually transmitted infection trichomoniasis. This ‘pathogroup’, which has undergone conditional differentiation to thrive in the presence of T. vaginalis, includes previously undescribed organisms and putative symbionts. Lastly in Chapter 4, we present the first characterization of BspA proteins, multi-modal virulence factors, in the vaginal microbiome and provide evidence of their extensive horizontal transfer across diverse microbial lineages. We use homology modeling to demonstrate conservation of structural and functional characteristics of these proteins between diverse bacterial taxa and identify structural variants, potentially indicative of subtypes. These findings further our understanding of the contributions of individual bacterial species, bacterial communities and virulence determinants in the health and disease. Furthermore, they lay the groundwork for future work characterizing coevolution of the human vaginal microbiome. These systems-level approaches will facilitate synergy between broad and reductive approaches and inform strategies for modulation of the microbiome and development of more effective therapeutics.

Cyclic-di-GMP, a common bacterial second messenger, has been thought to help develop virulence and biofilms in bacteria, most specifically in Salmonella Typhimurium. By being able to dysregulate cyclic-di-GMP production, virulence may be better combatted. STM1987, an L-arginine-responsive diguanylate cyclase with a periplasmic sensory domain, dimerizes and generates the bacterial second messenger cyclic-di-GMP in response to the amino acid L-arginine in a pathway that also requires the periplasmic L-arginine-binding protein ArtI. Their biochemical responses to L-arginine and when they dimerize could help clarify this pathway, so I sought to develop a periplasmic dimerization sensor to better monitor these biochemical interactions. Similar to STM1987, the ToxR transcriptional regulator from Vibrio cholera is also activated by dimerization. By switching out the periplasmic domain of ToxR for the periplasmic regions of interest, I can better evaluate the cyclic-di-GMP response to L-arginine. This research aims to find the specific responses in this pathway to be able to use this in combatting bacterial virulence. I was able to successfully show that the STM1987 periplasmic domain dimerizes in response to L-arginine, providing an important insight into this signaling pathway.

This study was performed to test the in vitro effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu”, a traditional Kenyan cultured milk, on 3T3-L1 and Caco-2 cell lines. Cultures of fully mature 3T3-L1 adipocytes were treated with bacterial isolate cell extract (CE), filtered spent broth (FSB) from overnight bacterial culture, or with a PBS control. Expression levels of PPAR³1 and 2, C/EBP±, and ATGL proteins in 3T3-L1 cells were upregulated by FSB treatment. CE treatment did not affect protein expression levels. Expression of MTTP and SREBP-1c proteins in Caco-2 cells showed no change with either treatment. Optical density measurements from Oil-Red-O stained 3T3-L1 adipocytes increased from PBS control cells to 25μl/ml FSB treated cells; measurements were reduced by treatments above 25μl/ml FSB. In conclusion, filtered spent broth prepared from a culture of Lactobacillus rhamnosus, isolated from “amabere amaruranu” cultured milk showed PPAR³1 and 2, C/EBP±, and ATGL agonistic properties.