Bacteriophage MooMoo is a temperate phage that was isolated and propagated on Mycobacterium smegmatis (M. smeg). It typically produces turbid plaques, however spontaneous clear plaque mutants can be readily isolated. Both turbid (MooMoo-T) and clear plaque (MooMoo-C) formers can establish stable lysogens, but the parental turbid plaque forming phage has a higher lysogenic frequency. The phage repressor protein typically plays the central role in regulating the lysis/lysogeny decision. Therefore, we expected that the mutation responsible for the clear plaque phenotype would be located in the repressor gene. Remarkably, whole genome sequencing detected a single base pair mutation in the minor tail protein gene (gp19). The regulatory role of the repressor protein could not be excluded considering it was unclear how the mutation in gp19 was leading to the altered plaque phenotype. To locate the phage repressor, we used bioinformatics to identify several candidate genes with helix-turn-helix and DNA binding motifs (gp42, gp43 and gp44). We also cloned the parental and mutant gp19 genes. Each candidate gene was cloned into a shuttle vector. The clones of gp43, gp44 and both derivatives of gp19 did not prevent MooMoo growth, whereas the clones of gp42 inhibited phage growth. Based on these results, we concluded that gp42 is the phage repressor for MooMoo. To determine if the presence of gp19 alters lysogenic frequency, lysogeny assays of wild-type (WT) and mutant gp19 clones were evaluated. Compared to the MooMoo-C lysate, the cloned copy of the mutant gp19 showed a slight increase in lysogeny efficiency. The lysogeny frequencies on strains that carry cloned copies of gp19 (WT or mutant) were similar to those obtained on strains that lacked the plasmids. From these results, we concluded, the presence of either parental or mutant gp19 clones does not affect the lysogeny frequency. To determine if host cell physiology was affected by lysogeny, carbon, nitrogen, phosphorus and sulfur utilization resources were screened using high-throughput phenotypic microarrays. From these results, we concluded the presence of the WT or mutant prophage had no significant effect on the utilization of the resources tested.

The unicellular cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism throughout the temperate and tropical open oceans, but it is difficult to grow in pure cultures. We developed a system for rendering spontaneous streptomycin-resistant mutants of Prochlorococcus axenic by diluting them to extinction in the presence of “helper” heterotrophic bacteria, allowing them to grow to high cell concentrations, and then killing the helpers with streptomycin. Using axenic strains obtained in this fashion, we demonstrated that Prochlorococcus experiences a number of growth defects in dilute axenic culture, including reduced growth rate, inability to form colonies on solid media, and higher incidence of mortality (i.e., catastrophic failure of liquid cultures). These defects were eliminated when Prochlorococcus was grown in co-culture with a phylogenetically diverse array of helper bacteria. The primary mechanism of helping was enzymatic removal of hydrogen peroxide (HOOH) from the culture medium. Axenic Prochlorococcus cultures were profoundly sensitive to HOOH additions in comparison with reported tolerance levels for all other wild-type aerobic bacteria, but in co-culture their resistance was similar to that of the helpers. Neither is dependence on helpers limited to the laboratory. Sterile-filtered seawater exposed to sunlight accumulated enough HOOH in 24h to kill ecologically relevant cell concentrations of Prochlorococcus. We also refined a method for delivering HOOH at a defined, steady rate using the buffer HEPES to more accurately simulate the steady accumulation of HOOH in natural waters. Even at the lowest production rates that could sustain the in situ HOOH concentration in the ocean, HEPES-generated HOOH was lethal to Prochlorococcus; again, co-culture with helpers prevented this effect. We speculate on the ecological consequences of Prochlorococcus’ dependency on other organisms for survival, as well as the evolutionary forces that have led to this lack of self-sufficiency.

Liquid chromatography tandem mass spectrometry has become a powerful tool for investigating biological systems. Herein we describe the development of both isotope dilution mass spectrometry methods and targeted metabolomics methods for the study of metabolic and cell-cell signaling applications. A putative yeast enzyme was characterized by discovery metabolite profiling, kinetic flux profiling, transcriptomics and structural biology. These experiments demonstrated that the enzyme shb17 was a sedoheptulose bisphosphatase that provides a thermodynamically dedicated step towards riboneogenesis, leading to the redefinition of the canonical pentose phosphate pathway. An extension of metabolic profiling and kinetic flux profiling methods was developed for a set of symbiotic marine microorganisms. Carbon flux from the most abundant photosynthetic organism, Prochlorococcus, to a symbiotic Alteromonas was observed in liquid coculture. These methods enable a more biologically relevant assay for marine species and will lead to a better understanding of carbon flux in the oceans. Energy taxis refers to the active migration of bacteria in response to electron transport system related signals. The second messenger cyclic-di-GMP provides a link between the metabolic signals and motility. Quantitation of c-di-GMP helped characterize the nature of this regulation. Autoinducer-2 is a small sugar produced by a large variety of bacteria that is proposed to be a universal quorum sensing signal. The quorum sensing function of autoinducer-2 is disputed because it is produced by an enzyme of the activated methyl cycle, leading to an alternate hypothesis that it is simply a metabolic byproduct. Herein a method for the detection of autoinducer-2 is developed to enable studies of its signaling role and biosynthetic regulation. These studies demonstrated that autoinducer-2 does not function as a signal in all species. Further, metabolic experiments indicated that the metabolic impact of LuxS dysfunction was small and could be mitigated by recycling oxidized glutathione. Together, these data indicate that neither hypothesis is adequate. Evidence is provided that autoinducer-2 suppresses the immune system, by the interruption of cytokine signaling, implying that autoinducer play a protective role during host colonization.

Protein-protein interactions (PPIs) are essential features of cellular processes including DNA replication, transcription, translation, recombination, and repair. In my study, the protein interactions of bacterial DNA topoisomerase I, an essential enzyme, were investigated. The topoisomerase I in bacteria relaxes excess negative supercoiling on DNA and maintains genomic stability. Investigating the PPI network of DNA topoisomerase I can further our understanding of the various functional roles of this enzyme. My study is focused on topoisomerase I of Escherichia coli and Mycobacterium smegmatis. Firstly, we have explored the biochemical mechanisms for an interaction between RNA Polymerase, and topoisomerase I in E. coli. Molecular docking and molecular dynamic simulations have predicted that the interactions are mediated through electrostatic, and hydrogen bonding. The predicted Lysine residues (K627, K664) of topoisomerase I that are involved in the electrostatic interactions were mutated to Alanine, and its effect on the binding efficiency with RNA polymerase was reported. In a separate study, PPI partners of topoisomerase I in mycobacteria were identified. Knowledge gained from the study can provide valuable insights into the physiological functions of a validated drug target, DNA topoisomerase I, in pathogenic mycobacteria. Co-immunoprecipitation and pull-down assays were coupled to mass spectrometry for identification of the protein partners of mycobacterial topoisomerase I. The study has identified RNA polymerase, and putative helicases (DEAD/DEAH BOX helicases) as potential protein partners of mycobacterial topoisomerase I. My results indicated that the tail region of the CTD-topoisomerase I was required for direct physical interaction with the RNAP beta’ subunit. My studies have also verified the physiological relevance of the topoisomerase I - RNA polymerase interactions for survival under antibiotic, and oxidative stress. Lastly, I report a direct physical interaction between E. coli topoisomerase I and RecA by pull-down assays. Previous studies have shown that RecA, a DNA repair protein, can stimulate the relaxation activity of E. coli topoisomerase I. Our new results showed that the stimulatory effect can be attributed to the physical interaction of topoisomerase I with RecA.

In a large agricultural operation, such as the one at Cal Poly San Luis Obispo, disposal of deceased animals is an immense issue. The cost of transporting and rendering every dead animal is inhibitory to the general function of the agricultural operations and their thin budget. Therefore, we propose that composting mortalities could be an economical alternative. Composting is a recognized method for taking animal waste products along with carbon waste and turning it into a pathogen-free, nutrient-rich topsoil. Carcass composting is in fact performed in other countries and states to varying degrees of success. However, the California EPA limits carcass composing to only private land. Therefore, the purpose of this work was to determine the efficacy of killing pathogens by composting using bench top composting models. Ultimately, our goal is to provide “proof of concept” data in order to gain permission for a full-scale carcass compost pile to be set up at Cal Poly San Luis Obispo. Using thermo tolerant Salmonella senftenberg as an indicator organism, we performed bench top trials of traditional and carcass compost in the lab. Samples were inoculated with S. senftenberg and kept at 55°C for 15 days in accordance with the California EPA and Test Method for the Examination of Composting and Compost (TMECC). Samples were then plated and processed for multiple tube analysis and most probable number. Samples were also partitioned for a viability qPCR with propidium monoazide (PMA) to compare to the classic techniques. Using these methods we were then able to track and produce thermal death time data for S. senftenberg in both traditional and carcass compost. By comparing the types of compost, we were able to determine that the composting method presented by the California EPA and the TMECC produces safe, pathogen free compost, even when inoculated carcasses were introduced. However, even with removal of dead cells by PMA, qPCR did not outperform the classical microbiological methods for as tracking pathogen killing.

Mycobacterium tuberculosis (Mtb) is one of the most virulent and prevalent bacterial pathogens across the world. As Mtb infects millions of people a year, it remains essential to study its physiology with the goal of developing new therapeutic interventions. A critical part of the bacteria’s ability to propagate is through successful cell division. Although the process of bacterial cell division and the key proteins therein are well understood in Escherichia coli, much remains to be understood about division in mycobacteria. Genetic and cell biological approaches have recently begun to identify key divisome components in Mycobacterium smegmatis. However, questions remain regarding the role and function of one divisome protein in particular, the DNA translocase FtsK. In this dissertation, I investigated the necessity of FtsK for the growth of mycobacteria. Using an inducible knockdown of FtsK, I present evidence that complete loss of FtsK is required to inhibit growth in both Mtb and M. smegmatis, and that these orthologs share a homologous function. Additional work suggests extended loss of FtsK may be lethal to bacteria. These observations support that FtsK is an essential member of the divisome in mycobacteria, facilitating the processes of growth and division.

Over time, “superbugs,” or bacteria that have become resistant to antibiotics, have become a great concern in modern medicine. Viable alternates are currently being looked into as effective and safe ways to prevent or treat infections caused by these superbugs. One such method is through the utilization of the second messenger molecule cyclic-di-GMP (c-di-GMP) that has been shown to regulate phenotypes within other bacteria that may control surface colonization in Acinetobacter baumannii. Through a series of experiments, the active enzymes that create c-di-GMP - diguanylate cyclases - and break down c-di- GMP - phosphodiesterases - have been inactivated in mutants to test phenotypes including biofilm formation, motility, antibiotic resistance, and desiccation survival. The research’s objective is to show that manipulation of c-di-GMP within the multi-drug resistant strain of Acinetobacter baumannii may serve as a means to control this bacteria.

The primary contributor to Vibrio fischeri aminopeptidase activity is aminopeptidase N, PepN. Colonization assays revealed the pepN mutant strain to be deficient at forming dense aggregates and populating the host’s light organ compared to wildtype within the first 12 hours of colonization; however the mutant competed normally at 24 hours. To address the role of PepN in colonization initiation and establish additional phenotypes for the pepN mutant strain, stress response and other physiological assays were employed. Marked differences were found between pepN mutant and wildtype strain in response to salinity, acidity, and antibiotic tolerance. This study has provided a foundation for future work on identifying a putative role for V. fischeri PepN in regulating stress response.

Pseudomonas aeruginosa is a ubiquitous, opportunistic pathogen that causes acute and chronic infections. Infection is typically initiated via motile and virulent strains. After exposure to stressors, acute infections make both genotypic and phenotypic switches to a chronic, sessile strain. This is due to intricate regulatory networks directing gene expression in response to stressors. One network, GacA/GacS, has been established to control virulence factors. The stringent response of bacteria is mediated by alarmones produced primarily by RelA which responds to starvation. To study the effect of the stringent response on the virulence switch. A series of experiments were run in both PAO1, a virulent strain, and PDO300, an acute strain, and RelA deletion mutants of each transcriptional fusions of GacA/GacA system were integrated in the wild-types and mutants. Alginate, swimming, twitching, and biofilm formation assays were performed on all. The preliminary data suggests that the stringent response influences the GacA/GacS system.

In the field of marine microbial ecology, many questions remain unanswered with regards to the physiological trade-offs made by phytoplankton to maximize growth (e.g., nutrient acquisition) and minimize loss (e.g., predation defenses). These tradeoffs, which occur at the cellular level, have wide reaching impacts on food web dynamics and global biogeochemical cycles. In the first chapter, we explored the use of a non-canonical amino acid (NCAA) technique, bioorthogonal non-canonical amino-acid tagging (BONCAT), in phytoplankton model systems. This technique has potential to work well in natural systems by enabling isolation of only newly synthesized proteins during an incubation period with the NCAA, reducing the complexity of natural proteomics and easing the elucidation of patterns. However, in testing BONCAT across several groups of cultured phytoplankton, we discovered that the NCAA molecule induced a stress response in the globally ubiquitous marine picocyanobacteria, Synechococcus sp. Therefore, in addition to confirming the uptake of modified amino acids by phytoplankton, chapter one investigated the implications of this stress response and limitations when using this technique to study marine microbial communities. In chapter two, we addressed our initial question by exploring tradeoffs at the protein level in a simplified culture system. This approach revealed insights into metabolic tradeoffs in response to predation pressure and nutrient stress. These insights into how phytoplankton negotiate these physiological tradeoffs at the protein level could ultimately allow for targeted proteomic studies in natural systems.