Acinetobacter baumannii is an increasingly multidrug-resistant pathogen contributing to hospital-acquired infections, necessitating a greater understanding of how it interacts with its surroundings. Many bacteria utilize different methods of bacterial motility to move about and interact with these surroundings. A bacterial second messenger, cyclic diguanosine monophosphate (c-di-GMP), can regulate various motility factors that are potentially advantageous for survival in and adaptation to their environment. Concentrations of c-di-GMP are regulated by specific synthesizing and degrading enzymes. Controlled levels of c-di-GMP allow interaction between the c-di-GMP and its binding effectors that induce changes in bacterial phenotypes such as biofilm formation and motility. A search of the A. baumannii genome identified two proteins that contain the c-di-GMP-binding PilZ domain. The PilZ protein for which this PilZ domain was named was initially discovered in Pseudomonas aeruginosa where it has been demonstrated to be a part of the type IV pilus machinery. Type IV pili play roles in twitching motility, adhering to surfaces, DNA uptake, protein secretion, and predation. One of the PilZ-containing proteins from A. baumannii resembled this original PilZ protein (PilZ), while the second PilZ-containing protein contained a hydrolase domain with unknown substrate specificity (HydP). I investigated whether these PilZ-containing proteins play a role in motility of A. baumannii by testing two strains: AB5075 that displays twitching motility, and ATCC17978 that displays an uncharacterized form of surface-associated motility. Results suggest PilZ plays a role in twitching motility, while its effect on surface-associated motility phenotypes3 is possibly due to polar effects from mutation. Results also suggest HydP plays a role in surface-associated motility, although its mechanism is not understood. Testing of both proteins’ PilZ domains indicates they may not bind c-di-GMP, implying they may be playing roles in motility regulation through other mechanisms outside of binding c-di-GMP. These findings give us greater insight into the regulatory mechanisms used by A. baumannii to move about its environment.

D-amino acids (D-AAs) are the α-carbon enantiomers of L-amino acids (L- AAs), the building blocks of proteins in known organisms. It was largely believed that D-AAs are unnatural and must be toxic to most organisms, as they would compete with the L-counterparts for protein synthesis. Recently, new methods have been developed that allow scientists to chromatographically separate the two AA stereoisomers. Since that time, it has been discovered that D-AAs are vital molecules and they have been detected in many organisms. The work of this dissertation focuses on their place in bacterial metabolism. This specific area was selected due to the abundance of D-AAs in bacteria-rich environments and the knowledge of their part in several processes, such as peptidoglycan synthesis, biofilm disassembly, and sporulation. We focused on the bacterium Pseudomonas putida KT2440 which inhabits the densely populated plant rhizosphere. Due to its versatility and cosmopolitan character, this bacterium has provided an excellent system to study D-AA metabolism. In the first chapter, we have developed a new approach to identify specific genes encoding enzymes acting on D-AAs, collectively known as amino acid racemases. Using this novel method, we identified three amino acid racemases encoded by the genome of P. putida KT2440. All of the enzymes were subsequently cloned and purified to homogeneity, followed by a complete biochemical characterization. The aim of the second chapter was to understand the specific role of the peculiar broad-spectrum amino acid racemase Alr identified in chapter one. After constructing a markerless deletion of the cognate gene, we conducted a variety of phenotypic assays that led to a model for a novel catabolic pathway that involves D-ornithine as an intermediate. The work in chapter three identifies for the first time numerous rhizosphere-dwelling bacteria capable of catabolizing D-AAs. Overall, the work in this dissertation contributes a novel understanding of D-AA catabolism in bacteria and aims to stimulate future efforts in this research area.

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.