Several bacteria isolated from the broccoli rhizosphere were assayed to compare their ability to solubilize phosphate and release pyrroloquinoline quinone (PQQ) into the surrounding media. Subsequently, their genomes were sequenced and analyzed for PQQ biosynthesis operon structure. PQQ biosynthesis genes pqqA-F were found in all isolates. The order of PQQ biosynthesis genes and predicted amino acid sequences were compared to each other and the host’s ability to solubilize phosphate and release PQQ. In all Pseudomonas species, two putative protease genes, pqqF, and pqqG, flanked the canonical pqqA-pqqE biosynthesis operon. No mechanistic studies have confirmed the function of pqqF and pqqG. Pseudomonas putida KT2440 is a versatile model organism, representing environmental, agronomical, and industrial interests. Like the broccoli isolates, P. putida KT2440 biosynthesizes and releases PQQ into its surroundings. To better understand their functions within PQQ synthesis in P. putida KT2440, ∆pqqF, ∆pqqG, and ∆pqqF/∆pqqG strains of P. putida KT2440 were generated and the resulting phenotypes were studied.

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.