Functional Genomics of D-Amino Acid Metabolism in Pseudomonas Aeruginosa

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Intellectual MeritD-amino acids are naturally synthesized in all living organisms to serve in a variety of specific functions. Examples include the well-known D-Ala and D-Glu as essential components of bacterial cell wall to the latest discovery of D-Ser as neurotransmitter in humans. The level of D-amino acids inside the cells is subjected to tight regulation to prevent any potential harmful effects. Up to date, the biochemistry of D-amino acid catabolism has not been intensively studied in comparison to those of L-amino acids, and therefore, the information is still fragmentary. Famous for its enormous catabolic capacity, P. aeruginosa is able to utilize many D-amino acids as nutrient, and hence serves as an excellent model organism to explore novel pathways and enzymes for D-amino acid metabolism. It is best evidenced by a recent report of a new type of D-to-L arginine racemization by coupled catabolic and anabolic dehydrogenases discovered by the PI's group on a previous NSF-funded project. The results of preliminary studies also uncovered several important features in D-amino acid utilization. D-amino acids have two potential physiological functions - inversion to L-amino acids for peptide synthesis or degradation to serve as nitrogen or carbon source. How each of these D-amino acids can be utilized depends on the presence as well as the induction level of specific racemases or catabolic enzymes/pathways. The goal of this research is to continue efforts in exploration of metabolic pathways for D-amino acids with a systemic approach. The specific aims of this project are: (i) to elucidate physiological functions and biochemical properties of DadAX and DauAB in D-amino acid deamination and racemization; and (ii) to explore catabolic pathways for D-glutamate/glutamine/asparagine and D-proline/hydroxyproline. Experiments will be conducted to test the proposed physiological and biochemical functions of thus identified genes and their encoding enzymes. Meanwhile, a more systemic transcriptome analysis will be conducted to have a better understanding of cellular responses to the presence of these D-amino acids. Genes and encoding enzymes thus identified from this study will be subjected to further characterization accordingly regarding physiological functions, genetic regulation, and biochemical properties in D-amino acid catabolism. Up till now, only limited effort has been devoted systemically to explore the biochemical diversity toward D-amino acids. Findings from this research are anticipated to further extend the current knowledge of amino acid metabolism.Broader ImpactsThe PI plans to continue the effort in maintaining an active research environment composed of post-doc associates, MS and PhD graduate students, and undergraduate students. In addition, the PI will extend the effort for recruiting promising minority students to research by serving as faculty mentor for the McNair Program in the summers. Scientifically, the PI lab will provide unique training opportunities with a combination of modern genomics/proteomics and conventional genetics/biochemistry approaches in the area of bacterial physiology and biochemistry. In addition, the ongoing collaborations between PI and colleagues in protein crystallography and mechanistic enzymology will be fostered to benefit trainees of different disciplines, by way of regular joint group meetings and encouragement of students taking courses offered by collaborating colleagues. The success of this proposed research will have broader impacts in training competitive researchers with multi-disciplinary coverage and in recruiting under-representative minority students into careers of biological research. All members of the Lu laboratory will participate in local, national, and international research symposia to disseminate the research and educational activities of the laboratory to a broader scientific community.