Files
Abstract
Bacteria generate and catabolize a number of non-proteinogenic amino acids, which serve many functions and some are best known as constituents of the peptidoglycan (PG) cell wall. PG is a distinctive, highly conserved Prokaryote-specific structure, and Eukaryotes have evolved to recognize PG along with other microbe-associated molecular patterns (MAMPs). For example, in the monospecific symbiotic relationship between the marine bacterium Vibrio fischeri and its squid host Euprymna scolopes, V. fischeri PG is important for triggering normal host development. While PG structure is largely conserved, there is natural variation among bacteria, and the identity of the non-proteinogenic amino acids in PG can affect the pathways by which this MAMP elicits responses in the host. In this dissertation, I describe a strategy to alter V. fischeris PG by generating auxotrophic mutants that require PG-specific amino acids and selecting spontaneous suppressor mutants able to overcome this auxotrophy. I report the characterization of two distinct types of suppressor mutants; one that had induced a novel pathway to generate wild-type PG and another that had evolved a new PG structure. In the former case, suppression of the D-Glu auxotrophy exhibited by a glutamate racemase (murI) mutant led me to discover a D-Asp responsive transcriptional regulator, DarR. I provide evidence that mutation of darR can render its encoded protein active in the absence of effector and compensate for the murI mutation by increasing expression of an Asp racemase gene with cryptic Glu racemase activity. In the second case, I describe a mutant of V. fischeri that has replaced diaminopimelate with lanthionine in the peptide side chain structure of its PG, a feature that appears to be enabled by alterations in cysteine metabolism. Despite this change in PG structure, cell surface fractions from the mutant with lanthionine-containing PG still induced morphogenesis in the host. Together, these data broaden our understanding of D-amino acid catabolism, PG evolution, and the flexibility of PG recognition in a model symbiosis.