In Escherichia coli, the integral outer membrane (OM) enzyme PagP covalently modifies lipid A by incorporating a phospholipid-derived palmitate chain to fortify the OM permeability barrier. We perturbed the bacterial OM in order to activate PagP and examined if it exerts transcriptional regulation through either of its extracellular or periplasmic active sites. Data from RNA-seq revealed the differential expression of 50 genes upon comparing the E. coli imp4213 (lptD4213) strain NR760∆pagPλInChpagP (shortened to NR760λp in this work), in which PagP is constitutively activated, and the mutant NR760λpY87F carrying the periplasmic residue mutation Y87F. 40 genes were upregulated, and encoded proteins related to anaerobic processes, whereas 10 genes were downregulated, and encoded proteins related to aerobic processes. RNA-seq was followed by a study of differential gene expression using the NanoString nCounter system. Results confirmed a 2.7-fold upregulation of dmsA when we compared the strains NR760λp to NR760λpY87F. We also found a 2.5-fold repression of dmsA transcription when we compared the lptD+ parental strain NR754λp to NR754λpS77A carrying the mutation S77A in the extracellular active site of PagP. We then investigated dmsA transcription using a lacZ reporter gene in plasmid pRS551-lacZ. High basal β-galactosidase activity became attenuated in pagP null mutants. OM perturbation using pentamidine showed that dmsA transcription was repressed. Complementation of the chromosomal ∆pagP deletion with a single copy pBADGr plasmid, expressing PagP under the control of an arabinose-inducible promoter, restored dmsA β-galactosidase activity. We observed partial complementation with the downstream cspE gene, which identified a polar effect of the ∆pagP allele. Through deletion of rpoS, rcsB, cpxA, cpxR, pmrA, pmrB, and fadD in E. coli MC4100, we showed that β-galactosidase activity of pdmsA-lacZ was affected by all of these regulators. Our results indicate that these regulators are involved in PagP-mediated regulation of dmsA transcription under aerobic conditions. / Thesis / Doctor of Philosophy (PhD)

The ability of Gram-negative bacteria to modulate outer membrane (OM) composition in response to stressful environments is essential for their survival and replication within host tissues. The OM enzyme PagP catalyzes the transfer of palmitate from a glycerophospholipid to lipid A. Lipid A is the endotoxic portion of LPS responsible for transmembrane signalling to initiate the immune response. Palmitoylation of lipid A can either attenuate or stimulate the immune response depending on where the palmitate chain is attached to a specific lipid A molecule. Here we report that the Escherichia coli PagP homolog is a multifunctional enzyme, which displays two distinct active sites exposed on either side of the bacterial OM. E. coli PagP converts phosphatidylglycerol (PG) to palmitoyl-PG (PPG) using the same cell surface active site involved in the palmitoylation of lipid A. PPG is then serially degraded to bis(monoacylglycero)phosphate (BMP) and either lyso-PG or lyso-BMP by a novel lipase active site located in PagP on the periplasmic side of the OM. The periplasmic lipase active site can be inactivated with the Y87F amino acid substitution. BMP is a novel glycerophosphoglycerol (GPG) that has not previously been reported in bacterial lipid metabolism. Not all PagP homologs have this ability to remodel GPGs. We have identified a divergent lipid A palmitoyltransferase in Pseudomonas aeruginosa that does not palmitoylate PG. The P. aeruginosa homolog also has different lipid A regiospecificity, adding palmitate on the opposite glucosamine at the 3’-position compared to the 2-position of the proximal sugar observed for the E. coli homolog. We have determined that P. aeruginosa PagP is representative of a distinct clade of PagP evolved to fulfill different functions. Although this minor clade is inclusive of homologs that lack obvious sequence similarity with the major clade enterobacterial PagP, we have identified conserved catalytic and structural elements within the minor clade that contribute to our growing understanding of homologous PagP structure/function relationships. A comparative analysis of all available sequences of minor clade PagP homologs has revealed invariant His, Ser, and Tyr residues that are necessary for catalysis. Additionally, a 4-amino acid conserved signature indel or CSI is unique to bacteria clustered phylogenetically within the γ-subclass of Proteobacteria. / Thesis / Doctor of Philosophy (PhD)

The role of membrane-intrinsic enzymes of lipid metabolism in complex biological processes is being realized through comprehensive structure function studies. Detailed analysis of substrate-enzyme interactions occurring within the restrictive membrane environment has proved to be exceedingly challenging. Using detergent micelles, we describe a detailed model for substrate recognition and binding by the outer-membrane intrinsic enzyme PagP from Escherichia coli. PagP is an 8-stranded antiparallel β-barrel that transfers a palmitoyl group from a phospholipid molecule to lipid A, the endotoxin component of lipopolysaccharide. This simple modification provides bacterial resistance to host antimicrobial peptides and attenuates the inflammatory response signalled through the host toll-like receptor 4 pathway. We describe a molecular embrasure and a crenel, which display weakened transmembrane β-strand hydrogen bonding, to provide site-specific routes for lateral entry of substrates into the PagP active site. A Tyr147 localized to the L4 loop gates the entry of the phospholipid substrate through the crenel, while lipid A enters via the embrasure. The side chains of the catalytic residues that are located in the extracellular loops point towards the central axis of the enzyme, directly above the active site. An acyl-chain binding pocket known as the hydrocarbon ruler is buried within the transmembrane β-barrel structure, and is optimized to accommodate a 16-carbon saturated palmitate chain. The hydrocarbon ruler, therefore, accounts for PagP's stringent selectivity for a palmitate chain. Substituting Gly88 lining the floor of the hydrocarbon ruler with residues possessing linear, unbranched, aliphatic side chains changes the selectivity of PagP to utilize shorter acyl chains. The serendipitous discovery of an exciton interaction between Trp66 and Tyr26 at the floor of the hydrocarbon ruler provides an intrinsic spectroscopic probe to monitor the methylene unit acyl-chain resolution of PagP. A compromised acyl chain resolution of the Gly88Cys mutant is attributed to an unexpected decrease of the Cys sulfhydryl group pKa within the β-barrel interior, resulting in a burying of a charged thiolate within the PagP core. The structural perturbation associated with the Cys thiolate extinguishes the exciton and expands the acyl-chain selectivity. These molecular details of lateral lipid diffusion and acyl-chain selection provide the first such example for any membrane-intrinsic enzyme of lipid metabolism. / Thesis / Doctor of Philosophy (PhD)

PagP is an integral outer membrane enzyme that transfers a palmitoyl group from a phospholipid to lipid A and the polar headgroup of phosphatidylglycerol (PG). Palmitoyl-lipid A and palmitoyl-PG (PPG) have been implicated in resistance to host immune defenses. PagP proteins are diverse, the E. coli PagP belongs to the major clade of PagP homologs and palmitoylates lipid A regiospecifically at the 2-position, whereas P. aeruginosa PagP belongs to the minor clade of PagP homologs and instead palmitoylates lipid A regiospecifically at the 3’-position. Our objective was to understand how PagP has been adapted in nature to interact with multiple lipid substrates and products. We investigated the structure-function relationships of key major clade homologs, to show that Bordetella PagP palmitoylates lipid A at the 3’-position and employs surface residue T29 in its palmitoyltransferase reaction. Legionella PagP palmitoylates lipid A at the 2-position and was confirmed to select a palmitate chain from a pool including iso-methyl branched phospholipids characteristic of this species. PagP is usually encoded as a single copy on the chromosome in most bacteria, but two copies of pagP are found in endophytic bacteria. These duplicated PagP homologs from the major clade branch into two subclades, namely chromosomal and plasmid-based PagP homologs. The chromosomal PagP homologs exhibit interacting periplasmic D61 and H67 residues, which are naturally mutated in plasmid-based PagP homologs, and are associated with a conformational change in the -barrel that determines its ability to palmitoylate PG. Chromosomal PagPs can convert PPG to bis(monoacylglycero)phosphate (BMP) and lysophosphatidylglycerol (LPG) through a periplasmic active site controlled by the invariant Y87 residue of E. coli PagP. Plasmid-based PagP homologs appear to have been adapted instead as monofunctional lipid A palmitoyltransferases. These results points to a common ancestor for PagP proteins. Knowledge gained from these studies can be applied to protein engineering. / Thesis / Doctor of Philosophy (PhD)