Lipopolysaccharide (LPS) is the glycolipid that comprises the outer leaflet of the Gram-negative outer membrane (OM). Because it is essential in nearly all Gram-negative species, and because it is responsible for making these bacteria impervious to many types of antibiotics, LPS biogenesis has become an important area of research. While its biosynthesis at the cytoplasmic face of the inner membrane (IM) is well studied, the process by which it is removed from the IM, transported across the aqueous periplasmic compartment, and specifically inserted into the outer leaflet of the OM is only beginning to be understood. This transport process is mediated by the essential seven-protein LPS transport (Lpt) complex, LptA/B/C/D/E/F/G. The OM portion of the exporter, LptD/E, is a unique plug-and-barrel protein complex in which LptE, a lipoprotein, sits inside of LptD, a β-barrel integral membrane protein. LptD is of particular interest, as it is the target of an antibiotic in Pseudomonas aeruginosa. Part I of this thesis investigates how the cell forms the two non-consecutive disulfide bonds that connect LptD's C-terminal β-barrel to its N-terminal soluble domain. These disulfides, one of which is almost universally conserved among Gram-negatives, are essential for cell viability. Here, we show that an intermediate oxidation state with non-native disulfide bonds accumulates in the absence of LptE and in strains defective in either LptE or LptD. We then demonstrate that this observed intermediate is on-pathway and part of the native LptD oxidative folding pathway. Using a defective mutant of DsbA, the protein that introduces disulfide bonds into LptD, we are able to identify additional intermediates in the LptD oxidative folding pathway. We ultimately demonstrate that the disulfide rearrangement that activates the LptD/E complex occurs following an exceptionally slow β-barrel assembly step and is dependent on the presence of LptE. Part II describes work towards obtaining X-ray crystal structures of the LptD N-terminal domain and LptD/E complex. Expression construct and purification optimization enabled the production of stable LptD/E in quantities that make crystallography feasible. Numerous precipitants, detergents, and additives were screened, ultimately resulting in protein crystals that diffract to a resolution of 3.85 Å. / Chemistry and Chemical Biology

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of Gram-negative bacteria and plays a fundamental role in protecting the bacteria from harsh environments and toxic compounds. The LPS transport system is responsible for transporting LPS from the periplasmic side of the inner membrane (IM) to the OM, in a process involving seven LptA-LptG proteins. The current model for lipopolysaccharide transport (Lpt) suggests that LPS is initially extracted by a four-protein complex, LptBCFG, from the inner membrane to the periplasm, where LptA mediates further transport to the OM. Another two protein complex, LptD/E, catalyses the assembly of LPS at the OM cell surface. However, the details of this transport mechanism have remained unknown, mainly due to a lack of structural information. In chapter 1 and 2 of this thesis, I report materials and methods for all LptD/E, and Schmallenberg virus (SBV) nucleoprotein (NP) experiments and the theories and software that were used in determining structures of LptD/E, SBV NP and the SBV NP/RNA complex. In chapter 3 of this thesis, I report the first crystal structure of the outer membrane protein LptD/E complex. LptD forms a 26-strand ß-barrel in a closed form and LptE is a roll-like structure located inside LptD to form “barrel and plug” architecture. Through structural analysis, function assay and molecular dynamics simulation, we proposed a mechanism in which the hydrophilic head of LPS molecule, including the oligosaccharide core and the O antigen, directly penetrates through the hydrophilic ß- barrel whilst the hydrophobic lipid A tail is inserted into an intramembrane hole, with a lateral opening between strand ß1 and ß26 of the LptD. LptE may assist this process. In chapter 4, I report the crystal structure of the SBV NP in two conformations: tetrameric when the protein was purified under native conditions, and trimeric when denatured and refolded during purification. The SBV NP has a novel fold and we have also identified that the N-terminal arm is crucial for RNA binding, and the N- and the C-terminal arm is essential for RNA multimerisation with adjacent protomers and for viral RNA encapsidation. Chapter 5 describes the crystal structure of SBV NP in complex with a 42 nucleotide long RNA (polyU). This ribonucleoprotein (RNP) complex was crystallized as a ring-like tetramer with each protomer bound to 11 ribonucleotides. Eight of these nucleotides are bound in a positively charged cleft between N- and C- terminal domains and three are bound in the N-terminal arm. I also compared the structure to that of other NPs from negative-sense RNA viruses, and found that SBV NP sequesters RNA using a different mechanism. Furthermore, the structure suggests that when RNA binds the protein, there are conformational changes in the RNA-binding cleft, and in the N- and C-terminal arms. Thus our results reveal a novel mechanism of RNA encapsidation by orthobunyaviruses NP.

Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.

β-barrel membrane proteins play important structural and functional roles in Gram negative bacteria and in mitochondria and chloroplasts of eukaryotes. A conserved machine is responsible for the folding and insertion of β-barrel membrane proteins but its mechanism remains largely unknown. In E. coli, a five protein β-barrel assembly machine (Bam) assembles β-barrel proteins into the outer membrane (OM). Among all β-barrel membrane proteins in E. coli , the β-barrel component of the OM LPS translocon is one of only two essential β-barrels, the other being the central component of the Bam machinery itself. The OM LPS translocon, which consists of OM β-barrel protein LptD (lipopolysaccharide transport) and OM lipoprotein LptE, is responsible for the final export of LPS molecules into the outer leaflet of the OM, resulting in an asymmetric bilayer that blocks the entry of toxic molecules such as antibiotics. This thesis describes the characterization of the biogenesis pathway of the OM LPS translocon and its folding and insertion into the OM by the Bam machinery. An in vivo S35-Methionine pulse-labeling assay was developed to identify intermediates along the biogenesis of the OM LPS translocon. Seven intermediates were identified along the pathway. We show that proper assembly of the OM LPS translocon involves an oxidative disulfide bond rearrangement from a nonfunctional intermediate containing non-native disulfides. We also found that the rate determining step of OM LPS translocon biogenesis is β-barrels folding process by the Bam machinery. Using in vivo chemical crosslinking, we accumulated and trapped a mutant form of LptD on BamA, the central component of the Bam machinery. We extended the S35-Methionine pulse-labeling method to allow chemical crosslinking of substrates on the Bam complex and trapped LptD while it was being folded on the Bam machine. We demonstrated that the interaction between LptD and BamA is independent of LptE, while that between LptD and BamD, the other essential component of the Bam complex beside BamA, is LptE dependent. Based on these findings, we proposed a model of Bam-assisted folding of the OM LPS translocon in which LptE templates the folding of LptD. / Chemistry and Chemical Biology