Omp85 family proteins and the BAM complex in Caulobacter crescentus.
2017-02-03T03:39:31Z (GMT) by
The outer membrane of Gram-negative bacteria is a compartment that houses many proteins involved in basic physiological functions, virulence and multi-drug resistance, and is therefore important for cell survival. Most proteins in the outer membrane adopt a β-barrel conformation, and require the β-barrel assembly machinery (BAM) for integration into the outer membrane. The BAM complex is a multi-subunit protein complex present in the outer membrane of all Gram-negative bacteria. It comprises of a core β-barrel protein, BamA and associated lipoproteins that collectively participate in the folding and insertion of β-barrel proteins. BamA has functional homologues in eukaryotes that form the SAM complex in mitochondria for insertion and assembly of β-barrel proteins into the mitochondrial outer membranes. Previous work by other groups in the field has used N. meningitidis (β-proteobacteria) and E. coli (y-proteobacteria) to study the function of BamA and the BAM complex. In this comparative study, we will use Caulobacter crescentus as a model α-proteobacterium to learn about differences and similarities in the BAM complexes across the three different classes of proteobacteria. The combined use of bioinformatics and biochemical experiments in C. crescentus have shown some key differences with the absence of the BamC partner lipoprotein and the presence of a novel OmpA-like protein as well as two other uncharacterized lipoprotein partners for BamA. In Chapter 2, we perform a comparative analysis of BamA and the BamA-like protein (Omp68) in C. crescentus. The Omp85 superfamily contains BamA, Omp68 and TpsB proteins and using bioinformatics analysis, we proposed that Omp68 is an evolutionary intermediate of BamA and TpsB proteins. We first established a method for preparing outer membranes from C. crescentus and demonstrated that like BamA, Omp68 forms an oligomeric complex in the outer membrane although it has characteristics that could classify it as a TpsB protein. The unusual nature of Omp68 is discussed and its possible functions. A method for effective purification of the BAM complex from outer membranes was also established for identification and analysis of the different components of the complex. In Chapter 3, we demonstrate that the BAM complex possesses modular characteristics and contains BamA and six outer membrane lipoproteins in the α-proteobacterium, C. crescentus. In addition to the three known lipoproteins (BamB, BamD and BamE), we identify three other subunits (Pal, BamF and BamG) of which only Pal is essential. We propose that Pal is a protein that anchors the BAM complex to the peptidoglycan layer and promotes proximity to the inner membrane Sec machinery for efficient outer membrane protein assembly. We also show BamF is a genuine component of the BAM complex and a potential homologue of the BamC protein found in all other proteobacteria. Both BamF and BamC contain a conserved motif that is possibly important for docking onto the BAM complex. In Chapter 4, we focus on biochemical and structural characterisations of BamD from C. crescentus. We show BamD is an outer membrane lipoprotein that forms the halo module of the BAM complex. We also demonstrate BamD contains TPR motifs that are essential for functioning of the BAM complex and that protrude into the periplasm for protein-protein interactions. Further experiments involved extensive optimisations of heterologous expression of BamD and purification with the aim of progressing to crystallisation trials. We were able to generate purified folded BamD that will be used for future experiments.