Protein secretion and outer membrane assembly in Alphaproteobacteria

The assembly of β-barrel proteins into membranes is a fundamental process that is essential in Gram-negative bacteria, mitochondria and plastids. Our understanding of the mechanism of β-barrel assembly is progressing from studies carried out in Escherichia coli and Neisseria meningitidis. Comparative sequence analysis suggests that while many components mediating β-barrel protein assembly are conserved in all groups of bacteria with outer membranes, some components are notably absent. The Alphaproteobacteria in particular seem prone to gene loss and show the presence or absence of specific components mediating the assembly of β-barrels: some components of the pathway appear to be missing from whole groups of bacteria (e.g. Skp, YfgL and NlpB), other proteins are conserved but are missing characteristic domains (e.g. SurA). This comparative analysis is also revealing important structural signatures that are vague unless multiple members from a protein family are considered as a group (e.g. tetratricopeptide repeat (TPR) motifs in YfiO, β-propeller signatures in YfgL). Given that the process of the β-barrel assembly is conserved, analysis of outer membrane biogenesis in Alphaproteobacteria, the bacterial group that gave rise to mitochondria, also promises insight into the assembly of β-barrel proteins in eukaryotes

A Modular BAM Complex in the Outer Membrane of the α-Proteobacterium Caulobacter crescentus

Mitochondria are organelles derived from an intracellular α-proteobacterium. The biogenesis of mitochondria relies on the assembly of β-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an α-proteobacterium, and the BAM (β-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A ∼150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane

Omp85 family proteins and the BAM complex in Caulobacter crescentus.

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

Omp85 family proteins and the BAM complex in Caulobacter crescentus.

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

DOI:10.1111/j.1574-6976.2008.00130.x

outer membrane assembly; membran

The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex

The β-barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α-, β-, γ-, δ- and ε-proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α-proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α-proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ-proteobacteria such as E. coli