Previous studies have indicated that the Bam complex is organised into two major subcomplexes, BamAB and BamACDE and that each of the subunits are essential for efficient OMP assembly (Wu et al., 2005, Gatsos et al., 2008, Malinverni et al., 2006, Anwari et al., 2010, Kim et al., 2007, Sklar et al., 2007a, Volokhina et al., 2009). The stoichiometry of the complex is thought to be a 1:1:1:1 ratio for BamA, B, C and D, although it is hard to determine how many subunits of BamE are present due to its small size (Hagan et al., 2010). However, in vitro reconstitution of the complex indicated that only one molecule of BamE was needed for activity of the full complex (Hagan et al., 2010, Gatsos et al., 2008). It should be noted that BamA tetramers have been observed (Robert et al., 2006). Due to this subcomplex organisation, it is possible that BamB and BamCDE perform separate steps in OMP assembly, or that they have discrete functions.
There is a wealth of information relating to the BamAB interaction. Mutational analysis has revealed five crucial residues required for BamB function. Mutations of these residues, that cluster to two adjacent β-blade interconnecting loops (IL4 and IL5), disrupt the physical association of BamB to BamA (Vuong et al., 2008). These mutations essentially yield a ΔbamB phenotype, although stable BamB is produced, suggesting that this interaction with BamA is needed for proper OMP assembly. Analysis of the BamB crystal suggests that these residues specify a BamA binding site (Albrecht and Zeth, 2011, Heuck et al., 2011). In addition to this, deletion of the propeller blade connected to IL5 abolishes BamB function (Ruiz et al., 2005).
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A re-ordering of the residues in the β-bulge of P3 has been shown to disrupt the BamA-B association, suggesting that there is a BamB binding site at the edge of the P3 β-sheet. Simulated protein docking has also suggested the binding of BamB IL4 and Bam P3 β-bulge by β-augmentation (Noinaj et al., 2011), which agrees with the data that demonstrates that both IL4 and the β-bulge are needed for the interaction (Vuong et al., 2008, Kim et al., 2007, Gatzeva-Topalova et al., 2008). It should also be noted that this interaction may be maintained electrostatically as P3 has been shown to be electropositive and the surface of IL4 and IL5 to be electronegative (Noinaj et al., 2011). However, these are not the only BamA-B associations as this interaction requires most of the POTRA domains for stable association. Removal of any POTRA domain except for P1 disrupts the BamA-B interaction.
The interaction of BamA-CDE seems to be dependent on P5 although from the current data, it is difficult to determine the nature of the interactions between BamA and BamC,D and E (Kim et al., 2007, Hagan et al., 2010). Data suggests that BamC and BamE interact with the complex through BamD in a manner that requires the C- terminus of BamD, as truncations in this region compromise the BamA-D interaction (Sklar et al., 2007a, Malinverni et al., 2006).
It has been suggested that BamC and BamE stabilise the BamA-D interaction. A co- crystal structure of the unstructured region and N-terminal domain of BamC and full length BamD revealed that the conserved unstructured region is important for the formation of the BamCD subcomplex (Kim et al., 2011a). Additionally, the
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unstructured region of BamC was shown to obscure the C-terminus of BamD, which is thought to bind to OMP substrates (Kim et al., 2011a). This data from this structure suggests the possibility of BamC playing a regulatory role in OMP assembly (Kim et al., 2011a, Sandoval et al., 2011, Albrecht and Zeth, 2010).
Genetic experiments can also give insight into Bam complex function. It has been shown that deletions of the individual non-essential lipoproteins yields OM defects (Hagan et al., 2011, Sklar et al., 2007a, Wu et al., 2005). However double knockout data can yield further information. Individual knockouts of bamC or bamE yield minor OM defects, however a double deletion of bamC and bamE, generates severe OM defects (Sklar et al., 2007a). Interestingly, a double bamB and bamE deletion is lethal (Sklar et al., 2007a). These genetic interactions may imply some functional redundancy between Bam complex components, but also highlight critical roles for the non-essential lipoproteins as part of the complex despite their individual dispensability.
Binding of the periplasmic chaperone SurA to BamA has been observed and is thought to be a transient interaction (Bennion et al., 2010). Although the nature of this interaction is not apparent, this data could suggest that it is involved in substrate handover from SurA to BamA, and this interaction is used as a docking site to facilitate this exchange. There is further biochemical evidence that suggests that P1 is crucial for this transient docking step (Bennion et al., 2010).
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The SurA-BamA interaction seems to be independent of BamB, despite some functional overlap between SurA and BamB. Both SurA and BamB depletions have shown altered efficiency of assembly of a subset of OMPs (Ureta et al., 2007, Vertommen et al., 2009) and that BamB is important for the assembly of Bam substrates delivered by SurA (Hagan et al., 2010). Additionally, the simultaneous deletion of bamB and surA leads to a synthetic lethal phenotype (Ureta et al., 2007). Since the loss of BamB does not affect the SurA-BamA interaction, it seems unlikely that SurA binds directly to BamB. BamB may somehow promote the assembly of SurA substrates in a manner that is independent of binding (Noinaj et al., 2011). It should also be noted that simultaneous removal of bamB and degP is also synthetically lethal (Charlson et al., 2006). These results suggest that BamB is involved in the earlier steps of OMP assembly.
So far, interactions of Skp or DegP to the Bam complex have not yet been observed (Sklar et al., 2007a). This could be due to the transient nature of these possible interactions, or that Skp and DegP are able to deliver Bam substrates in a manner that does not require docking at the Bam complex.
1.9.7 Concluding remarks
Although the individual contributions and interactions of the components of the Bam complex have been discussed, the actual process of OMP assembly still remains enigmatic. It is known that the periplasmic chaperones deliver Bam complex substrates that may bind to either the POTRA domains by β-augmentation or by the C-terminal of BamD (Kim et al., 2007, Albrecht and Zeth, 2011, Sandoval et al.,
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2011). BamB, BamC and BamE may have roles in regulation and generally increasing the efficiency of the process. Substrates could then be passed to the BamA β-barrel domain, which could act as a scaffold to aid the formation and assembly of β-barrels. During this process, BamC could act as a substrate binding regulator, by using its unstructured region to bind to BamD. BamB could provide substrate binding surfaces and BamE may recruit PG to increase folding efficiency.
The least understood aspect is the molecular nature of OMP assembly by the Bam complex. Data from in vitro studies suggest that the insertion of OMPs takes place in a concerted manner (Burgess et al., 2008, Kleinschmidt, 2003). Three different models of OMP assembly have been suggested.
The first model suggests that the substrate OMP is translocated through the β-barrel domain of BamA into the extracellular space in an unfolded form before being assembled into the OM. This model is based on structural studies of FhaC. FhaC has a pore diameter of ~3 Å, which is too small to accommodate unfolded polypeptides (Clantin et al., 2007). However, upon binding of the substrate (FHA), the pore diameter can increase to 16 Å (Clantin et al., 2007, Delattre et al., 2010), which could be enough to accommodate the substrate. A similar observation has been made for BamA, where its electrical conductivity has increased with substrate binding (Stegmeier and Andersen, 2006). It is feasible that this conformational change could allow translocation. A major caveat with this model is that it assumes that OMP folding and assembly would take place on the extracellular surface which could be an inefficient process without the direct aid of folding factors.
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The second model suggests that unfolded OMPs use the outer wall of BamA as a template, whereby the substrate is inserted between the BamA-lipid interface. This could lead to secondary structure formation, aided by the template of BamA and ultimately leading to formation of β-barrels. This model also takes into account the possible BamA tetramer, where the substrate could be contained in the space between BamA subunits. This enclosed space could drive the formation of β-barrels, which would then be released laterally into the bilayer.
The third model suggests that substrates are able to enter the β-barrel domain of BamA and that BamA is able to fold them into β-barrels and release them laterally into the bilayer (Bos and Tommassen, 2004). The major problems with this model are that the channel of BamA is not large enough to house a folded OMP and that lateral insertion would require breaking the hydrogen bonds of the β-barrel domain of BamA, which is very costly energetically. An additional model has been suggested by (Kim et al., 2012) based on this template. Instead of the substrate
folding within the β-barrel of BamA, the N and C-terminal β-strands of BamA (the two strands that hydrogen bond with each other to close the β-barrel), could act as folding templates of OMP substrates. Hydrogen bonds could form between these BamA terminal strands and the substrate, which leads to the formation of β-sheets in the substrate. This process is repeated which leads to the formation of more β- strands, until the β-barrel of the substrate is formed, which is then released laterally. This model requires the breakage of hydrogen bonds of the β-barrel of BamA, but
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the formation of hydrogen bonds between BamA and the substrate β-strands by β- augmentation is much more energetically favourable.
Within the next forthcoming years, the molecular events leading to assembly will hopefully be solved. Questions such as the structure of BamA, the role its periplasmic loops and the assembly of the complex would fill major gaps in understanding OMP biogenesis.