In Gram-negative bacteria, integral outer membrane β-barrel proteins (OMPs) are assembled by the beta-barrel assembly machine (Bam) complex. The essential components of this complex are the OMP BamA [which contains a carboxyl-terminal β-barrel and an amino-terminal periplasmic module composed of five polypeptide transport associated (POTRA) domains] and the lipoprotein BamD. In Escherichia coli, the Bam complex also contains three nonessential lipoproteins (BamBCE), all of which require the barrel-proximal POTRA domain (P5) for stable interactions with BamA. We have previously reported that the BamA β-barrel assumes two different conformations. A method for conformation-specific labeling of BamA described here reveals that these conformers reflect the degree of surface exposure of the conserved sixth extracellular loop (L6). L6 is surface accessible in one conformation but not in the other, likely because it occupies the lumen of the BamA β-barrel in the latter case. A gain-of-function mutation that promotes Bam activity (bamDR197L) and a loss-of-function mutation that decreases the activity of Bam (ΔbamE) both favor surface exposure of BamA L6, suggesting that BamD and BamE normally act to control L6 exposure through opposing functions. These results, along with the synthetic lethality of the bamDR197L ΔbamE double mutant, imply a cyclic mechanism in which the Bam lipoproteins regulate the conformation of BamA during the OMP assembly reaction. Our results further suggest that BamDE controls L6 exposure via conformational signals transmitted through P5 to L6.genetics | OM biogenesis | Omp85 | membrane protein folding |

Biogenesis of the outer membrane (OM) is an essential process in Gram-negative bacteria. One of the key steps of OM biogenesis is the assembly of integral outer membrane beta-barrel proteins (OMPs) by a protein machine called the Bam complex. In Escherichia coli, the Bam complex is composed of the essential proteins BamA and BamD and three nonessential lipoproteins, BamB, BamC, and BamE. Both BamC and BamE are important for stabilizing the interaction between BamA and BamD. We used comprehensive genetic analysis to clarify the interplay between BamA and the BamCDE subcomplex. Combining a ⌬bamE allele with mutations in genes that encode other OMP assembly factors leads to severe synthetic phenotypes, suggesting a critical function for BamE. These synthetic phenotypes are not nearly as severe in a ⌬bamC background, suggesting that the functions of BamC and BamE are not completely overlapping. This unique function of BamE is related to the conformational state of BamA. In wild-type cells, BamA is sensitive to externally added proteinase K. Strikingly, when ⌬bamE mutant cells are treated with proteinase K, BamA is degraded beyond detection. Taken together, our findings suggest that BamE modulates the conformation of BamA, likely through its interactions with BamD.

The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat

In bacteria, the majority of exported proteins are transported by the general Sec pathway from their site of synthesis in the cytoplasm across the cytoplasmic membrane. The essential SecA ATPase powers this Sec-mediated export. Mycobacteria possess two nonredundant SecA homologs: SecA1 and SecA2. In pathogenic Mycobacterium tuberculosis and the nonpathogenic model mycobacterium Mycobacterium smegmatis, SecA1 is essential for protein export and is the "housekeeping" SecA, whereas SecA2 is an accessory SecA that exports a specific subset of proteins. In M. tuberculosis the accessory SecA2 pathway plays a role in virulence. In this study, we uncovered basic properties of the mycobacterial SecA2 protein and its pathway for exporting select proteins. By constructing secA2 mutant alleles that encode proteins defective in ATP binding, we showed that ATP binding is required for SecA2 function. SecA2 mutant proteins unable to bind ATP were nonfunctional and dominant negative. By evaluating the subcellular distribution of each SecA, SecA1 was shown to be equally divided between cytosolic and cell envelope fractions, whereas SecA2 was predominantly localized to the cytosol. Finally, we showed that the canonical SecA1 has a role in the process of SecA2-dependent export. The accessory SecA2 export system is important to the physiology and virulence of mycobacteria. These studies help establish the mechanism of this new type of specialized protein export pathway.

SummaryThe export of proteins from their site of synthesis in the cytoplasm across the inner membrane is an important aspect of bacterial physiology. Because the location of extracytoplasmic proteins is ideal for host-pathogen interactions, protein export is also important to bacterial virulence. In bacteria, there are conserved protein export systems that are responsible for the majority of protein export: the general secretion (Sec) pathway and the twin-arginine translocation pathway. In some bacteria, there are also specialized export systems dedicated to exporting specific subsets of proteins. In this review, we discuss a specialized export system that exists in some Gram-positive bacteria and mycobacteria -the accessory Sec system. The common element to the accessory Sec system is an accessory SecA protein called SecA2. Here we present our current understanding of accessory Sec systems in Streptococcus gordonii, Streptococcus parasanguinis, Mycobacterium smegmatis, Mycobacterium tuberculosis and Listeria monocytogenes, making an effort to highlight apparent similarities and differences between the systems. We also review the data showing that accessory Sec systems can contribute to bacterial virulence.