The TAM, a Translocation and Assembly Module for protein assembly and potential conduit for phospholipid transfer.

The assembly of ß-barrel proteins into the bacterial outer membrane is an essential process enabling the colonization of new environmental niches. The TAM was discovered as a module of the ß-barrel protein assembly machinery; it is a heterodimeric complex composed of an outer membrane protein (TamA) bound to an inner membrane protein (TamB). The TAM spans the periplasm, providing a scaffold through the peptidoglycan layer and catalyzing the translocation and assembly of ß-barrel proteins into the outer membrane. Recently, studies on another membrane protein (YhdP) have suggested that TamB might play a role in phospholipid transport to the outer membrane. Here we review and re-evaluate the literature covering the experimental studies on the TAM over the past decade, to reconcile what appear to be conflicting claims on the function of the TAM.

Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa.

The outer membrane (OM) is an essential structure of Gram-negative bacteria that provides mechanical strength and protection from large and/or hydrophobic toxic molecules, including many antibiotics. The OM is composed of glycerophospholipids (GPLs) and lipopolysaccharide (LPS) in the inner and outer leaflets, respectively, and hosts integral ß-barrel proteins and lipoproteins. While the systems responsible for translocation and insertion of LPS and OM proteins have been elucidated, the mechanism(s) mediating transport of GPLs from the inner membrane to the OM has remained elusive for decades. Very recently, studies performed in Escherichia coli proposed a role in this process for AsmA-like proteins that are predicted to share structural features with eukaryotic lipid transporters. In this study, we provide the first systematic investigation of AsmA-like proteins in a bacterium other than E. coli, the opportunistic human pathogen Pseudomonas aeruginosa. Bioinformatic analyses revealed that P. aeruginosa possesses seven AsmA-like proteins. Deletion of asmA-like genes in many different combinations, coupled with conditional mutagenesis, revealed that four AsmA-like proteins are redundantly essential for growth and OM integrity in P. aeruginosa, including a novel AsmA-like protein (PA4735) that is not present in E. coli. Cells depleted of AsmA-like proteins showed severe defects in the OM permeability barrier that were partially rescued by lowering the synthesis or transport of LPS. Since fine balancing of GPL and LPS levels is crucial for OM integrity, this evidence supports the role of AsmA-like proteins in GPL transport toward the OM. IMPORTANCE: Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism Escherichia coli and demonstrate that the human pathogen Pseudomonas aeruginosa has an additional essential AsmA-like protein that is not present in E. coli, thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria.

Bacterial AsmA-Like Proteins: Bridging the Gap in Intermembrane Phospholipid Transport.

In eukaryotic cells, nonvesicular lipid transport between organelles is mediated by lipid-transfer proteins. Recently, a new class of these lipid transporters has been described to facilitate the bulk of inter-organelle lipid transport at contact sites by forming bridge-like structures with a hydrophobic groove through which lipids travel. Because their predicted structure is composed of repeating ß-groove (RBG) domains, they have been named the RBG protein superfamily. Early studies on RBG proteins VPS13 and ATG2 recognized the resemblance of their predicted structures to that of the bacterial Lpt system, which transports newly synthesized lipopolysaccharides (LPS) between the inner and the outer membranes (IMs and OMs) of Gram-negative bacteria. In these didermic bacteria, the IMs and OMs are separated by an aqueous periplasmic compartment that is traversed by a bridge-like structure built with ß-jelly roll domains from several Lpt proteins that provides a hydrophobic groove for LPS molecules to travel across the periplasm. Despite structural and functional similarities between RBG proteins and the Lpt system, the bacterial AsmA-like protein family has recently emerged as the likely ancestor of RBG proteins and long sought-after transporters that facilitate the transfer of phospholipids from the IM to the OM. Here, we review our current understanding of the structure and function of bacterial AsmA-like proteins, mainly focusing on recent studies that have led to the proposal that AsmA-like proteins mediate the bulk of phospholipid transfer between the IMs and OMs.

Remote homology searches identify bacterial homologues of eukaryotic lipid transfer proteins, including Chorein-N domains in TamB and AsmA and Mdm31p.

BACKGROUND: All cells rely on lipids for key functions. Lipid transfer proteins allow lipids to exit the hydrophobic environment of bilayers, and cross aqueous spaces. One lipid transfer domain fold present in almost all eukaryotes is the TUbular LIPid binding (TULIP) domain. Three TULIP families have been identified in bacteria (P47, OrfX2 and YceB), but their homology to eukaryotic proteins is too low to specify a common origin. Another recently described eukaryotic lipid transfer domain in VPS13 and ATG2 is Chorein-N, which has no known bacterial homologues. There has been no systematic search for bacterial TULIPs or Chorein-N domains. RESULTS: Remote homology predictions for bacterial TULIP domains using HHsearch identified four new TULIP domains in three bacterial families. DUF4403 is a full length pseudo-dimeric TULIP with a 6 strand ß-meander dimer interface like eukaryotic TULIPs. A similar sheet is also present in YceB, suggesting it homo-dimerizes. TULIP domains were also found in DUF2140 and in the C-terminus DUF2993. Remote homology predictions for bacterial Chorein-N domains identified strong hits in the N-termini of AsmA and TamB in diderm bacteria, which are related to Mdm31p in eukaryotic mitochondria. The N-terminus of DUF2993 has a Chorein-N domain adjacent to its TULIP domain. CONCLUSIONS: TULIP lipid transfer domains are widespread in bacteria. Chorein-N domains are also found in bacteria, at the N-terminus of multiple proteins in the intermembrane space of diderms (AsmA, TamB and their relatives) and in Mdm31p, a protein that is likely to have evolved from an AsmA/TamB-like protein in the endosymbiotic mitochondrial ancestor. This indicates that both TULIP and Chorein-N lipid transfer domains may have originated in bacteria.

The Structure of a Conserved Domain of TamB Reveals a Hydrophobic ß Taco Fold.

The translocation and assembly module (TAM) plays a role in the transport and insertion of proteins into the bacterial outer membrane. TamB, a component of this system spans the periplasmic space to engage with its partner protein TamA. Despite efforts to characterize the TAM, the structure and mechanism of action of TamB remained enigmatic. Here we present the crystal structure of TamB amino acids 963-1,138. This region represents half of the conserved DUF490 domain, the defining feature of TamB. TamB963-1138 consists of a concave, taco-shaped ß sheet with a hydrophobic interior. This ß taco structure is of dimensions capable of accommodating and shielding the hydrophobic side of an amphipathic ß strand, potentially allowing TamB to chaperone nascent membrane proteins from the aqueous environment. In addition, sequence analysis suggests that the structure of TamB963-1138 is shared by a large portion of TamB. This architecture could allow TamB to act as a conduit for membrane proteins.

The conserved carboxyl domain of MorC, an inner membrane protein of Aggregatibacter actinomycetemcomitans, is essential for membrane function.

Morphogenesis protein C (MorC) of Aggregatibacter actinomycetemcomitans is important for maintaining the membrane morphology and integrity of the cell envelope of this oral pathogen. The MorC sequence and operon organization were found to be conserved in Gammaproteobacteria, based on a bioinformatic analysis of 435 sequences from representative organisms. Functional conservation of MorC was investigated using an A. actinomycetemcomitans morC mutant as a model system to express MorC homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. The A. actinomycetemcomitans strains expressing the homologous proteins were assessed for sensitivity to bile salts, leukotoxin secretion, autoaggregation and membrane morphology. MorC from the most closely related organism (H. influenzae) was functionally identical to MorC from A. actinomycetemcomitans. However, the genes from more distantly related organisms restored some but not all A. actinomycetemcomitans mutant phenotypes. In addition, deletion mutagenesis indicated that the most conserved portion of the protein, the C-terminus DUF490 domain, was necessary to maintain the integrity of the membrane. Deletion of the last 10 amino acids of this domain of the A. actinomycetemcomitans MorC protein was sufficient to disrupt membrane stability and leukotoxin secretion. The data suggest that the MorC sequence is functionally conserved across Gammaproteobacteria and the C-terminus of the protein is essential for maintaining membrane physiology.

Evolution of the Translocation and Assembly Module (TAM).

Bacterial outer membrane proteins require the beta-barrel assembly machinery (BAM) for their correct folding and function. The central component of this machinery is BamA, an Omp85 protein that is essential and found in all Gram-negative bacteria. An additional feature of the BAM is the translocation and assembly module (TAM), comprised TamA (an Omp85 family protein) and TamB. We report that TamA and a closely related protein TamL are confined almost exclusively to Proteobacteria and Bacteroidetes/Chlorobi respectively, whereas TamB is widely distributed across the majority of Gram-negative bacterial lineages. A comprehensive phylogenetic and secondary structure analysis of the TamB protein family revealed that TamB was present very early in the evolution of bacteria. Several sequence characteristics were discovered to define the TamB protein family: A signal-anchor linkage to the inner membrane, beta-helical structure, conserved domain architecture and a C-terminal region that mimics outer membrane protein beta-strands. Taken together, the structural and phylogenetic analyses suggest that the TAM likely evolved from an original combination of BamA and TamB, with a later gene duplication event of BamA, giving rise to an additional Omp85 sequence that evolved to be TamA in Proteobacteria and TamL in Bacteroidetes/Chlorobi.

Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490(963-1138) domain of TamB from Escherichia coli.

TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963-1138 domain of TamB was overexpressed in Escherichia coli K-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space group P3121, with unit-cell parameters a = b = 57.34, c = 220.74 Å, and diffracted to 2.1 Šresolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future.