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.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'ß-signal' imprinted in the final ß-strand of the OMP engages the ß-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the ß-signal are repeated in other, internal ß-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the ß-signal, arranging several ß-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in Klebsiella spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of Klebsiella quasipneumoniae. A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes. Genetic reconstruction of the resistance phenotype confirmed that two distinct genetic loci are necessary in order for the strain to acquire carbapenem resistance. Experimental evolution of the carbapenem-resistant strains in growth conditions without the antibiotic revealed that both loci confer a significant cost and are readily lost by de novo mutations resulting in the rapid evolution of a carbapenem-sensitive phenotype. To explain how carbapenem resistance evolves via multiple, low-fitness single-locus intermediates, we hypothesised that one of these loci had previously conferred adaptation to another antibiotic. Fitness assays in a range of drug concentrations show how selection in the antibiotic ceftazidime can select for one gene (blaDHA-1) potentiating the evolution of carbapenem resistance by a single mutation in a second gene (ompK36). These results show how a patient's treatment history might shape the evolution of antibiotic resistance and could explain the genetic basis of carbapenem-resistance found in many enteric-pathogens.

Surveying membrane landscapes: a new look at the bacterial cell surface.

Recent studies applying advanced imaging techniques are changing the way we understand bacterial cell surfaces, bringing new knowledge on everything from single-cell heterogeneity in bacterial populations to their drug sensitivity and mechanisms of antimicrobial resistance. In both Gram-positive and Gram-negative bacteria, the outermost surface of the bacterial cell is being imaged at nanoscale; as a result, topographical maps of bacterial cell surfaces can be constructed, revealing distinct zones and specific features that might uniquely identify each cell in a population. Functionally defined assembly precincts for protein insertion into the membrane have been mapped at nanoscale, and equivalent lipid-assembly precincts are suggested from discrete lipopolysaccharide patches. As we review here, particularly for Gram-negative bacteria, the applications of various modalities of nanoscale imaging are reawakening our curiosity about what is conceptually a 3D cell surface landscape: what it looks like, how it is made and how it provides resilience to respond to environmental impacts.

A Structurally Characterized Staphylococcus aureus Evolutionary Escape Route from Treatment with the Antibiotic Linezolid.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance. IMPORTANCE Antibiotic resistance poses a critical problem to human health and decreases the utility of these lifesaving drugs. Of particular concern is the "superbug" methicillin-resistant Staphylococcus aureus (MRSA), for which treatment of infection requires the use of last-line antibiotics, including linezolid. In this paper, we characterize the atomic rearrangements which the ribosome, the target of linezolid, undergoes during its evolutionary journey toward becoming drug resistant. Using cryo-electron microscopy, we describe a particular molecular mechanism which MRSA uses to become resistant to linezolid.

A noncanonical chaperone interacts with drug efflux pumps during their assembly into bacterial outer membranes.

Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised. Complex phylogenetics support the concept that gene cassettes encoding the subunits for these pumps are commonly acquired by horizontal gene transfer. Using TolC as a model protein, we demonstrate that assembly of conduit subunits into the outer membrane uses the chaperone TAM to physically organise the membrane-embedded staves of the conduit subunit of the efflux pump. The characteristics of this assembly pathway have impact for the acquisition of efflux pumps across bacterial species and for the development of new antimicrobial compounds that inhibit efflux pump function.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

The Carbapenemase BKC-1 from Klebsiella pneumoniae Is Adapted for Translocation by Both the Tat and Sec Translocons.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding the periplasm and peptidoglycan layer. ß-Lactam antibiotics target the periplasmic penicillin-binding proteins that synthesize peptidoglycan, resulting in cell death. The primary means by which bacterial species resist the effects of ß-lactam drugs is to populate the periplasmic space with ß-lactamases. Resistance to ß-lactam drugs is spread by lateral transfer of genes encoding ß-lactamases from one species of bacteria to another. However, the resistance phenotype depends in turn on these "alien" protein sequences being recognized and exported across the cytoplasmic membrane by either the Sec or Tat protein translocation machinery of the new bacterial host. Here, we examine BKC-1, a carbapenemase from an unknown bacterial source that has been identified in a single clinical isolate of Klebsiella pneumoniae. BKC-1 was shown to be located in the periplasm, and functional in both K. pneumoniae and Escherichia coli. Sequence analysis revealed the presence of an unusual signal peptide with a twin arginine motif and a duplicated hydrophobic region. Biochemical assays showed this signal peptide directs BKC-1 for translocation by both Sec and Tat translocons. This is one of the few descriptions of a periplasmic protein that is functionally translocated by both export pathways in the same organism, and we suggest it represents a snapshot of evolution for a ß-lactamase adapting to functionality in a new host. IMPORTANCE Bacteria can readily acquire plasmids via lateral gene transfer (LGT). These plasmids can carry genes for virulence and antimicrobial resistance (AMR). Of growing concern are LGT events that spread ß-lactamases, particularly carbapenemases, and it is important to understand what limits this spread. This study provides insight into the sequence features of BKC-1 that exemplify the limitations on the successful biogenesis of ß-lactamases, which is one factor limiting the spread of AMR phenotypes by LGT. With a very simple evolutionary adaptation, BKC-1 could become a more effective carbapenemase, underscoring the need to understand the evolution, adaptability, and functional assessment of newly reported ß-lactamases rapidly and thoroughly.

Component Parts of Bacteriophage Virions Accurately Defined by a Machine-Learning Approach Built on Evolutionary Features.

Antimicrobial resistance (AMR) continues to evolve as a major threat to human health, and new strategies are required for the treatment of AMR infections. Bacteriophages (phages) that kill bacterial pathogens are being identified for use in phage therapies, with the intention to apply these bactericidal viruses directly into the infection sites in bespoke phage cocktails. Despite the great unsampled phage diversity for this purpose, an issue hampering the roll out of phage therapy is the poor quality annotation of many of the phage genomes, particularly for those from infrequently sampled environmental sources. We developed a computational tool called STEP3 to use the "evolutionary features" that can be recognized in genome sequences of diverse phages. These features, when integrated into an ensemble framework, achieved a stable and robust prediction performance when benchmarked against other prediction tools using phages from diverse sources. Validation of the prediction accuracy of STEP3 was conducted with high-resolution mass spectrometry analysis of two novel phages, isolated from a watercourse in the Southern Hemisphere. STEP3 provides a robust computational approach to distinguish specific and universal features in phages to improve the quality of phage cocktails and is available for use at http://step3.erc.monash.edu/. IMPORTANCE In response to the global problem of antimicrobial resistance, there are moves to use bacteriophages (phages) as therapeutic agents. Selecting which phages will be effective therapeutics relies on interpreting features contributing to shelf-life and applicability to diagnosed infections. However, the protein components of the phage virions that dictate these properties vary so much in sequence that best estimates suggest failure to recognize up to 90% of them. We have utilized this diversity in evolutionary features as an advantage, to apply machine learning for prediction accuracy for diverse components in phage virions. We benchmark this new tool showing the accurate recognition and evaluation of phage component parts using genome sequence data of phages from undersampled environments, where the richest diversity of phage still lies.

The TAM: A Translocation and Assembly Module of the ß-Barrel Assembly Machinery in Bacterial Outer Membranes.

Assembly of proteins into the outer membrane is an essential process in the cell biology of bacteria. The integration of ß-barrel proteins into the outer membrane is mediated by a system referred to as the ß-barrel assembly machinery (BAM) that includes two related proteins: BamA in the BAM complex and TamA in the TAM (translocation and assembly module). Here we review what is known about the TAM in terms of its function and the structural architecture of its two subunits, TamA and TamB. By linking the energy transduction possibilities in the inner membrane to TamA in the outer membrane, the TAM provides additional capability to the ß-barrel assembly machinery. Conservation of the TAM across evolutionary boundaries, and the presence of hybrid BAM/TAM complexes in some bacterial lineages, adds insight to our growing understanding of how bacterial outer membranes are built.

An investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis.

Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK. Here, we show that bamK is a conserved element in the core genome of Klebsiella, and its expression rescues a loss-of-function ∆bamA mutant. We developed an E. coli model system to measure and compare the specific activity of BamA and BamK in the assembly reaction for the critical substrate LptD, and find that BamK is as efficient as BamA in assembling the native LptDE complex. Comparative structural analysis revealed that the major distinction between BamK and BamA is in the external facing surface of the protein, and we discuss how such changes may contribute to a mechanism for resistance against infection by bacteriophage.

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane.

The ß-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.

Constraints on lateral gene transfer in promoting fimbrial usher protein diversity and function.

Fimbriae are long, adhesive structures widespread throughout members of the family Enterobacteriaceae. They are multimeric extrusions, which are moved out of the bacterial cell through an integral outer membrane protein called usher. The complex folding mechanics of the usher protein were recently revealed to be catalysed by the membrane-embedded translocation and assembly module (TAM). Here, we examine the diversity of usher proteins across a wide range of extraintestinal (ExPEC) and enteropathogenic (EPEC) Escherichia coli, and further focus on a so far undescribed chaperone-usher system, with this usher referred to as UshC. The fimbrial system containing UshC is distributed across a discrete set of EPEC types, including model strains like E2348/67, as well as ExPEC ST131, currently the most prominent multi-drug-resistant uropathogenic E. coli strain worldwide. Deletion of the TAM from a naive strain of E. coli results in a drastic time delay in folding of UshC, which can be observed for a protein from EPEC as well as for two introduced proteins from related organisms, Yersinia and Enterobacter We suggest that this models why the TAM machinery is essential for efficient folding of proteins acquired via lateral gene transfer.

Structural basis for substrate selection by the translocation and assembly module of the ß-barrel assembly machinery.

The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the ß-barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex. A peptide scan revealed that TamA and BamA bind the ß-strands of FimD, and do so selectively. Chemical cross-linking and molecular dynamics are consistent with this interaction taking place between the first and last strand of the TamA barrel domain, providing the first experimental evidence of a lateral gate in TamA: a structural element implicated in membrane protein assembly. We suggest that the lateral gates in TamA and BamA provide different environments for substrates to engage, with the differences observed here beginning to address how the TAM can be more effective than the BAM complex in the folding of some substrate proteins.

Conserved Features in the Structure, Mechanism, and Biogenesis of the Inverse Autotransporter Protein Family.

The bacterial cell surface proteins intimin and invasin are virulence factors that share a common domain structure and bind selectively to host cell receptors in the course of bacterial pathogenesis. The ß-barrel domains of intimin and invasin show significant sequence and structural similarities. Conversely, a variety of proteins with sometimes limited sequence similarity have also been annotated as "intimin-like" and "invasin" in genome datasets, while other recent work on apparently unrelated virulence-associated proteins ultimately revealed similarities to intimin and invasin. Here we characterize the sequence and structural relationships across this complex protein family. Surprisingly, intimins and invasins represent a very small minority of the sequence diversity in what has been previously the "intimin/invasin protein family". Analysis of the assembly pathway for expression of the classic intimin, EaeA, and a characteristic example of the most prevalent members of the group, FdeC, revealed a dependence on the translocation and assembly module as a common feature for both these proteins. While the majority of the sequences in the grouping are most similar to FdeC, a further and widespread group is two-partner secretion systems that use the ß-barrel domain as the delivery device for secretion of a variety of virulence factors. This comprehensive analysis supports the adoption of the "inverse autotransporter protein family" as the most accurate nomenclature for the family and, in turn, has important consequences for our overall understanding of the Type V secretion systems of bacterial pathogens.