Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen.

Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance.

Response regulator PorX coordinates oligonucleotide signalling and gene expression to control the secretion of virulence factors.

The PglZ family of proteins belongs to the alkaline phosphatase superfamily, which consists of metallohydrolases with limited sequence identity but similar metal-coordination architectures in otherwise divergent active sites. Proteins with a well-defined PglZ domain are ubiquitous among prokaryotes as essential components of BREX phage defence systems and two-component systems (TCSs). Whereas other members of the alkaline phosphatase superfamily are well characterized, the activity, structure and biological function of PglZ family proteins remain unclear. We therefore investigated the structure and function of PorX, an orphan response regulator of the Porphyromonas gingivalis TCS containing a putative PglZ effector domain. The crystal structure of PorX revealed a canonical receiver domain, a helical bundle, and an unprecedented PglZ domain, similar to the general organization of the phylogenetically related BREX-PglZ proteins. The PglZ domain of PorX features an active site cleft suitable for large substrates. An extensive search for substrates revealed that PorX is a phosphodiesterase that acts on cyclic and linear oligonucleotides, including signalling molecules such as cyclic oligoadenylates. These results, combined with mutagenesis, biophysical and enzymatic analysis, suggest that PorX coordinates oligonucleotide signalling pathways and indirectly regulates gene expression to control the secretion of virulence factors.

Purification, crystallization and crystallographic analysis of the PorX response regulator associated with the type IX secretion system.

Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane(s) and manipulate their infected host. To date, 11 secretion systems have been identified, including the type IX secretion system (T9SS) associated with human, avian and farmed-fish diseases. As a bacterial secretion system, the T9SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria. PorX is a highly conserved protein that regulates the transcription of essential T9SS components and additionally mediates the function of T9SS via direct interaction with PorL, the rotary motor protein of the T9SS. PorX is also a member of a two-component system regulatory cascade, where it serves as the response regulator that relays a signal transduced from a conserved sensor histidine kinase, PorY, to a designated sigma factor. Here, the recombinant expression and purification of PorX homologous proteins from the pathogenic bacterium Porphyromonas gingivalis and the nonpathogenic bacterium Flavobacterium johnsoniae are reported. A bioinformatical characterization of the different domains comprising the PorX protein is also provided, and the crystallization and X-ray analysis of PorX from F. johnsoniae are reported.

From conservation to structure, studies of magnetosome associated cation diffusion facilitators (CDF) proteins in Proteobacteria.

Magnetotactic bacteria (MTB) are prokaryotes that sense the geomagnetic field lines to geolocate and navigate in aquatic sediments. They are polyphyletically distributed in several bacterial divisions but are mainly represented in the Proteobacteria. In this phylum, magnetotactic Deltaproteobacteria represent the most ancestral class of MTB. Like all MTB, they synthesize membrane-enclosed magnetic nanoparticles, called magnetosomes, for magnetic sensing. Magnetosome biogenesis is a complex process involving a specific set of genes that are conserved across MTB. Two of the most conserved genes are mamB and mamM, that encode for the magnetosome-associated proteins and are homologous to the cation diffusion facilitator (CDF) protein family. In magnetotactic Alphaproteobacteria MTB species, MamB and MamM proteins have been well characterized and play a central role in iron-transport required for biomineralization. However, their structural conservation and their role in more ancestral groups of MTB like the Deltaproteobacteria have not been established. Here we studied magnetite cluster MamB and MamM cytosolic C-terminal domain (CTD) structures from a phylogenetically distant magnetotactic Deltaproteobacteria species represented by BW-1 strain, which has the unique ability to biomineralize magnetite and greigite. We characterized them in solution, analyzed their crystal structures and compared them to those characterized in Alphaproteobacteria MTB species. We showed that despite the high phylogenetic distance, MamBBW-1 and MamMBW-1 CTDs share high structural similarity with known CDF-CTDs and will probably share a common function with the Alphaproteobacteria MamB and MamM.

A Hybrid Secretion System Facilitates Bacterial Sporulation: A Structural Perspective.

Bacteria employ a number of dedicated secretion systems to export proteins to the extracellular environment. Several of these comprise large complexes that assemble in and around the bacterial membrane(s) to form specialized channels through which only selected proteins are actively delivered. Although typically associated with bacterial pathogenicity, a specialized variant of these secretion systems has been proposed to play a central part in bacterial sporulation, a primitive protective process that allows starving cells to form spores that survive in extreme environments. Following asymmetric division, the mother cell engulfs the forespore, leaving it surrounded by two bilayer membranes. During the engulfment process an essential channel apparatus is thought to cross both membranes to create a direct conduit between the mother cell and forespore. At least nine proteins are essential for channel formation, including SpoIIQ under forespore control and the eight SpoIIIA proteins (SpoIIIAA to -AH) under mother cell control. Presumed to form a core channel complex, several of these proteins share similarity with components of Gram-negative bacterial secretion systems, including the type II, III, and IV secretion systems and the flagellum. Based on these similarities it has been suggested that the sporulation channel represents a hybrid, secretion-like transport machinery. Recently, in-depth biochemical and structural characterization of the individual channel components accompanied by in vivo studies has further reinforced this model. Here we review and discuss these recent studies and suggest an updated model for the unique sporulation channel apparatus architecture.

A quantitative assessment of the membrane-integral sub-proteome of a bacterial magnetic organelle.

Magnetotactic bacteria produce chains of complex membrane-bound organelles that direct the biomineralization of magnetic nanoparticles and serve for magnetic field navigation. These magnetosome compartments have recently emerged as a model for studying the subcellular organization of prokaryotic organelles. Previous studies indicated the presence of specific proteins with various functions in magnetosome biosynthesis. However, the exact composition and stoichiometry of the magnetosome subproteome have remained unknown. In order to quantify and unambiguously identify all proteins specifically targeted to the magnetosome membrane of the Alphaproteobacterium Magnetospirillum gryphiswaldense, we analyzed the protein composition of several cellular fractions by semi-quantitative mass spectrometry. We found that nearly all genuine magnetosome membrane-integral proteins belong to a well-defined set of previously identified proteins encoded by gene clusters within a genomic island, indicating a highly controlled protein composition. Magnetosome proteins were present in different quantities with up to 120 copies per particle as estimated by correlating our results with available quantitative Western blot data. This high abundance suggests an unusually crowded protein composition of the membrane and a tight packing with transmembrane domains of integral proteins. Our findings will help to further define the structure of the organelle and contribute to the elucidation of magnetosome biogenesis. BIOLOGICAL SIGNIFICANCE: Magnetosomes are one of the most complex bacterial organelles and consist of membrane-bounded crystals of magnetic minerals. The exact composition and stoichiometry of the associated membrane integral proteins are of major interest for a deeper understanding of prokaryotic organelle assembly; however, previous proteomic studies failed to reveal meaningful estimations due to the lack of precise and quantitative data, and the inherently high degree of accumulated protein contaminants in purified magnetosomes. Using a highly sensitive mass spectrometer, we acquired proteomic data from several cellular fractions of a magnetosome producing magnetotactic bacterium and developed a comparative algorithm to identify all genuine magnetosome membrane-integral proteins and to discriminate them from contaminants. Furthermore, by combining our data with previously published quantitative Western blot data, we were able to model the protein copy number and density within the magnetosome membrane. Our results suggest that the magnetosome membrane is specifically associated with a small subset of integral proteins that are tightly packed within the lipid layer. Our study provides by far the most comprehensive estimation of magnetosomal protein composition and stoichiometry and will help to elucidate the complex process of magnetosome biogenesis.

The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization.

Magnetospirillum gryphiswaldense MSR-1 synthesizes membrane-enclosed magnetite (Fe3 O4 ) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome-associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome-directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi-disciplinary approach to define the role of MamB during magnetosome formation. Using site-directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo-electron tomography, we show that MamB is most likely an active magnetosome-directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport-independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C-terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation.

Near-atomic resolution cryoelectron microscopy structure of the 30-fold homooligomeric SpoIIIAG channel essential to spore formation in Bacillus subtilis.

Bacterial sporulation allows starving cells to differentiate into metabolically dormant spores that can survive extreme conditions. Following asymmetric division, the mother cell engulfs the forespore, surrounding it with two bilayer membranes. During the engulfment process, an essential channel, the so-called feeding tube apparatus, is thought to cross both membranes to create a direct conduit between the mother cell and the forespore. At least nine proteins are required to create this channel, including SpoIIQ and SpoIIIAA-AH. Here, we present the near-atomic resolution structure of one of these proteins, SpoIIIAG, determined by single-particle cryo-EM. A 3D reconstruction revealed that SpoIIIAG assembles into a large and stable 30-fold symmetric complex with a unique mushroom-like architecture. The complex is collectively composed of three distinctive circular structures: a 60-stranded vertical ß-barrel that forms a large inner channel encircled by two concentric rings, one ß-mediated and the other formed by repeats of a ring-building motif (RBM) common to the architecture of various dual membrane secretion systems of distinct function. Our near-atomic resolution structure clearly shows that SpoIIIAG exhibits a unique and dramatic adaptation of the RBM fold with a unique ß-triangle insertion that assembles into the prominent channel, the dimensions of which suggest the potential passage of large macromolecules between the mother cell and forespore during the feeding process. Indeed, mutation of residues located at key interfaces between monomers of this RBM resulted in severe defects both in vivo and in vitro, providing additional support for this unprecedented structure.

Correction: MamA as a Model Protein for Structure-Based Insight into the Evolutionary Origins of Magnetotactic Bacteria.

[This corrects the article DOI: 10.1371/journal.pone.0130394.].

MamA as a Model Protein for Structure-Based Insight into the Evolutionary Origins of Magnetotactic Bacteria.

MamA is a highly conserved protein found in magnetotactic bacteria (MTB), a diverse group of prokaryotes capable of navigating according to magnetic fields - an ability known as magnetotaxis. Questions surround the acquisition of this magnetic navigation ability; namely, whether it arose through horizontal or vertical gene transfer. Though its exact function is unknown, MamA surrounds the magnetosome, the magnetic organelle embedding a biomineralised nanoparticle and responsible for magnetotaxis. Several structures for MamA from a variety of species have been determined and show a high degree of structural similarity. By determining the structure of MamA from Desulfovibrio magneticus RS-1 using X-ray crystallography, we have opened up the structure-sequence landscape. As such, this allows us to perform structural- and phylogenetic-based analyses using a variety of previously determined MamA from a diverse range of MTB species across various phylogenetic groups. We found that MamA has remained remarkably constant throughout evolution with minimal change between different taxa despite sequence variations. These findings, coupled with the generation of phylogenetic trees using both amino acid sequences and 16S rRNA, indicate that magnetotaxis likely did not spread via horizontal gene transfer and instead has a significantly earlier, primordial origin.

Bacterial magnetosome biomineralization--a novel platform to study molecular mechanisms of human CDF-related Type-II diabetes.

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all organisms. CDFs were found to be involved in numerous human health conditions, such as Type-II diabetes and neurodegenerative diseases. In this work, we established the magnetite biomineralizing alphaproteobacterium Magnetospirillum gryphiswaldense as an effective model system to study CDF-related Type-II diabetes. Here, we introduced two ZnT-8 Type-II diabetes-related mutations into the M. gryphiswaldense MamM protein, a magnetosome-associated CDF transporter essential for magnetite biomineralization within magnetosome vesicles. The mutations' effects on magnetite biomineralization and iron transport within magnetosome vesicles were tested in vivo. Additionally, by combining several in vitro and in silico methodologies we provide new mechanistic insights for ZnT-8 polymorphism at position 325, located at a crucial dimerization site important for CDF regulation and activation. Overall, by following differentiated, easily measurable, magnetism-related phenotypes we can utilize magnetotactic bacteria for future research of CDF-related human diseases.

Cation diffusion facilitators transport initiation and regulation is mediated by cation induced conformational changes of the cytoplasmic domain.

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all domains of life. CDF's were shown to be involved in several human diseases, such as Type-II diabetes and neurodegenerative diseases. In this work, we employed a multi-disciplinary approach to study the activation mechanism of the CDF protein family. For this we used MamM, one of the main ion transporters of magnetosomes--bacterial organelles that enable magnetotactic bacteria to orientate along geomagnetic fields. Our results reveal that the cytosolic domain of MamM forms a stable dimer that undergoes distinct conformational changes upon divalent cation binding. MamM conformational change is associated with three metal binding sites that were identified and characterized. Altogether, our results provide a novel auto-regulation mode of action model in which the cytosolic domain's conformational changes upon ligand binding allows the priming of the CDF into its transport mode.

Crystallization and preliminary crystallographic analysis of the C-terminal domain of MamM, a magnetosome-associated protein from Magnetospirillum gryphiswaldense MSR-1.

MamM is a unique magnetosome-associated protein that shares substantial homology with cation diffusion facilitator (CDF) proteins, a group of heavy-metal-ion efflux transporters that participate in metal-ion homeostasis in all domains of life. Magnetotactic bacteria utilize CDF proteins in iron-oxide biomineralization and in magnetosome formation. Here, the crystallization and preliminary X-ray analysis of recombinant Magnetospirillum gryphiswaldense MamM is reported. The C-terminal domain of MamM was crystallized in the orthorhombic space group C222(1), with unit-cell parameters a = 37.1, b = 94.0, c = 53.3 Å. X-ray diffraction data were collected to a resolution of 2.0 Å.

Inter-phylum structural conservation of the magnetosome-associated TPR-containing protein, MamA.

Magnetotactic bacteria enclose the magnetosome, a unique prokaryotic sub-cellular organelle that allows the biomineralization of magnetic nano-crystals. Membrane-coated magnetosomes are arranged into a linear chain that permits magnetotactic bacteria to navigate geomagnetic fields. Magnetosome assembly and biomineralization are controlled by conserved magnetosome-associated proteins, including MamA, a tetra-trico-peptide repeat (TPR)-containing protein that was shown to coat the magnetosome membrane. In this study, two MamA structures from Candidatus Magnetobacterium bavaricum (Mbav) were determined via X-ray crystallography. These structures confirm that Mbav MamA folds as a sequential TPR protein and shares a high degree of structural similarity with homologous MamA proteins from Magnetospirillum species. Furthermore, the two TPR-containing domains of MamA are separated by an interphylum-conserved region containing a flexible hinge that is involved in ligand binding and recognition. Finally, substantial differences were found in the local stabilization of the MamA N-terminal domain as a result of the loss of an evolutionary conserved salt bridge.

Structural and functional discussion of the tetra-trico-peptide repeat, a protein interaction module.

Tetra-trico-peptide repeat (TPR) domains are found in numerous proteins, where they serve as interaction modules and multiprotein complex mediators. TPRs can be found in all kingdoms of life and regulate diverse biological processes, such as organelle targeting and protein import, vesicle fusion, and biomineralization. This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure. In addition, tools for predicting TPR-interacting partners are discussed, as are the abilities of TPR domains to serve as protein-protein interaction scaffolds in biotechnology and therapeutics.

Self-recognition mechanism of MamA, a magnetosome-associated TPR-containing protein, promotes complex assembly.

The magnetosome, a biomineralizing organelle within magnetotactic bacteria, allows their navigation along geomagnetic fields. Magnetosomes are membrane-bound compartments containing magnetic nanoparticles and organized into a chain within the cell, the assembly and biomineralization of magnetosomes are controlled by magnetosome-associated proteins. Here, we describe the crystal structures of the magnetosome-associated protein, MamA, from Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. MamA folds as a sequential tetra-trico-peptide repeat (TPR) protein with a unique hook-like shape. Analysis of the MamA structures indicates two distinct domains that can undergo conformational changes. Furthermore, structural analysis of seven crystal forms verified that the core of MamA is not affected by crystallization conditions and identified three protein-protein interaction sites, namely a concave site, a convex site, and a putative TPR repeat. Additionally, relying on transmission electron microscopy and size exclusion chromatography, we show that highly stable complexes form upon MamA homooligomerization. Disruption of the MamA putative TPR motif or N-terminal domain led to protein mislocalization in vivo and prevented MamA oligomerization in vitro. We, therefore, propose that MamA self-assembles through its putative TPR motif and its concave site to create a large homooligomeric scaffold which can interact with other magnetosome-associated proteins via the MamA convex site. We discuss the structural basis for TPR homooligomerization that allows the proper function of a prokaryotic organelle.

Crystallization and preliminary crystallographic analysis of the Magnetospirillum magneticum AMB-1 and M. gryphiswaldense MSR-1 magnetosome-associated proteins MamA.

MamA is a unique magnetosome-associated protein that is predicted to contain six sequential tetratricopeptide-repeat (TPR) motifs. The TPR structural motif serves as a template for protein-protein interactions and mediates the assembly of multi-protein complexes. Here, the crystallization and preliminary X-ray analysis of recombinant and purified Magnetospirillum magneticum and M. gryphiswaldense MamA are reported for the first time. M. gryphiswaldense MamADelta41 crystallized in the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 58.88, c = 144.09 A. M. magneticum MamADelta41 crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.75, b = 76.19, c = 105.05 A. X-ray diffraction data were collected to resolutions of 2.0 and 1.95 A, respectively.

Purification of the M. magneticum strain AMB-1 magnetosome associated protein MamADelta41.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamADelta41) from M. magneticum AMB-1.