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Drug-resistant Tuberculosis (TB) is a global public health problem. Resistance to rifampicin, the most effective drug for TB treatment, is a major growing concern. The etiological agent, Mycobacterium tuberculosis ( Mtb ), has a cluster of ATP-binding cassette (ABC) transporters which are responsible for drug resistance through active export. Here, we describe studies characterizing Mtb Rv1217c-1218c as an ABC transporter that can mediate mycobacterial resistance to rifampicin and have determined the cryo-electron microscopy structures of Rv1217c-1218c. The structures show Rv1217c-1218c has a type V exporter fold. In the absence of ATP, Rv1217c-1218c forms a periplasmic gate by two juxtaposed-membrane helices from each transmembrane domain (TMD), while the nucleotide-binding domains (NBDs) form a partially closed dimer which is held together by four salt-bridges. Adenylyl-imidodiphosphate (AMPPNP) binding induces a structural change where the NBDs become further closed to each other, which downstream translates to a closed conformation for the TMDs. AMPPNP binding results in the collapse of the outer leaflet cavity and the opening of the periplasmic gate, which was proposed to play a role in substrate export. The rifampicin-bound structure shows a hydrophobic and periplasm-facing cavity is involved in rifampicin binding. Phospholipid molecules are observed in all determined structures and form an integral part of the Rv1217c-1218c transporter system. Our results provide a structural basis for a mycobacterial ABC exporter that mediates rifampicin resistance, which can lead to different insights into combating rifampicin resistance., Competing Interests: Competing interests statement:The authors declare no competing interest.

SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in situ structures of the flagellar motor in wild-type and mutant cells of Borrelia burgdorferi , revealing that three flagellar proteins (FliL, MotA, and MotB) form a unique supramolecular complex in situ. Importantly, FliL not only enhances motor function by forming a ring around the stator complex MotA/MotB in its extended, active conformation but also facilitates assembly of the stator complex around the motor. Our in situ data provide insights into how cooperative remodeling of the FliL-stator supramolecular complex helps regulate the collective ion flux and establishes the optimal function of the flagellar motor to guide bacterial motility in various environments.

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections., Competing Interests: The authors declare no competing interest.

Type III secretion systems are multiprotein molecular machines required for the virulence of several important bacterial pathogens. The central element of these machines is the injectisome, a ∼5-Md multiprotein structure that mediates the delivery of bacterially encoded proteins into eukaryotic target cells. The injectisome is composed of a cytoplasmic sorting platform, and a membrane-embedded needle complex, which is made up of a multiring base and a needle-like filament that extends several nanometers from the bacterial surface. The needle filament is capped at its distal end by another substructure known as the tip complex, which is crucial for the translocation of effector proteins through the eukaryotic cell plasma membrane. Here we report the cryo-EM structure of the Salmonella Typhimurium needle tip complex docked onto the needle filament tip. Combined with a detailed analysis of structurally guided mutants, this study provides major insight into the assembly and function of this essential component of the type III secretion protein injection machine., Competing Interests: The authors declare no competing interest.

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Magnetotactic bacteria maneuver within the geomagnetic field by means of intracellular magnetic organelles, magnetosomes, which are aligned into a chain and positioned at midcell by a dedicated magnetosome-specific cytoskeleton, the "magnetoskeleton." However, how magnetosome chain organization and resulting magnetotaxis is linked to cell shape has remained elusive. Here, we describe the cytoskeletal determinant CcfM (curvature-inducing coiled-coil filament interacting with the magnetoskeleton), which links the magnetoskeleton to cell morphology regulation in Magnetospirillum gryphiswaldense Membrane-anchored CcfM localizes in a filamentous pattern along regions of inner positive-cell curvature by its coiled-coil motifs, and independent of the magnetoskeleton. CcfM overexpression causes additional circumferential localization patterns, associated with a dramatic increase in cell curvature, and magnetosome chain mislocalization or complete chain disruption. In contrast, deletion of ccfM results in decreased cell curvature, impaired cell division, and predominant formation of shorter, doubled chains of magnetosomes. Pleiotropic effects of CcfM on magnetosome chain organization and cell morphology are supported by the finding that CcfM interacts with the magnetoskeleton-related MamY and the actin-like MamK via distinct motifs, and with the cell shape-related cytoskeleton via MreB. We further demonstrate that CcfM promotes motility and magnetic alignment in structured environments, and thus likely confers a selective advantage in natural habitats of magnetotactic bacteria, such as aquatic sediments. Overall, we unravel the function of a prokaryotic cytoskeletal constituent that is widespread in magnetic and nonmagnetic spirilla-shaped Alphaproteobacteria., Competing Interests: The authors declare no competing interest.

Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca 2+ -activated K + channel from Methanobacterium thermoautotrophicum , the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K + concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF's four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K + affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K + , similar to KcsA, but that even a single K + binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K + concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK's S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes., Competing Interests: The authors declare no competing interest.

Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus : McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-β4 loop, pore loop 1, and the presensor 1-β hairpin (PS1-βH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO 2 fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures., Competing Interests: The authors declare no competing interest.

Host-pathogen interactions are central to understanding microbial pathogenesis. The staphylococcal pore-forming cytotoxins hijack important immune molecules but little is known about the underlying molecular mechanisms of cytotoxin-receptor interaction and host specificity. Here we report the structures of a staphylococcal pore-forming cytotoxin, leukocidin GH (LukGH), in complex with its receptor (the α-I domain of complement receptor 3, CD11b-I), both for the human and murine homologs. We observe 2 binding interfaces, on the LukG and the LukH protomers, and show that human CD11b-I induces LukGH oligomerization in solution. LukGH binds murine CD11b-I weakly and is inactive toward murine neutrophils. Using a LukGH variant engineered to bind mouse CD11b-I, we demonstrate that cytolytic activity does not only require binding but also receptor-dependent oligomerization. Our studies provide an unprecedented insight into bicomponent leukocidin-host receptor interaction, enabling the development of antitoxin approaches and improved animal models to explore these approaches., Competing Interests: Competing interest statement: A.B. and H.R. are employees of X4 Pharmaceuticals GmbH, the legal successor of Arsanis Biosciences GmbH, which has developed an antileukocidin-GH antibody., (Copyright © 2020 the Author(s). Published by PNAS.)

Type III protein secretion systems are essential virulence factors for many important pathogenic bacteria. The entire protein secretion machine is composed of several substructures that organize into a holostructure or injectisome. The core component of the injectisome is the needle complex, which houses the export apparatus that serves as a gate for the passage of the secreted proteins through the bacterial inner membrane. Here, we describe a high-resolution structure of the export apparatus of the Salmonella type III secretion system in association with the needle complex and the underlying bacterial membrane, both in isolation and in situ. We show the precise location of the core export apparatus components within the injectisome and bacterial envelope and demonstrate that their deployment results in major membrane remodeling and thinning, which may be central for the protein translocation process. We also show that InvA, a critical export apparatus component, forms a multiring cytoplasmic conduit that provides a pathway for the type III secretion substrates to reach the entrance of the export gate. Combined with structure-guided mutagenesis, our studies provide major insight into potential mechanisms of protein translocation and injectisome assembly., Competing Interests: The authors declare no competing interest.

Human gastric pathogen Helicobacter pylori ( H. pylori ) is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by H. pylori and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed β-helix made up of two domains with extensive domain-domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Heme A is an essential cofactor for respiratory terminal oxidases and vital for respiration in aerobic organisms. The final step of heme A biosynthesis is formylation of the C-8 methyl group of heme molecule by heme A synthase (HAS). HAS is a heme-containing integral membrane protein, and its structure and reaction mechanisms have remained unknown. Thus, little is known about HAS despite of its importance. Here we report the crystal structure of HAS from Bacillus subtilis at 2.2-Å resolution. The N- and C-terminal halves of HAS consist of four-helix bundles and they align in a pseudo twofold symmetry manner. Each bundle contains a pair of histidine residues and forms a heme-binding domain. The C-half domain binds a cofactor-heme molecule, while the N-half domain is vacant. Many water molecules are found in the transmembrane region and around the substrate-binding site, and some of them interact with the main chain of transmembrane helix. Comparison of these two domain structures enables us to construct a substrate-heme binding state structure. This structure implies that a completely conserved glutamate, Glu57 in B. subtilis , is the catalytic residue for the formylation reaction. These results provide valuable suggestions of the substrate-heme binding mechanism. Our results present significant insight into the heme A biosynthesis., Competing Interests: The authors declare no conflict of interest.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria and phosphorylates the sugar for metabolic consumption. The PTS is important for the survival of bacteria and thus a potential target for antibiotics, but its mechanism of sugar uptake and phosphorylation remains unclear. The PTS is composed of multiple proteins, and the membrane-embedded Enzyme IIC (EIIC) component transports sugars across the membrane. Crystal structures of two members of the glucose superfamily of EIICs, bcChbC and bcMalT, were solved in the inward-facing and outward-facing conformations, and the structures suggest that sugar translocation could be achieved by movement of a structured domain that contains the sugar-binding site. However, different conformations have not been captured on the same transporter to allow precise description of the conformational changes. Here we present a crystal structure of bcMalT trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. The structure allows direct comparison of the outward- and inward-facing conformations and reveals a large rigid-body motion of the sugar-binding domain and other conformational changes that accompany the rigid-body motion. All-atom molecular dynamics simulations show that the inward-facing structure is stable with or without the cross-linking. The conformational changes were further validated by single-molecule Föster resonance energy transfer (smFRET). Combined, these results establish the elevator-type mechanism of transport in the glucose superfamily of EIIC transporters., Competing Interests: The authors declare no conflict of interest.

Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partition systems, dependent on its unique dynamic properties. Here, we report the near-atomic resolution electron cryo-microscopy structure of the AlfA filament, which reveals a strikingly divergent filament architecture resulting from the loss of a subdomain conserved in all other actins and a mode of ATP binding. Its unusual assembly interfaces and nucleotide interactions provide insight into AlfA dynamics, and expand the range of evolutionary variation accessible to actin quaternary structure., Competing Interests: The authors declare no conflict of interest.

Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence parN Similar to the ParMRC partitioning system from Escherichia coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and parN Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être., Competing Interests: The authors declare no conflict of interest.

Magnetotactic bacteria produce iron-rich magnetic nanoparticles that are enclosed by membrane invaginations to form magnetosomes so they are able to sense and act upon Earth's magnetic field. In Magnetospirillum and other magnetotactic bacteria, to combine their magnetic moments, magnetosomes align along filaments formed by a bacterial actin homolog, MamK. Here, we present the crystal structure of a nonpolymerizing mutant of MamK from Magnetospirillum magneticum AMB-1 at 1.8-Å resolution, revealing its close similarity to actin and MreB. The crystals contain AMPPNP-bound monomeric MamK in two different conformations. To investigate conformational changes associated with polymerization, we used unmodified MamK protein and cryo-EM with helical 3D reconstruction in RELION to obtain a density map and a fully refined atomic model of MamK in filamentous form at 3.6-Å resolution. The filament is parallel (polar) double-helical, with a rise of 52.2 Å and a twist of 23.8°. As shown previously and unusually for actin-like filaments, the MamK subunits from each of the two strands are juxtaposed, creating an additional twofold axis along the filament. Compared with monomeric MamK, ADP-bound MamK in the filament undergoes a conformational change, rotating domains I and II against each other to further close the interdomain cleft between subdomains IB and IIB. The domain movement causes several loops to close around the nucleotide-binding pocket. Glu-143, a key residue for catalysis coordinating the magnesium ion, moves closer, presumably switching nucleotide hydrolysis upon polymerization-one of the hallmarks of cytomotive filaments of the actin type., Competing Interests: The authors declare no conflict of interest.

Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called "nucleoid occlusion" (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator-CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA-DNA-FtsZ CTD regulatory interaction and provide insight into FtsZ-regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA-DNA-FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA-DNA-FtsZ interaction with implications for SlmA's NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins.

Most bacteria that swim, including Escherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook.

The ability to predictably control the coassembly of multiple nanoscale building blocks, especially those with disparate chemical and physical properties such as biomolecules and inorganic nanoparticles, has far-reaching implications in catalysis, sensing, and photonics, but a generalizable strategy for engineering specific contacts between these particles is an outstanding challenge. This is especially true in the case of proteins, where the types of possible interparticle interactions are numerous, diverse, and complex. Herein, we explore the concept of trading protein-protein interactions for DNA-DNA interactions to direct the assembly of two nucleic-acid-functionalized proteins with distinct surface chemistries into six unique lattices composed of catalytically active proteins, or of a combination of proteins and DNA-modified gold nanoparticles. The programmable nature of DNA-DNA interactions used in this strategy allows us to control the lattice symmetries and unit cell constants, as well as the compositions and habit, of the resulting crystals. This study provides a potentially generalizable strategy for constructing a unique class of materials that take advantage of the diverse morphologies, surface chemistries, and functionalities of proteins for assembling functional crystalline materials.

Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix.

Bacterial type IV secretion systems are evolutionarily related to conjugation systems and play a pivotal role in infection by delivering numerous virulence factors into host cells. Using transmission electron microscopy, we report the native molecular structure of the core complex of the Dot/Icm type IV secretion system encoded by Legionella pneumophila, an intracellular human pathogen. The biochemically isolated core complex, composed of at least five proteins--DotC, DotD, DotF, DotG, and DotH--has a ring-shaped structure. Intriguingly, morphologically distinct premature complexes are formed in the absence of DotG or DotF. Our data suggest that DotG forms a central channel spanning inner and outer membranes. DotF, a component dispensable for type IV secretion, plays a role in efficient embedment of DotG into the functional core complex. These results highlight a common scheme for the biogenesis of transport machinery.

YiiP is a dimeric Zn(2+)/H(+) antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-Å resolution structure of a YiiP homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn(2+). Starting from the X-ray structure in the presence of Zn(2+), we used molecular dynamics flexible fitting to build a model consistent with our map. Comparison of the structures suggests a conformational change that involves pivoting of a transmembrane, four-helix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, our model is consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. Molecular dynamics simulation of YiiP in a lipid environment was used to address the feasibility of this conformational change. Association of the C-terminal domains is the same in both states, and we speculate that this association is responsible for stabilizing the dimer that, in turn, may coordinate the rearrangement of the transmembrane helices.

Small heat shock proteins (sHsps) are molecular chaperones that prevent the aggregation of nonnative proteins. The sHsps investigated to date mostly form large, oligomeric complexes. The typical bacterial scenario seemed to be a two-component sHsps system of two homologous sHsps, such as the Escherichia coli sHsps IbpA and IbpB. With a view to expand our knowledge on bacterial sHsps, we analyzed the sHsp system of the bacterium Deinococcus radiodurans, which is resistant against various stress conditions. D. radiodurans encodes two sHsps, termed Hsp17.7 and Hsp20.2. Surprisingly, Hsp17.7 forms only chaperone active dimers, although its crystal structure reveals the typical α-crystallin fold. In contrast, Hsp20.2 is predominantly a 36mer that dissociates into smaller oligomeric assemblies that bind substrate proteins stably. Whereas Hsp20.2 cooperates with the ATP-dependent bacterial chaperones in their refolding, Hsp17.7 keeps substrates in a refolding-competent state by transient interactions. In summary, we show that these two sHsps are strikingly different in their quaternary structures and chaperone properties, defining a second type of bacterial two-component sHsp system.

The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA kinase and the CheW coupling factor. However, a high-resolution structure of the array has been lacking, and the molecular basis of array assembly has thus remained elusive. Here, we use cryoelectron tomography of flagellated E. coli minicells to derive a 3D map of the intact array. Docking of high-resolution structures into the 3D map provides a model of the core signaling complex, in which a CheA/CheW dimer bridges two adjacent receptor trimers via multiple hydrophobic interactions. A further, hitherto unknown, hydrophobic interaction between CheW and the homologous P5 domain of CheA in an adjacent core complex connects the complexes into an extended array. This architecture provides a structural basis for array formation and could explain the high sensitivity and cooperativity of chemotaxis signaling in E. coli.

The chaplin proteins are functional amyloids found in the filamentous Streptomyces bacteria. These secreted proteins are required for the aerial development of Streptomyces coelicolor, and contribute to an intricate rodlet ultrastructure that decorates the surfaces of aerial hyphae and spores. S. coelicolor encodes eight chaplin proteins. Previous studies have revealed that only three of these proteins (ChpC, ChpE, and ChpH) are necessary for promoting aerial development, and of these three, ChpH is the primary developmental determinant. Here, we show that the model chaplin, ChpH, contains two amyloidogenic domains: one in the N terminus and one in the C terminus of the mature protein. These domains have different polymerization properties as determined using fluorescence spectroscopy, secondary structure analyses, and electron microscopy. We coupled these in vitro assays with in vivo genetic studies to probe the connection between ChpH amyloidogenesis and its biological function. Using mutational analyses, we demonstrated that both N- and C-terminal amyloid domains of ChpH were required for promoting aerial hypha formation, while the N-terminal domain was dispensable for assembly of the rodlet ultrastructure. These results suggest that there is a functional differentiation of the dual amyloid domains in the chaplin proteins.

The tubulin-like FtsZ protein initiates assembly of the bacterial cytokinetic machinery by polymerizing into a ring structure, the Z ring, at the prospective site of division. To block Z-ring formation over the nucleoid and help coordinate cell division with chromosome segregation, Escherichia coli employs the nucleoid-associated division inhibitor, SlmA. Here, we investigate the mechanism by which SlmA regulates FtsZ assembly. We show that SlmA disassembles FtsZ polymers in vitro. In addition, using chromatin immunoprecipitation (ChIP), we identified 24 SlmA-binding sequences (SBSs) on the chromosome. Remarkably, SlmA binding to SBSs dramatically enhanced its ability to interfere with FtsZ polymerization, and ChIP studies indicate that SlmA regulates FtsZ assembly at these sites in vivo. Because of the dynamic and highly organized nature of the chromosome, coupling SlmA activation to specific DNA binding provides a mechanism for the precise spatiotemporal control of its anti-FtsZ activity within the cell.

Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a polymerizing cytomotive filament. Together these components drive newly replicated plasmids to opposite ends of the dividing cell. The Bacillus thuringiensis plasmid pBToxis relies on a filament of the tubulin/FtsZ-like protein TubZ for its segregation. By combining crystallography and electron microscopy, we have determined the structure of this filament. We explain how GTP hydrolysis weakens the subunit-subunit contact and also shed light on the partitioning of the plasmid-adaptor complex. The double helical superstructure of TubZ filaments is unusual for tubulin-like proteins. Filaments of ParM, the actin-like partitioning protein, are also double helical. We suggest that convergent evolution shapes these different types of cytomotive filaments toward a general mechanism for plasmid separation.

Clostridium difficile toxins A and B are members of an important class of virulence factors known as large clostridial toxins (LCTs). Toxin action involves four major steps: receptor-mediated endocytosis, translocation of a catalytic glucosyltransferase domain across the membrane, release of the enzymatic moiety by autoproteolytic processing, and a glucosyltransferase-dependent inactivation of Rho family proteins. We have imaged toxin A (TcdA) and toxin B (TcdB) holotoxins by negative stain electron microscopy to show that these molecules are similar in structure. We then determined a 3D structure for TcdA and mapped the organization of its functional domains. The molecule has a "pincher-like" head corresponding to the delivery domain and two tails, long and short, corresponding to the receptor-binding and glucosyltransferase domains, respectively. A second structure, obtained at the acidic pH of an endosome, reveals a significant structural change in the delivery and glucosyltransferase domains, and thus provides a framework for understanding the molecular mechanism of LCT cellular intoxication.

Phytochromes are a collection of dimeric photoreceptors that direct a diverse array of responses in plants and microorganisms through photoconversion between a red light-absorbing ground state Pr, and a far-red light-absorbing photoactivated state Pfr. Photoconversion from Pr to Pfr is initiated by a light-driven rotation within the covalently attached bilin, which then triggers a series of protein conformational changes in the binding pocket. These movements ultimately affect an appended output module, which often has reversible protein kinase activity. Propagation of the light signal from the bilin to the output module likely depends on the dimerization interface but its architecture and response to phototransformation remain unclear. Here, we used single particle cryoelectron microscopy to determine the quaternary arrangement of the phytochrome dimer as Pr, using the bacteriophytochrome (BphP) from Deinococcus radiodurans. Contrary to the long-standing view that the two monomers are held together solely via their C-terminal region, we provide unambiguous evidence that the N-terminal bilin-binding region of BphP also provides a dimerization interface with the C-terminal kinase domain appearing as a more flexible appendage. The BphP monomers dimerize in parallel with the polypeptides intimately twisting around each other in a right-handed fashion. Based on this electron microscopic picture, we propose that the light-driven conformational changes transmitted from the chromophore to the output module along the spine of this extensive dimer interface is the central feature underpinning phytochrome signaling.

Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, we characterized the physicochemical properties of this protein. We report that purified TasA forms fibers of variable length and 10-15 nm in width. Biochemical analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymerized. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphology, indicating that the purified protein retained biological activity. We propose that TasA forms amyloid fibers that bind cells together in the biofilm.

Forces are important in biological systems for accomplishing key cell functions, such as motility, organelle transport, and cell division. Currently, known force generation mechanisms typically involve motor proteins. In bacterial cells, no known motor proteins are involved in cell division. Instead, a division ring (Z-ring) consists of mostly FtsZ, FtsA, and ZipA is used to exerting a contractile force. The mechanism of force generation in bacterial cell division is unknown. Using computational modeling, we show that Z-ring formation results from the colocalization of FtsZ and FtsA mediated by the favorable alignment of FtsZ polymers. The model predicts that the Z-ring undergoes a condensation transition from a low-density state to a high-density state and generates a sufficient contractile force to achieve division. FtsZ GTP hydrolysis facilitates monomer turnover during the condensation transition, but does not directly generate forces. In vivo fluorescence measurements show that FtsZ density increases during division, in accord with model results. The mechanism is akin to van der Waals picture of gas-liquid condensation, and shows that organisms can exploit microphase transitions to generate mechanical forces.

We demonstrate a method for generating discretely structured protein nanotubes from the simple ring-shaped building block, homohexameric Hcp1 from Pseudomonas aeruginosa. Our design exploited the observation that the crystal lattice of Hcp1 contains rings stacked in a repeating head-to-tail pattern. High-resolution detail of the ring-ring interface allowed the selection of sites for specific cysteine mutations capable of engaging in disulfide bond formation across rings, thereby generating stable Hcp1 nanotubes. Protein nanotubes containing up to 25 subunits ( approximately 100 nm in length) were self-assembled under simple conditions. Furthermore, we demonstrate that the tube ends and interior can be independently and specifically functionalized to generate nanocapsules.

Curli are functional amyloid fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In E. coli, the polymerization of the major curli fiber subunit protein CsgA into an amyloid fiber depends on the minor curli subunit protein, CsgB. The outer membrane-localized CsgB protein shares approximately 30% sequence identity with the amyloid-forming protein CsgA, suggesting that CsgB might also have amyloidogenic properties. Here, we characterized the biochemical properties of CsgB and the molecular basis for CsgB-mediated nucleation of CsgA. Deletion analysis revealed that a CsgB molecule missing 19 amino acids from its C terminus (CsgB(trunc)) was not outer membrane-associated, but secreted away from the cell. CsgB(trunc) was overexpressed and purified from the extracellular milieu of cells as an SDS-soluble, nonaggregated protein. Soluble CsgB(trunc) assembled into fibers that bound to the amyloid-specific dyes Congo red and thioflavin-T. CsgB(trunc) fibers were able to seed soluble CsgA polymerization in vitro. CsgB(trunc) displayed modest nucleator activity in vivo, as demonstrated by its ability to convert extracellular CsgA into an amyloid fiber. Unlike WT CsgB, CsgB(trunc) was only able to act as a nucleator when cells were genetically manipulated to secrete higher concentrations of CsgA. This work represents a unique demonstration of functional amyloid nucleation and it suggests an elegant model for how E. coli guides efficient amyloid fiber formation on the cell surface.

Trigger factor (TF), the first chaperone in eubacteria to encounter the emerging nascent chain, binds to the large ribosomal subunit in the vicinity of the protein exit tunnel opening and forms a sheltered folding space. Here, we present the 3.5-A crystal structure of the physiological complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with the N-terminal domain of TF (TFa) from the same organism. For anchoring, TFa exploits a small ribosomal surface area in the vicinity of proteins L23 and L29, by using its "signature motif" as well as additional structural elements. The molecular details of TFa interactions reveal that L23 is essential for the association of TF with the ribosome and may serve as a channel of communication with the nascent chain progressing in the tunnel. L29 appears to induce a conformational change in TFa, which results in the exposure of TFa hydrophobic patches to the opening of the ribosomal exit tunnel, thus increasing its affinity for hydrophobic segments of the emerging nascent polypeptide. This observation implies that, in addition to creating a protected folding space for the emerging nascent chain, TF association with the ribosome prevents aggregation by providing a competing hydrophobic environment and may be critical for attaining the functional conformation necessary for chaperone activity.

Trigger factor (TF) and signal recognition particle (SRP) bind to the bacterial ribosome and are both crosslinked to protein L23 at the peptide exit, where they interact with emerging nascent peptide chains. It is unclear whether TF and SRP exclude one another from their ribosomal binding site(s). Here we show that SRP and TF can bind simultaneously to ribosomes or ribosome nascent-chain complexes exposing a SRP-specific signal sequence. Based on changes of the crosslinking pattern and on results obtained by fluorescence measurements using fluorescence-labeled SRP, TF binding induces structural changes in the ribosome-SRP complex. Furthermore, we show that binding of the SRP receptor, FtsY, to ribosome-bound SRP excludes TF from the ribosome. These results suggest that TF and SRP sample nascent chains on the ribosome in a nonexclusive fashion. The decision for ribosome nascent-chain complexes exposing a signal sequence to enter SRP-dependent membrane targeting seems to be determined by the binding of SRP, which is stabilized by signal sequence recognition, and promoted by the exclusion of TF due to the binding of the SRP receptor to ribosome-bound SRP.

KaiA is a two-domain circadian clock protein in cyanobacteria, acting as the positive element in a feedback loop that sustains the oscillation. The structure of the N-terminal domain of KaiA is that of a pseudo-receiver, similar to those of bacterial response regulators, which likely interacts with components of the clock-resetting pathway. The C-terminal domain of KaiA is highly conserved among cyanobacteria and enhances the autokinase activity of KaiC. Here we present the NMR structure of the C-terminal domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. This domain adopts a novel all alpha-helical homodimeric structure. Several mutations known to affect the period of the circadian oscillator are shown to be located at an exposed groove near the dimer interface. This NMR structure and a 21-A-resolution electron microscopy structure of the hexameric KaiC particle allow us to postulate a mode of KaiA-KaiC interaction, in which KaiA binds a linker region connecting two globular KaiC domains.

The cap at the growing end of the bacterial flagellum is essential for its growth, remaining stably attached while permitting the insertion of flagellin transported from the cytoplasm through the narrow central channel. We analyzed the structure of the isolated cap in its frozen hydrated state by electron cryomicroscopy. The 3D density map now shows detailed features of domains and their connections, giving reliable volumes and masses, making assignment of the domains to the amino acid sequence possible. A model of the cap-filament complex built with an atomic model of the filament allows a quantitative analysis of the cap domain movements on cap binding and rotation that promotes the efficient self assembly of flagellin during the filament growth process.

Enteropathogenic Escherichia coli (EPEC) secretes several Esp proteins via the type III secretion system (secreton). EspA, EspB, and EspD are required for translocation of the effector proteins into host cells, in which EspB and EspD are thought to form a pore in the host membrane. Recent study has shown that EspA forms a filamentous structure that assembles as a physical bridge between bacteria and host cell surfaces, which then functions as a conduit for the translocation of bacterial effectors into host cells. To investigate the supermolecular structure of the type III secreton in EPEC, we partially purified it from the bacteria membrane and observed it via transmission electron microscopy. The EPEC type III secreton was composed of a basal body and a needle part and was similar to those of Salmonella and Shigella, except for a sheath-like structure at the tip of the needle. The length of sheath-like structures varied; it extended more than 600 nm and was 10 times longer than the Shigella needle part. The putative major needle component, EscF, was required for both secretion of Esp proteins and needle complex formation. Interestingly, elongation of the sheath-like structure was observed under constitutive expression of EspA but not of EscF. Furthermore, the transmission electron microscopy view with immunogold labeled anti-EspA antibodies clearly showed that EspA is a component of the sheath-like structure. This study revealed, to our knowledge for the first time, the supermolecular structure of the EPEC type III secreton and its direct association with the EspA-sheath-like structure.

RNase E isolated from Escherichia coli is contained in a multicomponent "degradosome" complex with other proteins implicated in RNA decay. Earlier work has shown that the C-terminal region of RNase E is a scaffold for the binding of degradosome components and has identified specific RNase E segments necessary for its interaction with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase. Here, we report electron microscopy studies that use immunogold labeling and freeze-fracture methods to show that degradosomes exist in vivo in E. coli as multicomponent structures that associate with the cytoplasmic membrane via the N-terminal region of RNase E. Whereas PNPase and enolase are present in E. coli in large excess relative to RNase E and therefore are detected in cells largely as molecules unlinked to the RNase E scaffold, immunogold labeling and biochemical analyses show that helicase is present in approximately equimolar amounts to RNase E at all cell growth stages. Our findings, which establish the existence and cellular location of RNase E-based degradosomes in vivo in E. coli, also suggest that RNA processing and decay may occur at specific sites within cells.

FliG, FliM, and FliN, key proteins for torque generation, are located in two rings. The first protein is in the M ring and the last two are in the C ring. The rotational symmetries of the C and M rings have been determined to be about 34 (this paper) and 26 (previous work), respectively. The mechanism proposed here depends on the symmetry mismatch between the rings: the C ring extends 34 levers, of which 26 can bind to the 26 equivalent sites on the M ring. The remaining 8 levers bind to proton-pore complexes (studs) to form 8 torque generators. Movement results from the swapping of stud-bound levers with M ring-bound levers. The model predicts that both the M and C rings rotate in the same direction but at different speeds.

Pathogenic strains of Helicobacter pylori secrete a cytotoxin, VacA, that in the presence of weak bases, causes osmotic swelling of acidic intracellular compartments enriched in markers for late endosomes and lysosomes. The molecular mechanisms by which VacA causes this vacuolation remain largely unknown. At neutral pH, VacA is predominantly a water-soluble dodecamer formed by two apposing hexamers. In this report, we show by using atomic force microscopy that below pH approximately 5, VacA associates with anionic lipid bilayers to form hexameric membrane-associated complexes. We propose that water-soluble dodecameric VacA proteins disassemble at low pH and reassemble into membrane-spanning hexamers. The surface contour of the membrane-bound hexamer is strikingly similar to the outer surface of the soluble dodecamer, suggesting that the VacA surface in contact with the membrane is buried within the dodecamer before protonation. In addition, electrophysiological measurements indicate that, under the conditions determined by atomic force microscopy for membrane association, VacA forms pores across planar lipid bilayers. This low pH-triggered pore formation is likely a critical step in VacA activity.

The establishment of differential gene expression in sporulating Bacillus subtilis involves four protein components, one of which, SpoIIAA, undergoes phosphorylation and dephosphorylation. We have used NMR spectroscopy to determine the solution structure of the nonphosphorylated form of SpoIIAA. The structure shows a fold consisting of a four-stranded beta-sheet and four alpha-helices. Knowledge of the structure helps to account for the phenotype of several strains of B. subtilis that carry known spoIIAA mutations and should facilitate investigations of the conformational consequences of phosphorylation.

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and/or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for protein-protein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces.

The major subassemblies of virulence-associated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were reconstituted from purified chaperone-subunit complexes in vitro. Subunits are held in assembly-competent conformations in chaperone-subunit complexes prior to their assembly into mature pili. The PapD chaperone binds, in part, to a conserved motif present at the C terminus of the subunits via a beta zippering interaction. Amino acid residues in this conserved motif were also found to be essential for subunit-subunit interactions necessary for the formation of pili, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit-subunit interactions in the periplasm. Uncapping of the chaperone-protected C terminus of PapA and PapE was mimicked in vitro by freeze-thaw techniques and resulted in the formation of pilus rods and tip fibrillae, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimuthal disorder. This change in the number of subunits per turn of the helix most likely reflects involvement of the leading edge of the beta zipper in forming a right-handed helical cylinder. Organelle development is a fundamental process in all living cells, and these studies shed new light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.

Haemophilus influenzae is a Gram-negative bacterium that represents a common cause of human disease. Disease due to this organism begins with colonization of the upper respiratory mucosa, a process facilitated by adhesive fibers called pili. In the present study, we investigated the structure and assembly of H. influenzae pili. Examination of pili by electron microscopy using quick-freeze, deep-etch and immunogold techniques revealed the presence of two distinct subassemblies, including a flexible two-stranded helical rod comprised of HifA and a short, thin, distal tip structure containing HifD. Genetic and biochemical studies demonstrated that the biogenesis of H. influenzae pili is dependent on a periplasmic chaperone called HifB, which belongs to the PapD family of immunoglobulin-like chaperones. HifB bound directly to HifA and HifD, forming HifB-HifA and HifB-HifD complexes, which were purified from periplasmic extracts by ion-exchange chromatography. Continued investigation of the biogenesis of H. influenzae pili should provide general insights into organelle development and may suggest novel strategies for disease prevention.

The Fur (ferric uptake regulation) protein is a global regulator that, in the presence of Fe2+, represses the expression of a number of iron-acquisition genes and virulence determinants such as toxins. Dark-field electron microscopy of positively stained Fur-DNA complexes in addition to atomic force microscopy allowed direct visualization of Fur interactions with the regulatory regions of aerobactin and hemolysin operons and provided complementary information about the structure of the complexes. According to the DNA used and the protein/DNA ratio, Fur binding to DNA results in partial or total covering of the fragments, indicating that the protein initiates polymerization along the DNA molecules at specific sites. Negative staining of Fur-DNA complexes revealed a well-ordered structure of the polymer suggesting a helical arrangement. Local rigidification of the DNA molecules resulting from Fur binding could be involved in the repression process.

The Thermus aquaticus DNA methyltransferase M.Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of --CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethionine shows alpha/beta folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide cleft suitable to accommodate the DNA substrate. The N-terminal domain is dominated by a nine-stranded beta-sheet; it contains the two conserved segments typical for N-methyltransferases which form a pocket for cofactor binding. The C-terminal domain is formed by four small beta-sheets and alpha-helices. The three-dimensional folding of M.Taq I is similar to that of the cytosine-specific Hha I methyltransferase, where the large beta-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.