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In the process of natural transformation bacteria import extracellular DNA molecules for integration into their genome. One strand of the incoming DNA molecule is degraded, whereas the remaining strand is transported across the cytoplasmic membrane. The DNA transport channel is provided by the protein ComEC. Many ComEC proteins have an extracellular C‐terminal domain (CTD) with homology to the metallo‐β‐lactamase fold. Here we show that this CTD binds Mn2+ ions and exhibits Mn2+‐dependent phosphodiesterase and nuclease activities. Inactivation of the enzymatic activity of the CTD severely inhibits natural transformation in Bacillus subtilis. These data suggest that the ComEC CTD is a nuclease responsible for degrading the nontransforming DNA strand during natural transformation and that this process is important for efficient DNA import., During natural transformation in bacteria one DNA strand is transported into the cytoplasm via the membrane protein ComEC while the other strand is degraded. Here we show that the C‐terminal domain of ComEC has a nuclease activity that is important for natural transformation in B. subtilis. Thus, this domain is likely to be responsible for digesting the non‐transforming DNA strand.

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor and a proton-driven motor that powers gliding motility and the type 9 protein secretion system in Bacteroidetes bacteria. Here, we present cryo-electron microscopy structures of the gliding motility/type 9 protein secretion system motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identify protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane. Using cryo-electron microscopy, the authors describe the structure and function of a molecular motor powering both the Bacteroidetes type 9 protein secretion system and the associated gliding motility apparatus.

SummaryIon-driven motors are rare in biology. The archetypes of the three classes identified to date are ATP synthase, the bacterial flagellar motor, and a proton-driven motor that powers gliding motility and protein secretion in Bacteroidetes bacteria. Whilst the molecular mechanism of ATP synthase is now well understood, structural information is lacking for the other two classes of motor. Here we present the structure of the Bacteroidetes gliding motility motor determined by cryo-electron microscopy. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM proteins are positioned within a ring of five GldL proteins. Combining mutagenesis and single-molecule tracking, we identify protonatable amino acid residues within the transmembrane domain of the complex that are important for motor function. Our data imply a mechanism in which proton flow leads the periplasm-spanning GldM dimer to rotate with respect to the intra-membrane GldL ring to drive processes at the bacterial outer membrane. This work provides a molecular basis for understanding how the gliding motility motor is able to transduce the energy of the inner membrane protonmotive force across the bacterial cell envelope.

SummaryThe bacterial flagellum is the proto-typical protein nanomachine and comprises a rotating helical propeller attached to a membrane-embedded motor complex1. The motor consists of a central rotor surround by stator units that couple ion flow across the cytoplasmic membrane to torque generation. Here we present the structures of stator complexes from multiple bacterial species, allowing interpretation of the extensive body of data on stator mechanism. The structures reveal an unexpected asymmetric A5B2 subunit assembly in which the five A subunits enclose the two B subunits. Comparison to novel structures of other ion-driven motors indicates that this A5B2 architecture is fundamental to bacterial systems that couple energy from ion-flow to generate mechanical work at a distance, and suggests that such events involve rotation in the motor structures.

Ion-driven motors are amongst the most fascinating protein nanomachines known. The archetypes of the three classes identified to date are ATP synthase, the bacterial flagellar motor, and a proton-driven motor that powers gliding motility and protein secretion in Bacteroidetes bacteria. Here we present the structure of the Bacteroidetes gliding motility motor. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM proteins are positioned within a ring of five GldL proteins. Combining mutagenesis and single-molecule tracking, we identify protonatable amino acid residues within the transmembrane domain of the complex that are important for motor function. Our data imply a mechanism in which proton flow leads the periplasm-spanning GldM dimer to rotate with respect to the intra-membrane GldL ring to drive processes at the bacterial outer membrane.

The twin arginine translocation (Tat) system moves folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Signal peptide-bearing substrates of the Tat pathway (precursor proteins) are recognized at the membrane by the TatBC receptor complex. The only established preparation of the TatBC complex uses the detergent digitonin, rendering it unsuitable for biophysical analysis. Here we show that the detergent glyco-diosgenin (GDN) can be used in place of digitonin to isolate homogeneous TatBC complexes that bind precursor proteins with physiological specificity. We use this new preparation to quantitatively characterize TatBC–precursor interactions in a fully defined system. Additionally, we show that the GDN-solubilized TatBC complex co-purifies with substantial quantities of phospholipids.

Background: The hemoprotein TsdA catalyzes the oxidation of two thiosulfate molecules to form tetrathionate. Results: The mechanism of TsdA has been probed using biochemical and structural methods. Conclusion: The TsdA reaction proceeds via a cysteine S-thiosulfonate intermediate formed on a cysteine ligand to the active site heme. Significance: TsdA provides a catalytic model for other members of the SoxA enzyme family., Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.

The twin-arginine translocase (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat translocation site is formed by substrate-triggered oligomerization of the protein TatA. Walther and co-workers have proposed a structural model for the TatA oligomer in which TatA monomers self-assemble using electrostatic ‘charge zippers’ (Cell (2013) 132: 15945). This model was supported by in vitro analysis of the oligomeric state of TatA variants containing charge-inverting substitutions. Here we have used live cell assays of TatA assembly and function in Escherichia coli to re-assess the roles of the charged residues of TatA. Our results do not support the charge zipper model. Instead, we observe that substitutions of charged residues located in the TatA amphipathic helix lock TatA in an assembled state, suggesting that these charged residues play a critical role in the protein translocation step that follows TatA assembly.

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA-YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.

Cofactors linked to nutrient limitation Microbes require inventive ways to acquire scarce nutrients from the environment. Enzymes that catalyze the acquisition of phosphorus from dissolved organic matter, for example, rely on complex metal cofactors in the active site. Yong et al. determined the crystal structure of the PhoX alkaline phosphatase from Pseudomonas fluorescens (see the Perspective by Moore). The metal centers arrange themselves in a triangular structure of two iron atoms and one calcium atom, bridged together by an oxide ion. The presence of iron, which itself is a trace nutrient in most environments, suggests that it limits phosphorus acquisition. Science , this issue p. 1170 ; see also p. 1120

The Tat protein transport system is found in the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Unusually, the Tat system translocates proteins only after they have folded. Proteins are targeted to the Tat system by specific N-terminal signal peptides. High resolution structures have recently been determined for the TatA and TatC proteins that form the Tat translocation site. These structures provide a molecular framework for understanding the mechanism of Tat transport. The interactions between TatC and the signal peptide of the substrate protein can be provisionally modelled. However, the way that TatA and TatC combine in the active translocation site remains to be definitively established.

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes. DOI: http://dx.doi.org/10.7554/eLife.20718.001

The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of approximately 25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains approximately 15 mobile TatA complexes and a pool of approximately 100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.

BACKGROUND: SoxAX enzymes initiate microbial oxidation of reduced inorganic sulfur compounds. Their catalytic mechanism is unknown. RESULTS: Cyanide displaces the CysS(-) ligand to the active site heme following reduction by S(2)O(4)(2-) but not Eu(II). CONCLUSION: An active site heme ligand becomes labile on exposure to substrate analogs. SIGNIFICANCE: Elucidation of SoxAX mechanism is necessary to understand a widespread pathway for sulfur compound oxidation. SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E(m)) at pH 7.0 of approximately +210, -340, and -400 mV for the His/Met, His/Cys(-), and active site His/CysS(-)-ligated heme, respectively. Exposing SoxAX to S(2)O(4)(2-), a substrate analog with E(m) ~-450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E(m) ~-1140 mV), allows cyanide to displace the cysteine persulfide (CysS(-)) ligand to the active site heme. This provides the first evidence for the dissociation of CysS(-) that has been proposed as a key event in SoxAX catalysis.

The twin-arginine translocation (Tat) machinery transports folded substrate proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. This mechanistically challenging machinery deploys three essential proteins, TatA, TatB, and TatC. TatA is known to form the protein translocating element of the Tat system. The size and the stoichiometry of the Tat translocation complex are thought to be dependent on the size of the substrate. Current models for Tat transport only predict the oligomeric state of TatA and if, and whether, this state changes during the transport cycle. We use single-molecule fluorescence techniques to probe into the stoichiometry of the translocase.

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins. © 2012 Blackwell Publishing Ltd.

The twin-arginine translocation (Tat) protein export system is present in the cytoplasmic membranes of most bacteria and archaea and has the highly unusual property of transporting fully folded proteins. The system must therefore provide a transmembrane pathway that is large enough to allow the passage of structured macromolecular substrates of different sizes but that maintains the impermeability of the membrane to ions. In the Gram-negative bacterium Escherichia coli, this complex task can be achieved by using only three small membrane proteins: TatA, TatB and TatC. In this Review, we summarize recent advances in our understanding of how this remarkable machine operates. © 2012 Macmillan Publishers Limited. All rights reserved.

Thiosulfate respiration in Salmonella enterica serovar Typhimurium is catalyzed by the membrane-bound enzyme thiosulfate reductase. Experiments with quinone biosynthesis mutants show that menaquinol is the sole electron donor to thiosulfate reductase. However, the reduction of thiosulfate by menaquinol is highly endergonic under standard conditions (Δ E °′ = −328 mV). Thiosulfate reductase activity was found to depend on the proton motive force (PMF) across the cytoplasmic membrane. A structural model for thiosulfate reductase suggests that the PMF drives endergonic electron flow within the enzyme by a reverse loop mechanism. Thiosulfate reductase was able to catalyze the combined oxidation of sulfide and sulfite to thiosulfate in a reverse of the physiological reaction. In contrast to the forward reaction the exergonic thiosulfate-forming reaction was PMF independent. Electron transfer from formate to thiosulfate in whole cells occurs predominantly by intraspecies hydrogen transfer.

The twin arginine protein transport (Tat) system transports folded proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of plant chloroplasts. In Escherichia coli, the TatB and TatC components form a multivalent receptor complex that binds Tat substrates. Here, we have used a genetic fusion approach to construct covalent TatC oligomers in order to probe the organisation of TatC. A fused dimer of TatC supported Tat transport activity and was fully stable in vivo. Inactivating point mutations in one or other of the TatC units in the fused TatC dimer did not inactivate TatC function, indicating that only one TatC protomer in the TatC fused dimer needs to be active. Larger covalent fusions of TatC also supported Tat transport activity but were degraded in vivo to release smaller TatC forms. Taken together, these results strongly suggest that TatC forms a functional dimer, and support the idea that there is an even number of TatC protomers in the TatBC complex.

The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile12 and Val14 are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile12 on one subunit and Val14 on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport.

SoxB is an essential component of the bacterial Sox sulfur oxidation pathway. SoxB contains a di-manganese(II) site and is proposed to catalyze the release of sulfate from a protein-bound cysteine S-thiosulfonate. A direct assay for SoxB activity is described. The structure of recombinant Thermus thermophilus SoxB was determined by x-ray crystallography to a resolution of 1.5 A. Structures were also determined for SoxB in complex with the substrate analogue thiosulfate and in complex with the product sulfate. A mechanistic model for SoxB is proposed based on these structures.

The Tat (twin arginine translocation) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The integral membrane proteins TatA, TatB, and TatC are essential components of the Tat pathway. TatA forms high order oligomers and is thought to constitute the protein-translocating unit of the Tat system. Cysteine scanning mutagenesis was used to systematically investigate the functional importance of residues in the essential N-terminal transmembrane and amphipathic helices of Escherichia coli TatA. Cysteine substitutions of most residues in the amphipathic helix, including all the residues on the hydrophobic face of the helix, severely compromise Tat function. Glutamine 8 was identified as the only residue in the transmembrane helix that is critical for TatA function. The cysteine variants in the transmembrane helix were used in disulfide mapping experiments to probe the oligomeric arrangement of TatA protomers within the larger TatA complex. Residues in the center of the transmembrane helix (including residues 10-16) show a distinct pattern of cross-linking indicating that this region of the protein forms well defined interactions with other protomers. At least two interacting faces were detected. The results of our TatA studies are compared with analogous data for the homologous, but functionally distinct, TatB protein. This comparison reveals that it is only in TatA that the amphipathic helix is sensitive to amino acid substitutions. The TatA amphipathic helix may play a role in forming and controlling the path of substrate movement across the membrane.

Summary The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in E scherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate‐bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild‐type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue‐native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild‐type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate‐induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag-coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size-exclusion chromatography and shown to have apparent molecular masses of ∼ 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin-arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin-arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins.

The Tat system mediates Sec-independent transport of folded precursor proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. Tat transport involves distinct high-molecular-weight TatA and TatBC complexes. Here we report the 3D architecture of the TatA complex from Escherichia coli obtained by single-particle electron microscopy and random conical tilt reconstruction. TatA forms ring-shaped structures of variable diameter in which the internal channels are large enough to accommodate known Tat substrate proteins. This morphology strongly supports the proposal that TatA forms the protein-conducting channel of the Tat system. One end of the channel is closed by a lid that might gate access to the channel. On the basis of previous protease accessibility measurements, the lid is likely to be located at the cytoplasmic side of the membrane. The observed variation in TatA diameter suggests a model for Tat transport in which the number of TatA protomers changes to match the size of the channel to the size of the substrate being transported. Such dynamic close packing would provide a mechanism to maintain the membrane permeability barrier during transport.

The twin-arginine (Tat) protein translocase is a highly unusual protein transport machine that is dedicated to the movement of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-terminal signal peptides harbouring a distinctive twin-arginine motif. In the model organism Escherichia coli, many of the Tat substrates bind redox cofactors that are inserted into apo-proteins before they engage with the Tat machinery. Here we review recent advances in understanding the events involved in the coordination of cofactor insertion with the export process. Current models for Tat protein transport are also discussed.

The respiratory formate dehydrogenase N (FDH-N) enzyme of Escherichia coli is a seleno-molybdoenzyme that is synthesized when the bacterium grows anaerobically with nitrate as exogenous electron acceptor. FDH-N can comprise up to 10% of the total membrane protein (9). Together with nitrate reductase-A, it forms a respiratory chain transferring electrons from formate to nitrate and results in the generation of a protonmotive force (10). FDH-N has a number of cofactors, including bis-molybdopterin guanine dinucleotide cofactor, selenocysteine, and a single [4Fe-4S] cluster. Consequently, synthesis of this enzyme requires careful control. Transcription of both the fdnGHI operon, which encodes FDH-N, and narGHJI, which encodes nitrate reductase-A, is coordinately controlled. Expression of both operons is maximal anaerobically in the presence of nitrate and is controlled by the transcription factors Fnr and NarL (8). The high-resolution X-ray structure of FDH-N has revealed that it adopts an (αβγ)3 “trimer-of-trimers ” architecture, with the active site of the enzyme located in the periplasm (13). FDH-N is translocated across the membrane by the twin arginine translocation (Tat) pathway (21). The Tat translocase is dedicated to the transport of prefolded proteins, which bear an N-terminal signal peptide with the conserved S/T-R-R-x-F-L-K twin arginine motif (2, 3). Translocation of FDH-N is mediated by virtue of a Tat signal peptide on the FdnG subunit. Stanley et al. have shown previously that the FdnG signal peptide is able to mediate export of the reporter proteins β-lactamase and chloramphenicol acetyltransferase to the periplasm in a Tat-dependent fashion (24). During the course of these fusion studies we noted that point mutations in the first or second arginine codons of the twin arginine motif resulted in a dramatic overproduction of the fusion protein. The mRNA specifying the first 17 codons of the fdnG gene is predicted to fold into a stable stem-loop structure, and we demonstrate here that this stem-loop mediates translational control of FDH-N synthesis. As shown in Table ​Table1,1, substitution of Arg5, either conservatively for Lys or nonconservatively for Ser, resulted in a marked increase (up to 60-fold) in fusion protein synthesis, regardless of the nature of the reporter protein. In contrast, substitution of Lys10 for Glu had no significant effect on levels of fusion protein (Table ​(Table1).1). No significant differences in fusion protein synthesis were noted in a tat mutant background, suggesting that these observations were not directly related to operation of the Tat pathway. When the mfold program (25) was used, the mRNA covering the start codon and signal peptide-coding region of fdnG could be folded into a stem-loop structure (Fig. ​(Fig.1B).1B). The ΔG value (at 37°C and physiological pH) associated with this structure was calculated to be −12.6 kCal/mol, suggesting that the folded mRNA would be relatively stable. Such a folded mRNA structure would be consistent with the results seen in Table ​Table1,1, since mutations at codon 5, which fall within one arm of the predicted stem, would be expected to disrupt the fold, whereas the mutation at codon 10, which is located within the putative loop region, would not be expected to disrupt the structure. FIG. 1. The mRNA specifying the fdnG signal peptide coding region can be folded into a stem-loop structure. The position of the initiation codon is indicated in bold type; the Shine-Dalgarno ribosome-binding sequence is underlined. Numbering is shown relative ... TABLE 1. Total cellular activity of reporter proteins fused to the wild-type or amino acid-substituted FdnG signal sequencea To test whether a hairpin in the fdnG mRNA was controlling expression at a translational level, we constructed a number of additional mutations in codons 3 to 14 of fdnG. As shown in Fig. ​Fig.2A,2A, substitutions that were predicted to severely disrupt the mRNA fold (R5S [CGC→AGC], R5K [CGC→AAG], R5Rhigh [CGC→CGT], and R5Rlow [CGC→AGG], where the natural Arg codon was replaced with Arg codons of higher and lower usage) (18) led to significant upregulation of β-galactosidase activity. Mutations in codon 6 that were predicted to have more modest effects on the mRNA secondary structure gave less-dramatic increases in β-galactosidase activity. As predicted by the model, substitutions of codons that fell within the putative loop region (Fig. ​(Fig.2B)2B) did not have significant effects on the activity of β-galactosidase, with the exception of the I11V mutation, which resulted in a marked decrease in the activity of β-galactosidase. Interestingly, this substitution is predicted to result in a significant increase in the stability of the stem-loop (from −12.6 to −16.4 kCal/mol) due to the formation of an extra base pair at the top of the stem. The results of substitutions in the putative second arm of the stem (Fig. ​(Fig.2C)2C) were also consistent with the premise that a stem-loop structure was controlling translation of fdnG-lacZ. A plot of ΔG value for the structures associated with the wild-type sequence and each of the mutations against the observed β-galactosidase activity (Fig. ​(Fig.2E)2E) shows a linear relationship. Indeed, a straight line can be drawn through the points with a correlation factor (R2) of 0.82, providing strong support for the model. As a final test, we looked at the effect of introducing a compensatory mutation into the second arm of the stem, to restore base pairing, with a mutation in the first arm of the stem that disrupts the stem-loop structure. When introduced singly, both the AGA→AAG mutations at codon 6 and the ATC→ACT mutations at codon 11 led to increased β-galactosidase activity (Fig. ​(Fig.2D).2D). However, when these two sets of mutations were combined, β-galactosidase activity was restored to the wild-type level, indicating that the two mutations had a compensatory effect. FIG. 2. The effects of mutations in the stem and loop regions of the fdnG mRNA on expression of fdnG-lacZ. Constructs were transformed into strain MC4100-P (4), which carries the pcnB allele, providing more careful control of plasmid copy number (14, 15). (A) ... In addition to the substantial transcriptional regulation already reported (1), our findings strongly suggest that a further, possibly up to 10-fold, level of control of fdnGHI expression was exerted at the translational level. To confirm the physiological relevance of these findings, we used published methods (11) to introduce two sets of mutations (R5Rhigh [CGC→CGT] and R5Rlow [CGC→AGG]) in the fdnG gene in the chromosome. In each case we made a substitution that retained an Arg codon at position 5 so that the cellular location of FDH-N would not be compromised. To circumvent possible translational errors associated with use of rare arginine codons, such as AGG, in E. coli (see, e.g., reference 5), we additionally introduced into each strain plasmid pUt-AGA/AGG, which carries the genes encoding tRNAArgUCU and tRNAArgUCC (22). In the presence of this plasmid, but not in its absence, we saw an approximate fourfold increase in FDH-N activity from the strain with the R5Rlow chromosomal replacement of fdnG (Fig. ​(Fig.3A)3A) and a corresponding overproduction of the FdnG polypeptide (Fig. ​(Fig.3C).3C). This result strongly suggests that the translational control of fdnG expression is of physiological significance, since we have clearly demonstrated that the cell has the capacity to synthesize, assemble, and export fourfold-higher levels of FDH-N. Interestingly, as shown in Fig. ​Fig.3B3B the fourfold overproduction of FDH-N activity was associated with a marked increase in NAR activity. This suggests that there might be a further level of coordinate control of FDH-N and NAR synthesis not previously observed. FIG. 3. The stem-loop structure regulates FDH-N activity in vivo. FDH-N (A) and NAR (B) enzyme assays were performed with crude cell extracts derived from strains MC4100, NRS9 (as MC4100; FdnG R5R [CGC→CGT]), and CMP1 (as MC4100; FdnG R5R [CGC→AGG]) ... In conclusion, we have demonstrated that there is control of FDH-N synthesis at the translational level. Interestingly, the stem-loop structure seen here for the E. coli mRNA is also predicted to be conserved in the fdnG coding regions of several other bacteria, including Salmonella enterica serovar Typhimurium, Haemophilus influenzae, and Pseudomonas aeruginosa. Notably, however, the stem-loop structure is apparently not conserved over the similar coding region of E. coli fdoG. While the mechanism underlying modulation of FDH-N translation is presently unclear, it may potentially serve as a quality control feature ensuring that only the fully assembled complex is transported to the periplasm.

The Tat protein export system is located in the bacterial cytoplasmic membrane and operates in parallel to the well-known Sec pathway. While the Sec system only transports unstructured substrates, the function of the Tat pathway is to translocate folded proteins. The Tat translocase thus faces the formidable challenge of moving structured macromolecular substrates across the bacterial cytoplasmic membrane without rendering the membrane freely permeable to protons and other ions. The substrates of the Tat pathway are often proteins that bind cofactor molecules in the cytoplasm, and are thus folded, prior to export. Such periplasmic cofactor-containing proteins are essential for most types of bacterial respiratory and photosynthetic energy metabolism. In addition, the Tat pathway is involved in outer membrane biosynthesis and in bacterial pathogenesis. Substrates are targeted to the Tat pathway by amino-terminal signal sequences harbouring consecutive, essentially invariant, arginine residues, and movement of proteins through the Tat system is energized by the transmembrane proton electrochemical gradient. The TatA protein probably forms the transport channel while the TatBC proteins act as a receptor complex that recognizes the signal peptide of the substrate protein.

The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a "suicide enzyme" strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein-protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway.

The hyf locus ( hyfABCDEFGHIJ-hyfR-focB ) of Escherichia coli encodes a putative 10-subunit hydrogenase complex (hydrogenase-4 [Hyf]); a potential σ 54 -dependent transcriptional activator, HyfR (related to FhlA); and a putative formate transporter, FocB (related to FocA). In order to gain insight into the physiological role of the Hyf system, we investigated hyf expression by using a hyfA-lacZ transcriptional fusion. This work revealed that hyf is induced under fermentative conditions by formate at a low pH and in an FhlA-dependent fashion. Expression was σ 54 dependent and was inhibited by HycA, the negative transcriptional regulator of the formate regulon. Thus, hyf expression resembles that of the hyc operon. Primer extension analysis identified a transcriptional start site 30 bp upstream of the hyfA structural gene, with appropriately located −24 and −12 boxes indicative of a σ 54 -dependent promoter. No reverse transcriptase PCR product could be detected for hyfJ-hyfR , suggesting that hyfR-focB may be independently transcribed from the rest of the hyf operon. Expression of hyf was strongly induced (∼1,000-fold) in the presence of a multicopy plasmid expressing hyfR from a heterologous promoter. This induction was dependent on low pH, anaerobiosis, and postexponential growth and was weakly enhanced by formate. The hyfR- expressing plasmid increased fdhF-lacZ transcription just twofold but did not influence the expression of hycB-lacZ . Interestingly, inactivation of the chromosomal hyfR gene had no effect on hyfA-lacZ expression. Purified HyfR was found to specifically interact with the hyf promoter/operator region. Inactivation of the hyf operon had no discernible effect on growth under the range of conditions tested. No Hyf-derived hydrogenase or formate dehydrogenase activity could be detected, and no Ni-containing protein corresponding to HyfG was observed.

The tetrathionate (Ttr) and thiosulfate (Phs) reductases of Salmonella enterica LT2, together with the polysulfide reductase (Psr) of Wolinella succinogenes, are unusual examples of enzymes containing a molybdopterin active-site cofactor since all formally catalyse sulfur-sulfur bond cleavage. This is in contrast to the oxygen or hydrogen transfer reactions exhibited by other molybdopterin enzymes. Here the catalytic specificity of Ttr and Phs has been compared using both physiological and synthetic electron-donor systems. Ttr is shown to catalyse reduction of trithionate but not sulfur or thiosulfate. In contrast, Phs cannot reduce tetrathionate or trithionate but allows whole cells to utilize elemental sulfur as an electron acceptor. Mechanisms are proposed by which the bacterium is able to utilize an insoluble sulfur substrate by means of reactions at the cytoplasmic rather than the outer membrane.

The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli . C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport. These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted.

The Tat protein-export system serves to translocate folded proteins, often containing redox cofactors, across the bacterial inner membrane. Substrate proteins are directed to the Tat apparatus by distinctive N-terminal signal peptides containing a consensus SRRxFLK 'twin-arginine' motif. Here we review recent studies of the Tat system with particular emphasis on the assembly of membrane-bound respiratory complexes. We discuss the connection between Tat targeting and topological organisation of the complexes and consider the role of chaperone proteins in cofactor insertion and Tat targeting. The crystal structure of Escherichia coli formate dehydrogenase-N demonstrates that some Tat substrates are integral membrane proteins. Sequence analysis suggests that one-quarter of all traffic on the E. coli Tat pathway is inner-membrane proteins.

Proteins of the CcdA/DsbD family have previously been found to be involved in the protein disulfide isomerase and cytochrome c maturation pathways of bacteria. SoxV is a CcdA homologue encoded by a genetic locus involved in lithotrophic thiosulfate oxidation in Rhodovulum sulfidophilum. Mutagenesis studies demonstrate an essential and specific role for SoxV in thiosulfate oxidation. Another protein encoded by the same locus, SoxW, is a periplasmic thioredoxin. SoxW was found to be in the reduced state during growth of R. sulfidophilum in the presence of thiosulfate. Maintenance of SoxW in the reduced state was shown to require SoxV. Nevertheless, SoxW was found to be dispensible for thiosulfate oxidation suggesting that SoxV reduces more than one periplasmic partner protein.

The first electron nuclear double resonance (ENDOR) study of a member of the Mo-bis-molybdopterin guanine dinucleotide family of molybdoenzymes is presented, using the periplasmic nitrate reductase from Paracoccus pantotrophus. Rapid freeze-quenched time-resolved EPR revealed that during turnover the intensity of a Mo(V) EPR signal known as High-g [resting] increases. This signal is split by two interacting protons that are not solvent-exchangeable. X-band proton-ENDOR analysis resolved broad symmetrical resonance features that arose from four classes of protons weakly coupled to the Mo(V). Signals from two of these were lost upon exchange into deuterated buffer, suggesting that they may originate from OH− or H2O groups. One of these signals was also lost when the enzyme was redox-cycled in the presence of azide. Since these protons are very weakly coupled OH/H2O groups, they are not likely to be ligated directly to the Mo(V). This suggests that protonation of a Mo(VI)=O group does not occur on reduction to Mo(V), but most probably accompanies reduction of Mo(V) to Mo(IV). A resonance feature from a more strongly coupled proton, that was not lost following exchange into deuterated buffer, could also be resolved at 22–24MHz. The anisotropy of this feature, determined from ENDOR spectra collected at a range of field positions, indicated a Mo—proton distance of approx. 3.2Å, consistent with this being one of the β-methylene protons of a Mo—Cys ligand.

The twin-arginine translocation (Tat) system, found in prokaryotes, chloroplasts, and some mitochondria, allows folded proteins to be moved across membranes. How this transport is achieved without significant ion leakage is an intriguing mechanistic question. Tat transport is mediated by complexes formed from small integral membrane proteins from just two protein families. Atomic-resolution structures have recently been determined for representatives of both these protein families, providing the first molecular-level glimpse of the Tat machinery. I review our current understanding of the mechanism of Tat transport in light of these new structural data.

Many photosynthetic bacteria use inorganic sulfur compounds as electron donors for carbon dioxide fixation. A thiosulfate-induced cytochrome c has been purified from the photosynthetic α-proteobacterium Rhodovulum sulfidophilum . This cytochrome c 551 is a heterodimer of a diheme 30-kDa SoxA subunit and a monoheme 15-kDa SoxX subunit. The cytochrome c 551 structural genes are part of an 11-gene sox locus. Sequence analysis suggests that the ligands to the heme iron in SoxX are a methionine and a histidine, while both SoxA hemes are predicted to have unusual cysteine-plus-histidine coordination. A soxA mutant strain is unable to grow photoautotrophically on or oxidize either thiosulfate or sulfide. Cytochrome c 551 is thus essential for the metabolism of both these sulfur species. Periplasmic extracts of wild-type R. sulfidophilum exhibit thiosulfate:cytochrome c oxidoreductase activity. However, such activity can only be measured for a soxA mutant strain if the periplasmic extract is supplemented with purified cytochrome c 551 . Gene clusters similar to the R. sulfidophilum sox locus can be found in the genome of a green sulfur bacterium and in phylogenetically diverse nonphotosynthetic autotrophs.

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin-arginine signal peptides. The essential Tat pathway components TatA, TatB and TatC are shown to be integral membrane proteins. Upon removal of the predicted N-terminal transmembrane helix TatA becomes a water-soluble protein. In contrast the homologous TatB protein retains weak peripheral interactions with the cytoplasmic membrane when the analogous helix is deleted. Chemical crosslinking studies indicate that TatA forms at least homotrimers, and TatB minimally homodimers, in the native membrane environment. The presence of such homo-oligomeric interactions is supported by size exclusion chromatography.

The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co-overexpressing tatABCDE show an increased rate of export of a signal peptide-defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild-type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.

Currently described substrates of the bacterial Tat protein transport system are directed for export by signal peptides containing a pair of invariant arginine residues. The signal peptide of the TtrB subunit of Salmonella enterica tetrathionate reductase contains a single arginine residue but is nevertheless able to mediate Tat pathway transport. This naturally occurring example of a Tat signal peptide lacking a consensus arginine pair expands the range of sequences that can target a protein to the Tat pathway. The possible implications of this finding for the assembly of electron transfer complexes containing Rieske proteins in plant organelles are discussed.

The transcription start sites for the tatABCD and tatE loci, encoding components of the Tat (twin-arginine translocase) protein export pathway, have been identified. Expression studies indicate that the tatABCD and tatE transcription units are expressed constitutively. Translational fusion experiments suggest that TatA is synthesized at a much higher level than the other Tat proteins.

Prokaryotic nitrate reduction can serve a number of physiological roles and can be catalysed by a number of biochemically distinct nitrate reductases. Three distinct nitrate reductase classes can be indentified in prokaryotes, NAS, NAR and NAP. NAS is located in the cytoplasmic compartment and participates in nitrogen assimilation. NAR is usually a three-subunit complex anchored to the cytoplasmic face of the membrane with its active site located in the cytoplasmic compartment and is involved in anaerobic nitrate respiration. NAP is a two-subunit complex, located in the periplasmic compartment, that is coupled to quinol oxidation via a membrane anchored tetraheme cytochrome. It shows considerable functional flexibility by participating in anaerobic respiration or redox energy dissipation depending on the organism in which it is found. The members of all three classes of enzymes bind the bis-molybdopterin guanine dinucleotide cofactor at the active site, but they differ markedly in the number and nature of cofactors used to transfer electrons to this site. Analysis of prokaryotic genome sequences available at the time of writing reveals that the different nitrate reductases are phylogenetically widespread.