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Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed coevolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three supersecreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than those observed for wild-type TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of more efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.

Summary Neoagarobiose (NA2) derived from agar marine biomass is a rare reagent that acts as an anti‐melanogenesis reagent and moisturizer. Here, for the economical manufacturing of NA2, we developed the co‐secretory production system of endo‐type β‐agarases (DagA) and exo‐type β‐agarases (EXB3) in Corynebacterium glutamicum. For this purpose, we first developed a secretory system of DagA via Tat pathway. To improve the secretion efficiency, we coexpressed two Tat pathway components (TatA and TatC), and to improve the purity of secreted DagA in the culture supernatant, two endogenous protein genes (Cg2052 and Cg1514) were removed. Using the engineered strain (C. glutamicum SP002), we confirmed that DagA as high as 1.53 g l‐1 was successfully produced in the culture media with high purity (72.7% in the supernatant protein fraction). Next, we constructed the expression system (pHCP‐CgR‐DagA‐EXB3) for the simultaneous secretion of EXB3 via Sec‐pathway together with DagA, and it was clearly confirmed that DagA and EXB3 were successfully secreted as high as 54% and 24.5%, respectively. Finally, using culture medium containing DagA and EXB3, we successfully demonstrated the conversion of high‐concentration agar (40 g l‐1) into NA2 via a two‐stage hydrolysis process.

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoids, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits: TatA, TatB, and TatC. TatA and TatB possess short transmembrane alpha helices (TMHs), both of which are only 15 residues long in Escherichia coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer, although the functional significance of this mismatch is unclear. Here, we sought to address the functional importance of the hydrophobic mismatch in the Tat transport mechanism in E.coli. We conducted three different assays to evaluate the effect of TMH length mutants on Tat activity and observed that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off following any modification of this length. Surprisingly, TatA and TatB with as few as 11 residues in their TMHs can still insert into the membrane bilayer, albeit with a decline in membrane integrity. These findings support a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, we conclude that the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, we examined a complete set of 19-amino-acid substitutions. Unexpectedly, although the polar amino acid is preferred overall, our experiments suggest that it is not necessary for a functional TatA. Hydrophilicity and helix-stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophilicity, which was demonstrated to impact the Tat transport activity. Furthermore, we identified a four-residue motif at the N terminus of the TatA TMH by sequence alignment. Using a biochemical approach, we found that the N-terminal motif was functionally significant, with evidence indicating a potential role in the preference for utilizing different proton-motive force components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

Phenazine-1-carboxylic acid (PCA), a member of phenazines secreted by microorganisms, inhibits the growth of many bacteria and fungi. Xanthomonas campestris pv. campestris is the causal agent of black rot, the most important disease of cruciferous crops worldwide, and is more tolerant to PCA than other Xanthomonas species. Previous studies reported that reactive oxygen species (ROS) scavenging ability is involved in regulating the PCA tolerance of Xanthomonas species. Additionally, the cytochrome c maturation (CCM) system has been found to play a more important role in tolerance to phenazines than the ROS scavenging system. In this study, a highly PCA-sensitive insertion mutant of X. campestris pv. campestris, X-5, was identified and studied. The insertion site of X-5 was found to be in tatB gene (XC_4183), which encodes a subunit of the twin-arginine translocation (TAT) complex. Disruption of the three genes of TAT pathway resulted in decreased biological fitness and reduced tolerance to phenazines in comparison with the wild-type strain 8004. These results imply that the tolerance mechanism of the TAT pathway to phenazines is related to the CCM system, but not due to the ROS scavenging system. Furthermore, respiration-related characteristic tests and peptide analysis suggested that disruption of the TAT complex causes a defect in the cytochrome bc1 complex, which may be involved in the tolerance to phenazines. In summary, this study sheds new light on the critical role of the TAT pathway in influencing the fitness and phenazines tolerance of Xanthomonas species.

The twin‐arginine protein transport (Tat pathway) is found in prokaryotes and plant organelles and transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N‐terminal signal sequence containing a highly conserved twin‐arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. Assembly of the Tat translocon is dynamic and is triggered by the interaction of a Tat substrate with the Tat receptor complex. This review will summarise recent advances in our understanding of Tat transport, focusing in particular on the roles played by Tat signal peptides in protein targeting and translocation., The twin‐arginine protein transport (Tat) pathway transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N‐terminal signal sequence containing a highly conserved twin‐arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. This review will summarise recent advances in our understanding of Tat transport.

Discovery of multidrug resistance (MDR) in environmental microorganisms provides unique resources for uncovering antibiotic resistomes, which could be vital to predict future emergence of MDR pathogens. Our previous studies indicated that Lysobacter sp. conferred intrinsic resistance to multiple antibiotics at high levels, especially ampicillin, the first broad-spectrum β-lactam antibiotics against both Gram-positive and Gram-negative bacteria. However, the underlying molecular mechanisms for resistance to ampicillin in Lysobacter enzymogenes strain C3 (LeC3) remain unknown. In this study, screening a Tn5 transposon mutant library of LeC3 recovered 12 mutants with decreased ampicillin resistance, and three mutants (i.e., tatC, lebla, and lpp) were selected for further characterization. Our results revealed that genes encoding β-lactamase (lebla) and twin-arginine translocation (tatC) system for β-lactamase transport played a pivotal role in conferring ampicillin resistance in L. enzymogenes. It was also demonstrated that the lpp gene was not only involved in resistance against β-lactams but also conferred resistance to multiple antibiotics in L. enzymogenes. Permeability assay results indicated that decreased MDR in the lpp mutant was in part due to its higher cellular permeability. Furthermore, our results showed that the difference of LeC3 and L. antibioticus strain LaATCC29479 in ampicillin susceptibility was partly due to their differences in cellular permeability, but not due to β-lactamase activities.

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (pmf) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, a complete set of 19-amino-acid substitutions was examined. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophobicity and helix stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophobicity, which was demonstrated to impact the Tat transport activity. Furthermore, a four-residue motif at the N-terminus of the TatA TMH was identified by sequence alignment. Using a biochemical approach, the N-terminal motif was found to be functionally significant, with evidence indicating a potential role in the preference for utilizing different pmf components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargodependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable, but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typical low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.

A genome-wide investigation of the anhydrobiotic cyanobacterium Chroococcidiopsis sp. CCMEE 029 identified three genes coding superoxide dismutases (SODs) annotated as MnSODs (SodA2.1 and SodA2.2) and Cu/ZnSOD (SodC) as suggested by the presence of metal-binding motifs and conserved sequences. Structural bioinformatics analysis of the retrieved sequences yielded modeled MnSODs and Cu/ZnSOD structures that were fully compatible with their functional role. A signal-peptide bioinformatics prediction identified a Tat signal peptide at the N-terminus of the SodA2.1 that highlighted its transport across the thylakoid/cytoplasmic membranes and release in the periplasm/thylakoid lumen. Homologs of the Tat transport system were identified in Chroococcidiopsis sp. CCMEE 029, and the molecular docking simulation confirmed the interaction between the signal peptide of the SodA2.1 and the modeled TatC receptor, thus supporting the SodA2.1 translocation across the thylakoid/cytoplasmic membranes. No signal peptide was predicted for the MnSOD (SodA2.2) and Cu/ZnSOD, thus suggesting their occurrence as cytoplasmic proteins. No FeSOD homologs were identified in Chroococcidiopsis sp. CCMEE 029, a feature that might contribute to its desiccation tolerance since iron produces hydroxyl radical via the Fenton reaction. The overall-overexpression in response to desiccation of the three identified SOD-coding genes highlighted the role of SODs in the antioxidant enzymatic defense of this anhydrobiotic cyanobacterium. The periplasmic MnSOD protected the cell envelope against oxidative damage, the MnSOD localized in the thylakoid lumen scavengered superoxide anion radical produced during the photosynthesis, while the cytoplasmic MnSOD and Cu/ZnSOD reinforced the defense against reactive oxygen species generated at the onset of desiccation. Results contribute to decipher the desiccation-tolerance mechanisms of this cyanobacterium and allow the investigation of its oxidative stress response during future space experiments in low Earth orbit and beyond.

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we assess the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.

The Tat system has the unique purpose to translocate folded proteins across energy-transducing membranes. It occurs in bacteria and archaea, as well as in eukaryotic organelles of bacterial origin. In the bacterial model system Escherichia coli, the three components TatA, TatB, and TatC assemble to functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) that is followed by an amphipathic helix (APH). The TMHs of TatA and TatB generate a hydrophobic mismatch with only 12 consecutive hydrophobic residues that span the membrane. We shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both, and analyzed effects on transport functionality and translocon assembly. The wild type length functioned best but was not an absolute requirement, as some variation was clearly tolerated. Defects of shortenings or extensions were enhanced by simultaneous mutations in TatA and TatB, indicating partial compensations of mutations in TatA by wild type TatB or vice versa. Length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residues-length is important for Tat component interactions and translocon assembly. To also address potential effects on TatA associations, we characterized these by metal tagging transmission electron microscopy and carried out molecular dynamics simulations. In these simulations, interacting short TMHs of larger TatA assemblies were thinning the membrane together with laterally aligned tilted APHs that generated a deep V-shaped groove. The conserved length of 12 hydrophobic residues may thus not only be important for translocon interactions, but also for a membrane destabilization during Tat transport. If this is the case, the specific short length could be a compromise between functionality and proton leakage minimization.

Cytochrome c6 is a redox carrier in the thylakoid lumen of cyanobacteria and some eukaryotic algae. Although the isofunctional plastocyanin is present in land plants and the green alga Chlamydomonas reinhardtii, these organisms also possess a cytochrome c6-like protein designated as cytochrome c6A. Two other cytochrome c6-like groups, c6B and c6C, have been identified in cyanobacteria. In this study, we have identified a novel c6-like cytochrome called PetJ2, which is encoded in the nuclear genome of Cyanophora paradoxa, a member of the glaucophytes – the basal branch of the Archaeplastida. We propose that glaucophyte PetJ2 protein is related to cyanobacterial c6B and c6C cytochromes, and that cryptic green algal and land plant cytochromes c6A evolved from an ancestral archaeplastidial PetJ2 protein. In vitro import experiments with isolated muroplasts revealed that PetJ2 is imported into plastids. Although it harbors a twin-arginine motif in its thylakoid-targeting peptide, which is generally indicative of thylakoid import via the Tat import pathway, our import experiments with isolated muroplasts and the heterologous pea thylakoid import system revealed that PetJ2 uses the Sec pathway instead of the Tat import pathway.

Twin-arginine-dependent translocases transport folded proteins across bacterial, archaeal, and chloroplast membranes. Upon substrate binding, they assemble from hexahelical TatC and single-spanning TatA and TatB membrane proteins. Although structural and functional details of individual Tat subunits have been reported previously, the sequence and dynamics of Tat translocase assembly remain to be determined. Employing the zero-space cross-linker N,N′-dicyclohexylcarbodiimide (DCCD) in combination with LC-MS/MS, we identified as yet unknown intra- and intermolecular contact sites of TatB and TatC. In addition to their established intramembrane binding sites, both proteins were thus found to contact each other through the soluble N terminus of TatC and the interhelical linker region around the conserved glutamyl residue Glu49 of TatB from Escherichia coli. Functional analyses suggested that by interacting with the TatC N terminus, TatB improves the formation of a proficient substrate recognition site of TatC. The Glu49 region of TatB was found also to contact distinct downstream sites of a neighboring TatB molecule and to thereby mediate oligomerization of TatB within the TatBC receptor complex. Finally, we show that global DCCD-mediated cross-linking of TatB and TatC in membrane vesicles or, alternatively, creating covalently linked TatC oligomers prevents TatA from occupying a position close to the TatBC-bound substrate. Collectively, our results are consistent with a circular arrangement of the TatB and TatC units within the TatBC receptor complex and with TatA entering the interior TatBC-binding cavity through lateral gates between TatBC protomers.

The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations.

We cloned a set of genes encoding alcohol oxidase from Ochrobactrum sp. AIU 033 (OcAOD), which exhibits the appropriate substrate specificity for glyoxylic acid production from glycolic acid. The set of genes for OcAOD contained two open reading frames consisting of 555-bp (aodB) and 1572-bp (aodA) nucleotides, which encode the precursor for the β-subunit and α-subunit of OcAOD, respectively. We expressed the cloned genes as an active product in Escherichia coli BL21(DE3). The recombinant OcAOD oxidized glycolic acid and primary alcohols with C2–C8 but not glyoxylic acid (as is the case for native OcAOD), whereas the Km and Vmax values for glycolic acid and the pH stability were higher than those of native OcAOD. A consensus sequence for the twin-arginine translocation (Tat) pathway was identified in the N-terminal region of the precursor for the β-subunit, and the active form of OcAOD was localized in the periplasm of recombinant E. coli, which indicated that OcAOD would be transported from the cytoplasm to the periplasm by the hitchhiker mechanism through the Tat pathway. The OcAOD productivity of the recombinant E. coli was 24-fold higher than that of Ochrobactrum sp. AIU 033, and it was further enhanced by 1.2 times by the co-expression of additional tatABC from E. coli BL21(DE3). Our findings thus suggest a function of the β-subunit of OcAOD in membrane translocation, and that the recombinant OcAOD has characteristics that are suitable for the enzymatic synthesis of glyoxylic acid as well as native OcAOD.

Plant-pathogenic Ralstonia spp. colonize plant xylem and cause wilt diseases on a broad range of host plants. To identify genes that promote growth of diverse Ralstonia strains in xylem sap from tomato plants, we performed genome-scale genetic screens (random barcoded transposon mutant sequencing screens [RB-TnSeq]) in three strains spanning the genetic, geographical, and physiological range of plant-pathogenic Ralstonia: Ralstonia solanacearum IBSBF1503, Ralstonia pseudosolanacearum GMI1000, and Ralstonia syzygii PSI07. Contrasting mutant fitness phenotypes in culture media versus in xylem sap suggest that Ralstonia strains are adapted to ex vivo xylem sap and that culture media impose foreign selective pressures. Although wild-type Ralstonia grew in sap and in rich medium with similar doubling times and to a similar carrying capacity, more genes were essential for growth in sap than in rich medium. Each strain required many genes associated with envelope remodeling and repair processes for full fitness in xylem sap. These genes were associated with peptidoglycan peptide formation (murI), secretion of periplasmic proteins (tatC), periplasmic protein folding (dsbA), synthesis of osmoregulated periplasmic glucans (mdoGH), and lipopolysaccharide (LPS) biosynthesis. Mutant strains with mutations in four genes had strong, sap-specific fitness defects in all strain backgrounds: murI, thiC, purU, and a lipoprotein (RSc2007). Many amino acid biosynthesis genes were required for fitness in both minimal medium and xylem sap. Multiple mutants with insertions in virulence regulators had gains of fitness in culture media and neutral fitness in sap. Our genome-scale genetic screen identified Ralstonia fitness factors that promote growth in xylem sap, an ecologically relevant condition. IMPORTANCE Traditional transposon mutagenesis genetic screens pioneered molecular plant pathology and identified core virulence traits like the type III secretion system. TnSeq approaches that leverage next-generation sequencing to rapidly quantify transposon mutant phenotypes are ushering in a new wave of biological discovery. Here, we have adapted a genome-scale approach, random barcoded transposon mutant sequencing (RB-TnSeq), to discover fitness factors that promote growth of three related bacterial strains in a common niche, tomato xylem sap. Fitness of the wild type and mutants show that Ralstonia spp. are adapted to grow well in xylem sap from their natural host plant, tomato. Our screen identified multiple sap-specific fitness factors with roles in maintaining the bacterial envelope. These factors include putative adaptations to resist plant defenses that may include antimicrobial proteins and specialized metabolites that damage bacterial membranes.

Persisters are metabolically quiescent phenotypic variants of the wild type that are tolerant to cidal antibiotics, and the mechanisms of persister formation and survival are complex and not completely understood. To identify genes involved in persistence to tosufloxacin, which has higher activity against persisters than most other quinolones, we screened the E. coli KEIO mutant library using a different condition from most persister mutant screens (6 h) with a longer exposure of 18 h with tosufloxacin. We identified 18 mutants (acrA, acrB, ddlB, dnaG, gltI, hlpA, lpcA, recG, recN, rfaH, ruvC, surA, tatC, tolQ, uvrD, xseA, and ydfI) that failed to form tosufloxacin tolerant persisters. Among them, gltI, hlpA, ruvC, ddlB, ydfI, and tatC are unique genes involved in E. coli persistence to tosufloxacin which have not been reported before. Furthermore, deletion mutants in genes coding periplasmic proteins (surA, lpcA, hlpA, and gltI) had more defect in persistence to tosufloxacin than the other identified mutants, with surA and lpcA mutants being the most prominent. The “deep” persister phenotype of surA and lpcA mutants was further confirmed both in vitro and in vivo. Compared with the wild type strain E. coli BW25113 in vitro, the persister phenotype of the surA and lpcA mutants was decreased more than 100–1,000-fold in persistence to various antibiotics, acidic, hyperosmotic and heat conditions. In addition, in both stationary phase bacteria and biofilm bacteria infection mouse models, the surA and lpcA mutants had lower survival and persistence than the parent uropathogenic strain UTI89, suggesting that the in vitro identified persister mechanisms (surA and lpcA) are operative and valid for in vivo persistence. Our findings provide new insight into the mechanisms of persister formation and maintenance under tosufloxacin and will likely provide novel therapeutic and vaccine targets for developing more effective treatment and prevention of persistent E. coli infections.

Consistent with a role in catalyzing rate-limiting step of protein folding, removal of genes encoding cytoplasmic protein folding catalysts belonging to the family of peptidyl-prolyl cis/trans isomerases (PPIs) in Escherichia coli confers conditional lethality. To address the molecular basis of the essentiality of PPIs, a multicopy suppressor approach revealed that overexpression of genes encoding chaperones (DnaK/J and GroL/S), transcriptional factors (DksA and SrrA), replication proteins Hda/DiaA, asparatokinase MetL, Cmk and acid resistance regulator (AriR) overcome some defects of &Delta, 6ppi strains. Interestingly, viability of &Delta, 6ppi bacteria requires the presence of transcriptional factors DksA, SrrA, Cmk or Hda. DksA, MetL and Cmk are for the first time shown to exhibit PPIase activity in chymotrypsin-coupled and RNase T1 refolding assays and their overexpression also restores growth of a &Delta, (dnaK/J/tig) strain, revealing their mechanism of suppression. Mutagenesis of DksA identified that D74, F82 and L84 amino acid residues are critical for its PPIase activity and their replacement abrogated multicopy suppression ability. Mutational studies revealed that DksA-mediated suppression of either &Delta, 6ppi or &Delta, dnaK/J is abolished if GroL/S and RpoE are limiting, or in the absence of either major porin regulatory sensory kinase EnvZ or RNase H, transporter TatC or LepA GTPase or Pi-signaling regulator PhoU.

Cyanobacteria are ubiquitous oxygenic photosynthetic bacteria with a versatile metabolism that is highly dependent on effective protein targeting. Protein sorting in diderm bacteria is not trivial and, in cyanobacteria, even less so due to the presence of a complex membrane system: the outer membrane, the plasma membrane and the thylakoid membrane. In cyanobacteria, protein import into the thylakoids is essential for photosynthesis, export to the periplasm fulfills a multifunctional role in maintaining cell homeostasis, and secretion mediates motility, DNA uptake and environmental interactions. Intriguingly, only one set of genes for the general secretory and the twin-arginine translocation pathways seem to be present. However, these systems have to operate in both plasma and thylakoid membranes. This raises the question of how substrates are recognized and targeted to their correct, final destination. Additional complexities arise when a protein has to be secreted across the outer membrane, where very little is known regarding the mechanisms involved. Given their ecological importance and biotechnological interest, a better understanding of protein targeting in cyanobacteria is of great value. This review will provide insights into the known knowns of protein targeting, propose hypotheses based on available genomic sequences and discuss future directions.

The biogenesis of membrane-bound electron transport chains requires membrane translocation pathways for folded proteins carrying complex cofactors, like the Rieske Fe/S proteins. Two independent systems were developed during evolution, namely the Twin-arginine translocation (Tat) pathway, which is present in bacteria and chloroplasts, and the Bcs1 pathway found in mitochondria of yeast and mammals. Mitochondria of plants carry a Tat-like pathway which was hypothesized to operate with only two subunits, a TatB-like protein and a TatC homolog (OrfX), but lacking TatA. Here we show that the nuclearly encoded TatA has dual targeting properties, i.e., it can be imported into both, chloroplasts and mitochondria. Dual targeting of TatA was observed within organelloexperiments employing isolated chloroplasts and mitochondria as well as after transient expression of suitable reporter constructs in epidermal leaf cells. The extent of transport of these constructs into mitochondria of transiently transformed leaf cells was relatively low, causing a demand for highly sensitive methods to be detected, like thesasplitGFP approach. Yet, the dual import of TatA into mitochondria and chloroplasts observed here points to a common mechanism of Tat transport for folded proteins within both endosymbiotic organelles.

Certain methanogens deteriorate steel surfaces through a process called microbiologically influenced corrosion (MIC). However, the mechanisms of MIC, whereby methanogens oxidize zerovalent iron (Fe0), are largely unknown. In this study, Fe0-corroding Methanococcus maripaludis strain OS7 and its derivative (strain OS7mut1) defective in Fe0-corroding activity were isolated. Genomic analysis of these strains demonstrated that the strain OS7mut1 contained a 12-kb chromosomal deletion. The deleted region, termed “MIC island”, encoded the genes for the large and small subunits of a [NiFe] hydrogenase, the TatA/TatC genes necessary for the secretion of the [NiFe] hydrogenase, and a gene for the hydrogenase maturation protease. Thus, the [NiFe] hydrogenase may be secreted outside the cytoplasmic membrane, where the [NiFe] hydrogenase can make direct contact with Fe0, and oxidize it, generating hydrogen gas: Fe0 + 2 H+ → Fe2+ + H2. Comparative analysis of extracellular and intracellular proteomes of strain OS7 supported this hypothesis. The identification of the MIC genes enables the development of molecular tools to monitor epidemiology, and to perform surveillance and risk assessment of MIC-inducing M. maripaludis.

The Gram-negative pathogenic bacterium Dickeya zeae causes soft rot in rice, corn and many other plants. The molecular mechanisms underlying D. zeae virulence are not clearly understood. In this study, we characterized the twin-arginine translocation (Tat) system of this pathogen. The mutants of tatA, tatB, tatC and tatE of D. zeae EC1 were generated through the use of the suicide vector pKNG101 and their phenotypic characters were determined. Our results showed that mutations in D. zeae Tat system have no effect on the bacterial growth. △tatA, △tatB and △tatE mutants reduced the motility distinctly, and could not form biofilm, while the △tatC mutant did not show a distinctive decrease on the motility and biofilm formation. Less production of proteases, pectate lyases and cellulases were shown in △tatA, △tatB, △tatC and △tatE mutants as compared with the wild type EC1, whereas the toxin in Tat mutants was reduced, as compared with the wild type. The Tat mutants △tatA, △tatB, △tatC and △tatE could cause soft rot in potato and Chinese cabbage and hypersensitive reactions (HR) in tobacco, that were not different significantly from those of the wild type EC1. However, △tatA, △tatB and △tatE were not significantly effective in the inhibition of rice seed germination compared to the wild type. Furthermore, the disease incidence of the Tat mutants decreased significantly when they were inoculated in rice plants. This reveals that the Tat system has a different role in the virulence of D. zeae in rice, potato and cabbage and it is required for the virulence of D. zeae in rice plants.

The twin-arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram-negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C-terminally truncated 'core TatA' from Arabidopsis thaliana using solution-state NMR. Our results show that TatA consists of a short N-terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using 15 N relaxation NMR in combination with model-free analysis. The TMH has order parameters characteristic of a well-structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast-tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N-terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments.

Twin-arginine translocation (Tat) systems transport folded proteins that harbor a conserved arginine pair in their signal peptides. They assemble from hexahelical TatC-type and single-spanning TatA-type proteins. Many Tat systems comprise two functionally diverse, TatA-type proteins, denominated TatA and TatB. Some bacteria in addition express TatE, which thus far has been characterized as a functional surrogate of TatA. For the Tat system of Escherichia coli we demonstrate here that different from TatA but rather like TatB, TatE contacts a Tat signal peptide independently of the proton-motive force and restricts the premature processing of a Tat signal peptide. Furthermore, TatE embarks at the transmembrane helix five of TatC where it becomes so closely spaced to TatB that both proteins can be covalently linked by a zero-space cross-linker. Our results suggest that in addition to TatB and TatC, TatE is a further component of the Tat substrate receptor complex. Consistent with TatE being an autonomous TatAB-type protein, a bioinformatics analysis revealed a relatively broad distribution of the tatE gene in bacterial phyla and highlighted unique protein sequence features of TatE orthologs.

Pentatricopeptide repeat (PPR) proteins constitute the largest family of proteins in angiosperms, and most members are predicted to play roles in the maturation of organellar RNAs. Here we describe the novel mitochondrial editing factor 31 (MEF31), an E-PPR protein involved in editing at two close sites in the same transcript encoding subunit C of the twin-arginine translocation (tat) pathway. MEF31 is essential for editing at site tatC-581 and application of the recently proposed amino acid code for RNA recognition by PPR proteins supports the view that MEF31 directly targets this site by recognizing its cis sequence. In contrast, editing at site tatC-586 five nucleotides downstream is only partially affected in plants lacking MEF31, being restored to wild-type levels in complemented plants. Application of the amino acid code and analysis of individual RNA molecules for editing at sites 581 and 586 suggest that MEF31 does not directly target site tatC-586, and only indirectly influences editing at this site. It is likely that editing at site tatC-581 improves recognition of the site tatC-586 cis sequence by a second unknown PPR protein.

Twin-arginine translocation (Tat) systems transport folded proteins across cellular membranes with the concerted action of mostly three membrane proteins: TatA, TatB, and TatC. Hetero-oligomers of TatB and TatC form circular substrate–receptor complexes with a central binding cavity for twin-arginine–containing signal peptides. After binding of the substrate, energy from an electro-chemical proton gradient is transduced into the recruitment of TatA oligomers and into the actual translocation event. We previously reported that Tat-dependent protein translocation into membrane vesicles of Escherichia coli is blocked by the compound N,N′-dicyclohexylcarbodiimide (DCCD, DCC). We have now identified a highly conserved glutamate residue in the transmembrane region of E. coli TatC, which when modified by DCCD interferes with the deep insertion of a Tat signal peptide into the TatBC receptor complex. Our findings are consistent with a hydrophobic binding cavity formed by TatB and TatC inside the lipid bilayer. Moreover, we found that DCCD mediates discrete intramolecular cross-links of E. coli TatC involving both its N- and C-tails. These results confirm the close proximity of two distant sequence sections of TatC proposed to concertedly function as the primary docking site for twin-arginine signal peptides.

Microorganisms secrete proteins to carry out different types of biological functions including biogenesis of flagella or pili, sequestration of nutrients, pathogenicity, etc. Translocation of these proteins across the plasma membrane to the periplasm or external environment takes place via two main pathways viz. Sec (secretory) and Tat (twin arginine translocation). Sec pathway translocates proteins in their unfolded state, whereas, Tat pathway favors the translocation of folded proteins. However, the selection of a particular translocation pathway must be accomplished during or immediately after the completion of protein biosynthesis. This suggests that the primary structure of proteins itself must have some specific signatures for Sec- and Tat-mediated translocation. To identify and further verify the signature motifs and properties of proteins translocated through Sec and Tat pathways, we took up a case study of periplasmic UgpB protein. UgpB is a periplasmic solute (or substrate) binding protein of UgpABCE ATP-binding cassette (ABC) transporter, which mediates the sequestration of either sn -glycerol-3-phosphate (G3P) or glycerophosphocholine (GPC) molecules in the periplasmic space. Recently, UgpB protein from Mycobacterium tuberculosis ( Mt UgpB) was reported to follow the Tat pathway for its translocation to the periplasm during its pathogenesis; hence it has been proposed as a promising candidate for drug target. Thus, in this study, we attempted to identify the translocation pathway for all UgpB proteins from different organisms available in protein databases. Our computational analysis of these proteins reveals that most of the UgpB proteins follow Sec pathway rather than Tat pathway, unlike Mt UgpB. Moreover, this study reveals that the selection of pathway by UgpB proteins mainly depends on the characteristics of signal peptide. In addition, it suggests that the selection of the translocation pathway can also be influenced by the environment in which the organism thrives.

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in their binding to the Tat translocase, but some facets of this interaction remain unclear. Here, we investigated the role of the hydrophobic (h-) region of the Escherichia coli trimethylamine N-oxide reductase (TorA) signal peptide in TatBC receptor binding in vivo and in vitro. We show that besides the RR motif, a minimal, functional h-region in the signal peptide is required for Tat-dependent export in Escherichia coli. Furthermore, we identified mutations in the h-region that synergistically suppressed the export defect of a TorA[KQ]-30aa-MalE Tat reporter protein in which the RR motif was replaced with a lysine-glutamine pair. Strikingly, all suppressor mutations increased the hydrophobicity of the h-region. By systematically replacing a neutral residue in the h-region with various amino acids, we detected a positive correlation between the hydrophobicity of the h-region and the translocation efficiency of the resulting reporter variants. In vitro cross-linking of residues located in the periplasmically-oriented part of the TatBC receptor to TorA[KQ]-30aa-MalE reporter variants harboring a more hydrophobic h-region in their signal peptides confirmed that unlike in TorA[KQ]-30aa-MalE with an unaltered h-region, the mutated reporters moved deep into the TatBC-binding cavity. Our results clearly indicate that, besides the Tat motif, the h-region of the Tat signal peptides is another important binding determinant that significantly contributes to the productive interaction of Tat precursor proteins with the TatBC receptor complex.

Most chloroplast proteins are synthesized in the cytosol and imported into chloroplasts. Many imported proteins are further targeted to the thylakoid membrane and lumen by the SEC1, TAT, or SRP/ALB3 translocases. Others are targeted to the inner chloroplast envelope membrane by undescribed translocases. Recently, a second SEC system (SEC2) consisting of SCY2, SECE2, and SECA2 was found in the chloroplast envelope. Null mutants of SCY2 in Arabidopsis (Arabidopsis thaliana) exhibit a severe embryo-lethal phenotype. To investigate the function of the SEC2 system in plants, we used inducible RNA interference to knock down SCY2 in Arabidopsis. Seedlings cultured with inducer were chlorotic with aberrant chloroplasts and undeveloped thylakoids, indicating an essential role for SCY2 in chloroplast biogenesis beyond embryo development. In SCY2 down-regulated seedlings, several thylakoid membrane proteins, including SCY1, ALB3, and TATC, and inner envelope membrane proteins, including TIC40, TIC110, and FTSH12, were reduced substantially, suggesting that they may be SEC2 substrates. Additional insight was achieved by the in vitro reconstitution of protein integration into chloroplast membranes. The results show that SCY1 and ALB3 target directly to the thylakoid membrane and are likely independent of SEC2. FTSH12 was integrated into the envelope membrane in a coupled import-integration reaction that was impaired by the SECA inhibitor sodium azide. The stromal intermediate of TIC40 integrated into the envelope in a reaction that was largely inhibited when antibodies against epitope-tagged SCY2 or SECE2 were applied. These data demonstrate that the SEC2 translocase likely integrates a subset of inner envelope membrane proteins, such as FTSH12 and TIC40.