Tat System Research Articles

Mechanistic Insights into the Twin-Arginine Translocation Cycle of Escherichia Coli by an In Vivo Single-Molecule Approach

The twin-arginine translocation (Tat) system is a unique protein targeting pathway which has been found in bacteria, archea and chloroplasts as well as in plant mitochondria. Its ability to transport fully folded proteins across the cytoplasmic membrane distinguishes it from other translocation pathways. In E. coli the essential components of the system, TatA, TatB, and TatC, have been isolated in complexes of different size, suggesting that a fully active Tat complex forms only transiently. Furthermore, the precise steps in the translocation cycle remain mostly unknown.We perform fluorescence microscopy and single-particle tracking to gain deeper insight into the dynamics of the Tat machinery. To this end, bacteria expressing low levels of GFP-fused Tat subunits are imaged with sensitive laser-illuminated wide-field fluorescence microscopy. Mobile fluorescent spots are observed, and their intensity and location determined by fitting them with a 2D Gaussian function. The trajectories of these spots are then established by linking the Gaussian fits in successive frames.Our data shows that diffusion of TatA-eGFP is heterogeneous, and that its average diffusion coefficient decreases when translocation substrate is over-expressed. Moreover, TatA-eGFP mobility depends on the existence of an electrochemical potential, which is the driving force behind the Tat translocation system. This could suggest that TatA-eGFP complexes undergo a topological transition upon becoming ‘translocation active’.

Engineering Antibody Fitness and Function Using Membrane-Anchored Display of Correctly Folded Proteins

A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.

Investigation of the impact of Tat export pathway enhancement on E. coli culture, protein production and early stage recovery

The twin arginine translocation (Tat) pathway occurs naturally in E. coli and has the distinct ability to translocate folded proteins across the inner membrane of the cell. It has the potential to export commercially useful proteins that cannot be exported by the ubiquitous Sec pathway. To better understand the bioprocess potential of the Tat pathway, this article addresses the fermentation and downstream processing performances of E. coli strains with a wild-type Tat system exporting the over-expressed substrate protein FhuD. These were compared to strains cell-engineered to over-express the Tat pathway, since the native export capacity of the Tat pathway is low. This low capacity makes the pathway susceptible to saturation by over-expressed substrate proteins, and can result in compromised cell integrity. However, there is concern in the literature that over-expression of membrane proteins, like those of the Tat pathway, can impact negatively upon membrane integrity itself. Under controlled fermentation conditions E. coli cells with a wild-type Tat pathway showed poor protein accumulation, reaching a periplasmic maximum of only 0.5 mg L⁻¹ of growth medium. Cells over-expressing the Tat pathway showed a 25% improvement in growth rate, avoided pathway saturation, and showed 40-fold higher periplasmic accumulation of FhuD. Moreover, this was achieved whilst conserving the integrity of cells for downstream processing: experimentation comparing the robustness of cells to increasing levels of shear showed no detrimental effect from pathway over-expression. Further experimentation on spheroplasts generated by the lysozyme/osmotic shock method--a scaleable way to release periplasmic protein--showed similar robustness between strains. A scale-down mimic of continuous disk-stack centrifugation predicted clarifications in excess of 90% for both intact cells and spheroplasts. Cells over-expressing the Tat pathway performed comparably to cells with the wild-type system. Overall, engineering E. coli cells to over-express the Tat pathway allowed for greater periplasmic yields of FhuD at the fermentation scale without compromising downstream processing performance.

The role of the twin-arginine translocation pathway in Escherichia coli K1 pathogenicity in the African migratory locust, Locusta migratoria

Escherichia coli K1 infection is a major cause of neonatal meningitis, with high rates of mortality and disability. Despite years of research, only a small number of factors contributing to E. coli K1 virulence have been identified. The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane of E. coli and is involved in the transport of folded proteins. In vivo and ex vivo models using the African migratory locust, Locusta migratoria, were employed to explore the role of Tat pathway in E. coli K1 virulence using tat-deletion mutants. Groups of locusts were infected and mortality was recorded at 24-h intervals. The findings revealed that ΔtatA, ΔtatAC and Δtat produced levels of mortality similar to wild-type E. coli K1, with >78% mortality recorded within 72 h. Bacteraemia was determined from haemolymph obtained 3 and 24 h postinfection. Again, wild-type and ΔtatA produced similar levels of bacteraemia. In contrast, ΔtatAC and Δtat produced lower levels of bacteraemia. Following injection of bacteria into isolated head capsules ex vivo, all mutants invaded the CNS. Overall, these studies showed no evidence of involvement of the Tat pathway in locust mortality but suggest its possible role in bacteraemia.

The Tat-dependent protein translocation pathway.

The twin-arginine translocation (Tat) pathway is found in bacteria, archaea, and plant chloroplasts, where it is dedicated to the transmembrane transport of fully folded proteins. These proteins contain N-terminal signal peptides with a specific Tat-system binding motif that is recognized by the transport machinery. In contrast to other protein transport systems, the Tat system consists of multiple copies of only two or three usually small (∼8-30 kDa) membrane proteins that oligomerize to two large complexes that transiently interact during translocation. Only one of these complexes includes a polytopic membrane protein, TatC. The other complex consists of TatA. Tat systems of plants, proteobacteria, and several other phyla contain a third component, TatB. TatB is evolutionarily and structurally related to TatA and usually forms tight complexes with TatC. Minimal two-component Tat systems lacking TatB are found in many bacterial and archaeal phyla. They consist of a 'bifunctional' TatA that also covers TatB functionalities, and a TatC. Recent insights into the structure and interactions of the Tat proteins have various important implications.

Salt Sensitivity of Minimal Twin Arginine Translocases

Bacterial twin arginine translocation (Tat) pathways have evolved to facilitate transport of folded proteins across membranes. Gram-negative bacteria contain a TatABC translocase composed of three subunits named TatA, TatB, and TatC. In contrast, the Tat translocases of most Gram-positive bacteria consist of only TatA and TatC subunits. In these minimal TatAC translocases, a bifunctional TatA subunit fulfils the roles of both TatA and TatB. Here we have probed the importance of conserved residues in the bifunctional TatAy subunit of Bacillus subtilis by site-specific mutagenesis. A set of engineered TatAy proteins with mutations in the cytoplasmic hinge and amphipathic helix regions were found to be inactive in protein translocation under standard growth conditions for B. subtilis or when heterologously expressed in Escherichia coli. Nevertheless, these mutated TatAy proteins did assemble into TatAy and TatAyCy complexes, and they facilitated membrane association of twin arginine precursor proteins in E. coli. Interestingly, most of the mutated TatAyCy translocases were salt-sensitive in B. subtilis. Similarly, the TatAC translocases of Bacillus cereus and Staphylococcus aureus were salt-sensitive when expressed in B. subtilis. Taken together, our present observations imply that salt-sensitive electrostatic interactions have critical roles in the preprotein translocation activity of certain TatAC type translocases from Gram-positive bacteria.

Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis

To overcome the limitations of serological diagnosis, including false positive reactions caused by other pathogens, specific antigens for diagnosis of brucellosis other than LPS have been required. The present study was conducted to separate and identify immuno-dominant insoluble proteins of Brucella abortus against the antisera of cattle infected with B. abortus, or/and Yersinia enterocolitica, or the sera of non-infected cattle. After separating insoluble proteins of B. abortus by two dimensional electrophoresis (2-DE), their immuno-reactivity was determined by western blotting. A portion of the immunogenic spots against the positive antisera of B. abortus that have the potential for use as specific antigens were identified by MS/MS analysis. Overall, 18 immunogenic insoluble proteins of B. abortus 1119-3 showed immuno-reactivity against only the positive antisera of B. abortus, but failed to have immunogenicity toward both the positive sera of Y. enterocolitica and the negative sera of B. abortus. Identification of these proteins revealed the following: F0F1 ATP synthase subunit β, solute-binding family 5 protein, 28kDa OMP, Leu/Ile/Val-binding family protein, Histidinol dehyddrogenase, Hypothetical protein, Twin-arginine translocation pathway signal sequence domain-containing protein, Dihydroorotase, Serine protease family protein, β-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA, Short-chain dehydrogenase-/reductase carbonic anhydrase, Orinithine carbamoyltransferase, Leucyl aminopeptidase, Cold shock DNA-binding domain-containing protein, Cu/Zn superoxide dismutase, and Methionine aminopeptidase. The 18 immunogenic proteins separated in the present study can be considered candidate antigens to minimize cross reaction in the diagnosis of brucellosis and useful sources for Brucella vaccine development.

The Twin-Arginine Translocation System: Contributions to the Pathobiology of Campylobacter Jejuni

The twin-arginine translocation (Tat) system is specialized in the transport of folded proteins across the cytoplasmic membrane. Although the mechanisms that govern the Tat transport and its scope are not well understood, this system and its cognate substrates are involved in important functions that facilitate the adaptation and survival of bacteria. Evidence also exists that connects the Tat system to virulent traits of clinically relevant pathogens. Of interest is Campylobacter jejuni, an important foodborne pathogen that is capable of surviving in different hosts and environmental niches. Recent studies have shown that the Tat system in this bacterium mediates key metabolic and stress resistance traits. Furthermore, the majority of the identified Tat substrates in C. jejuni are cofactor-containing redox proteins that contribute to the bacterium?s branched electron transport chain, a component essential for survival under differing conditions. These studies as well as the absence of Tat homologs in the sequenced genomes of animals suggest that the Tat system might pose an attractive target for therapeutics against C. jejuni.

Erratum to: Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

Erratum to: Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

Contribution of TAT system translocated PhoX to Campylobacter jejuni phosphate metabolism and resilience to environmental stresses.

Campylobacter jejuni is a common gastrointestinal pathogen that colonizes food animals; it is transmitted via fecal contamination of food, and infections in immune-compromised people are more likely to result in serious long-term illness. Environmental phosphate is likely an important sensor of environmental fitness and the ability to obtain extracellular phosphate is central to the bacteria's core metabolic responses. PhoX is the sole alkaline phosphatase in C. jejuni, a substrate of the TAT transport system. Alkaline phosphatases mediate the hydrolytic removal of inorganic phosphate (Pi) from phospho-organic compounds and thereby contribute significantly to the polyphosphate kinase 1 (ppk1) mediated formation of poly P, a molecule that regulates bacterial response to stresses and virulence. Similarly, deletion of the tatC gene, a key component of the TAT system, results in diverse phenotypes in C. jejuni including reduced stress tolerance and in vivo colonization. Therefore, here we investigated the contribution of phoX in poly P synthesis and in TAT-system mediated responses. The phoX deletion mutant showed significant decrease (P<0.05) in poly P accumulation in stationary phase compared to the wild-type, suggesting that PhoX is a major contributor to the inorganic phosphate pool in the cell which is essential for poly P synthesis. The phoX deletion is sufficient for a nutrient stress defect similar to the defect previously described for the ΔtatC mutant. Additionally, the phoX deletion mutant has increased resistance to certain antimicrobials. The ΔphoX mutant was also moderately defective in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization. Further, the ΔphoX mutant produced increased biofilm that can be rescued with 1 mM inorganic phosphate. The qRT-PCR of the ΔphoX mutant revealed transcriptional changes that suggest potential mechanisms for the increased biofilm phenotype.

Novel GFP expression using a short N-terminal polypeptide through the defined twin-arginine translocation (Tat) pathway.

Escherichia coli is frequently used as a convenient host organism for soluble recombinant protein expression. However, additional strategies are needed for proteins with complex folding characteristics. Here, we suggested that the acidic, neutral, and alkaline isoelectric point (pI) range curves correspond to the channels of the E. coli type-II cytoplasmic membrane translocation (periplasmic translocation) pathways of twin-arginine translocation (Tat), Yid, and general secretory pathway (Sec), respectively, for unfolded and folded target proteins by examining the characteristic pI values of the N-termini of the signal sequences or the leader sequences, matching with the known diameter of the translocation channels, and analyzing the N-terminal pI value of the signal sequences of the Tat substrates. To confirm these proposed translocation pathways, we investigated the soluble expression of the folded green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting pI and hydrophilicity separately or collectively. This, in turn, revealed the existence of an anchor function with a specific directionality based on the N-terminal pI value (termed as N-terminal pI-specific directionality) and distinguished the presence of the E. coli type-II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for the unfolded and folded target proteins. We concluded that the pI value and hydrophilicity of the short N-terminal polypeptide, and the total translational efficiency of the target proteins based on the ΔGRNA value of the N-terminal coding regions are important factors for promoting more efficient translocation (secretion) through the largest diameter of the Tat channel. These results show that the short N-terminal polypeptide could substitute for the Tat signal sequence with improved efficiency.

The Outcomes of Treatment and the Etiology of Lymphoceles With a Focus on Hemostasis in Kidney Recipients: A Preliminary Report

Background The etiopathogenesis of lymphoceles remains incompletely understood. The aim of our work was to analyze the perturbations of blood coagulation process for their possible impact on the etiology of lymphoceles. Additionally we performed an evaluation of the incidence and effectiveness of treatment methods for lymphoceles. Materials and methods During 2004 to 2010, we performed 242 kidney transplantations in 92 female and 150 male patients. The hemostatic parameters included concentrations of: antithrombin, plasminogen, thrombin/antithrombin complexes (TAT), prothrombin products F1+2 (F1+2), d-dimers, and plasmin/antiplasmin complexes. Results At 7 years follow-up 27 (11%) recipients had developed symptomatic lymphoceles, namely abdominal discomfort, a palpable mess in the lower abdomen, arterial hypertension, infection of the operative site with fever, lymphorrhoea with surgical wound dehiscence, decreased diurnal urine output with an elevated plasma creatinine, voiding problems of urgency and vesical tenesmus, and/or symptoms of deep vein thrombosis. We applied the following methods of treatment aspiration alone, percutaneous drainage, laparoscopic fenestration or open surgery. In two only patients did perform open surgery. Since 2008 we have not performed an aspiration alone because of high rate of recurrence (almost 100%) and abandoned open surgery in favor of a laparoscopic approach. Our minimally invasive surgery includes percutaneous drainage guided by ultrasound and a laparoscopic procedure with 100% effectiveness. The examined hemostatic parameters revealed decreased concentrations of TAT complexes and F1+2 in subjects with lymphocele showing positive predictive values of 33% and 41% respectively. The negative predictive values for TAT complexes and F1+2 were 14% and 10%, respectively, suggesting decreased blood coagulation activity among effected recipients. Altered blood coagulation processes may explain some aspects of the disturbances of postoperative obliteration of damaged lymphatic vessels and formation of pathological lymph collection afterward. Conclusions Perturbations of blood coagulation may be one cause for a lymphocele.

The Effect of Cause of Cadaveric Kidney Donors Death on Fibrinolysis and Blood Coagulation Processes

Background Organ donors can be generally divided into two groups according to the cause of their death. The first group is composed of those who died because of physical injuries, especially road traffic injury, and the second group, those who died from central nervous system (CNS) stroke or bleeding. The aim of our work was to examine hemostatic processes among kidney donors. Materials and methods The 38 deceased kidney donors (KD) included 11 women and 27 men of overall average age of 37 ± 12 years. The donor group of according to the cause of death, included 14 injured donors (ID) (41%) and 24 noninjured donors (ND) donors (59%). The control group consisted of 25 healthy volunteers matched for sex and age. We determined the following concentrations: antithrombin (AT), thrombin/antithrombin complexes (TAT), and prothrombin F1 + 2 fragments. The fibrinolytic parameter concentrations were: plasminogen (PL), plasmin/antiplasmin complexes (PAP), and D-dimers. Results Deceased kidney donors showed an increased plasma concentrations of TAT complexes ( P < .000001) and prothrombin fragments F1 + 2 ( P < .0000001); however, the protein C concentration was decreased ( P < .000001). The antithrombin activity was similar to the control group. The concentrations of PAP complexes and d-dimers were higher (both P < .000001), but the level of PL lower among KD compared with controls ( P < .0000001). The higher of TAT, PAP complexes, d-dimers, and F1 + 2 concentrations as well and as lower plasminogen and PC concentrations were evidence for increased activation of blood coagulation and fibrinolysis in cadaveric KD. However, analysis compairing ID versus ND donors revealed increased concentrations of PAP complexes ( P < .05) and decreased amounts of TAT complexes ( P < .01) among ID subgroup. The positive predictive value (PPV) and negative (NPV) for PAP complexes were 75% and 68% and for TAT, 71% and 57%, respectively. On the basis of these observations, we concluded that an intensive activation of fibrinolytic process occurs among the ID. In contrast, ND show intensive activation of blood coagulation.

The superoxide dismutase SodA is targeted to the periplasm in a SecA‐dependent manner by a novel mechanism

The manganese/iron-type superoxide dismutase (SodA) of Rhizobium leguminosarum bv. viciae 3841 is exported to the periplasm of R. l. bv. viciae and Escherichia coli. However, it does not possess a hydrophobic cleaved N-terminal signal peptide typically present in soluble proteins exported by the Sec-dependent (Sec) pathway or the twin-arginine translocation (TAT) pathway. A tatC mutant of R. l. bv. viciae exported SodA to the periplasm, ruling out export of SodA as a complex with a TAT substrate as a chaperone. The export of SodA was unaffected in a secB mutant of E. coli, but its export from R. l. bv. viciae was inhibited by azide, an inhibitor of SecA ATPase activity. A temperature-sensitive secA mutant of E. coli was strongly reduced for SodA export. The 10 N-terminal amino acid residues of SodA were sufficient to target the reporter protein alkaline phosphatase to the periplasm. Our results demonstrate the export of a protein lacking a classical signal peptide to the periplasm by a SecA-dependent, but SecB-independent targeting mechanism. Export of the R. l. bv. viciae SodA to the periplasm was not limited to the genus Rhizobium, but was also observed in other proteobacteria.

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron-sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

Functional Diversification of the Twin-Arginine Translocation Pathway Mediates the Emergence of Novel Ecological Adaptations

Microorganisms occupy a myriad of ecological niches that show an astonishing diversity. The molecular mechanisms underlying microbes' ecological diversity remain a fundamental conundrum in evolutionary biology. Evidence points to that the secretion of a particular set of proteins mediates microbes' interaction with the environment. Several systems are involved in this secretion, including the Sec secretion system and the Tat pathway. Shifts in the functions of proteins from the secretion systems may condition the set of secreted proteins and can, therefore, mediate adaptations to new ecological niches. In this manuscript, we have investigated processes of functional divergence (FD)-a term used here to refer to the emergence of novel functions by the modification of ancestral ones-of Tat pathway proteins using a large set of microbes with different lifestyles. The application of a novel approach to identify FD allowed us to distinguish molecular changes in the three Tat proteins among different groups of archaea and bacteria. We found these changes as well as the composition of secreted proteins to be correlated with differences in microbe's lifestyles. We identified major signatures of FD in halophilic and thermophilic archaea as well as in pathogenic bacteria. The location of amino acids affected by FD in functionally important domains of Tat proteins made it possible to find the link between the molecular changes in Tat, the set of secreted proteins and the environmental features of the microbes. We present evidence that links specific molecular changes in secretion mediating proteins of microbes to their ecological adaptations.

Towards understanding the Tat translocation mechanism through structural and biophysical studies of the amphipathic region of TatA from Escherichia coli

The twin-arginine translocase (Tat) system is used by many bacteria and plants to move folded proteins across the cytoplasmic or thylakoid membrane. In most bacteria, the TatA protein is believed to form a defined pore in the membrane through homo-oligomerization with other TatA protomers. The predicted secondary structure of TatA includes a transmembrane helix, an amphipathic helix, and an unstructured C-terminal region. Here biophysical and structural investigations were performed on a synthetic peptide representing the amphipathic region of TatA (residues 22 to 44, abbreviated TatAH2). The C-terminal region of TatA (residues 44–89) was previously shown to be accessible from both the cytoplasmic and periplasmic sides of the membrane only when the membrane potential was intact, suggesting dependence of its topology on an energized membrane (Chan et al. 2007 Biochemistry 46: 7396–404). Such observation suggests that the TatAH2 region would have unique lipid interactions that may be related to the function of TatA during translocation and thus warranted further investigations. NMR and CD spectroscopy of TatAH2 show that it adopts a predominantly helical structure in a membrane environment while remaining unstructured in aqueous solution. Differential scanning calorimetry studies also reveal that TatAH2 interacts with DPPG lipids but not with DPPC, suggesting that negatively charged phospholipid head groups contribute to the membrane interactions with TatA.

Changes in localization of cellular vesicular apparatus during differentiation of myoblasts into myotubules in cell culture

The study of changes in the intracellular processes during differentiation of myoblasts into myotubules is of great importance for understanding several fundamental problems of cell biology. At first, this concerns the spatial organization of vacuolar apparatus that reflects the alterations in the properties of cell membranes, cytoskeleton elements and dynamics of vesicular transport in the course of differentiation. The distribution of acidic membrane organelles (lysosomes, late endosomes, Golgi cisternae) during the myotubule formation was revealed. It was shown that perinuclear localization of acidic organelles in myoblasts was replaced by diffuse distribution of these structures in the whole volume of myotubules. Using lipophilic fluorescent dyes, RH 414 and di-8-ANEPPS, the process of formation and dynamics of endocytic vesicles in myoblasts and myotubules was investigated. In the present work, semiconductive nanocrystals, quantum dots (QDs), conjugated with TAT-peptide, which belongs to cell-penetrating peptides, were used to characterize nonspecific endocytosis. It was shown that QDs--TAT complexes penetrate myoblasts but do not penetrate myotubules even after 24 h incubation, which might be connected with plasma membrane changes during the process of skeletal muscle differentiation.

Redundancy and specificity of multiple trigger factor chaperones in Desulfitobacteria

The ribosome-bound trigger factor (TF) chaperone assists folding of newly synthesized polypeptides and participates in the assembly of macromolecular complexes. In the present study we showed that multiple distinct TF paralogues are present in genomes of Desulfitobacteria, a bacterial genus known for its ability to grow using organohalide respiration. Two full-length TF chaperones and at least one truncated TF (lacking the N-terminal ribosome-binding domain) were identified, the latter being systematically linked to clusters of reductive dehalogenase genes encoding the key enzymes in organohalide respiration. Using a well-characterized heterologous chaperone-deficient Escherichia coli strain lacking both TF and DnaK chaperones, we demonstrated that all three TF chaperones were functional in vivo, as judged by their ability to partially suppress bacterial growth defects and protein aggregation in the absence of both major E. coli chaperones. Next, we found that the N-terminal truncated TF-like protein PceT functions as a dedicated chaperone for the cognate reductive dehalogenase PceA by solubilizing and stabilizing it in the heterologous system. Finally, we showed that PceT specifically interacts with the twin-arginine signal peptide of PceA. Taken together, our data define PceT (and more generally the new RdhT family) as a class of TF-like chaperones involved in the maturation of proteins secreted by the twin-arginine translocation pathway.

Dissecting the complete lipoprotein biogenesis pathway in Streptomyces scabies

Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol transferase (Lgt) and cleaved of their signal peptides by lipoprotein signal peptidase (Lsp). In Gram-negative bacteria and mycobacteria, lipoproteins are further lipidated by lipoprotein N-acyl transferase (Lnt), to give triacylated lipoproteins. Streptomyces are unusual amongst Gram-positive bacteria because they export large numbers of lipoproteins via the twin arginine protein transport (Tat) pathway. Furthermore, some Streptomyces species encode two Lgt homologues and all Streptomyces species encode two homologues of Lnt. Here we characterize lipoprotein biogenesis in the plant pathogen Streptomyces scabies and report that lgt and lsp mutants are defective in growth and development while only moderately affected in virulence. Lipoproteins are lost from the membrane in an S. scabies lgt mutant but restored by expression of Streptomyces coelicolor lgt1 or lgt2 confirming that both encode functional Lgt enzymes. Furthermore, lipoproteins are N-acylated in Streptomyces with efficient N-acylation dependent on Lnt1 and Lnt2. However, deletion of lnt1 and lnt2 has no effect on growth, development or virulence. We thus present a detailed study of lipoprotein biogenesis in Streptomyces, the first study of Lnt function in a monoderm bacterium and the first study of bacterial lipoproteins as virulence factors in a plant pathogen.