The polar amino acid in the TatA transmembrane helix is not strictly necessary for protein function

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

Hydrophobic mismatch is a key factor in protein transport across lipid bilayer membranes via the Tat pathway.

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

In-situ investigation of the interaction between hydrogen and stacking faults in a bulk austenitic steel

The interaction between hydrogen (H) atoms and various microstructural defects remains a key to understand the H-induced damage and the subsequent premature failure of high-strength metallic materials. Previous studies on this subject are mainly focused on the in-situ probing of dislocations in a thin foil placed in an environmental transmission electron microscopy (TEM) cell. Here, a three-point bending test coupled with electron channeling contrast imaging (ECCI) has been applied to investigate the interaction of H with stacking faults (SFs) in a bulk high Mn austenitic steel. The expansion of some SFs, in terms of one partial dislocation movement within a partial dislocation pair, was observed on the H pre-charged sample when kept at a constant loading (i.e., a continuous H migration and likely build-up close to the pre-prepared notch tip). A temporal-resolved cross-correlation EBSD (CC-EBSD) measurement shows that the migration of H towards the notch tip region has a minor effect on the internal stress evolution. However, the local shear modulus (μ) and stacking fault energy (γSF) can be reduced by a local H segregation. Further theoretical calculation of SF ribbon width indicates that the reduction of μ by H results in the shrinkage of SF, while the H-induced reduction of γSF results in SF expansion at a lower resolved shear stress. The observed expansion of SF ribbons can be interrupted by a combined reduction of both μ and γSF due to H.</p

A real-time analysis of protein transport via the twin arginine translocation pathway in response to different components of the protonmotive force

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in&nbsp;vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E.&nbsp;coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH&nbsp;= 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in&nbsp;vivo in E.&nbsp;coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway

A human stool-derived Bilophila wadsworthia strain caused systemic inflammation in specific-pathogen-free mice

Abstract Background Bilophila wadsworthia is a major member of sulfidogenic bacteria in human gut, it was originally recovered from different clinical specimens of intra-abdominal infections and recently was reported potentially linked to different chronic metabolic disorders. However, there is still insufficient understanding on its detailed function and mechanism to date. Methods A B. wadsworthia strain was isolated from fresh feces of a latent autoimmune diabetes in adults patient and we investigated its pathogenicity by oral administration to specific-pathogen-free mice. Tissue samples and serum were collected after sacrifice. Stool samples were collected at different time points to profile the gut microbiota. Results Bilophila wadsworthia infection resulted in the reduction of body weight and fat mass, apparent hepatosplenomegaly and elevated serum inflammatory factors, including serum amyloid A and interleukin-6, while without significant change of the overall gut microbiota structure. Conclusions These results demonstrated that higher amount of B. wadsworthia caused systemic inflammatory response in SPF mice, which adds new evidence to the pathogenicity of this bacterium and implied its potential role to the chronic inflammation related metabolic diseases like diabetes

Chemical heterogeneity enhances hydrogen resistance in high-strength steels

The antagonism between strength and resistance to hydrogen embrittlement in metallic materials is an intrinsic obstacle to the design of lightweight yet reliable structural components operated in hydrogen-containing environments. Economical and scalable microstructural solutions to this challenge must be found. Here, we introduce a counterintuitive strategy to exploit the typically undesired chemical heterogeneity within the material’s microstructure that enables local enhancement of crack resistance and local hydrogen trapping. We use this approach in a manganese-containing high-strength steel and produce a high dispersion of manganese-rich zones within the microstructure. These solute-rich buffer regions allow for local micro-tuning of the phase stability, arresting hydrogen-induced microcracks and thus interrupting the percolation of hydrogen-assisted damage. This results in a superior hydrogen embrittlement resistance (better by a factor of two) without sacrificing the material’s strength and ductility. The strategy of exploiting chemical heterogeneities, rather than avoiding them, broadens the horizon for microstructure engineering via advanced thermomechanical processing