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Nitroreductases are important enzymes for a variety of applications, including cancer therapy and bioremediation. They often require encapsulation to improve stability and activity. We focus on genetically encoded encapsulation of nitroreductases within protein capsids, like encapsulins. Our study showcases the encapsulation of nitroreductase NfsB as functional dimers within encapsulins, which enhances protein activity and stability in diverse conditions. Mutations within the pore region are beneficial for activity of the encapsulated enzyme, potentially by increasing diffusion rates. Cryogenic electron microscopy reveals the overall architecture of the encapsulated dimeric NfsB within the nanoreactor environment and identifies multiple pore states in the shell. These findings highlight the potential of encapsulins as versatile tools for enhancing enzyme performance across various fields., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

About 30% of all bacterial proteins execute their function outside of the cytosol and must be inserted into or translocated across the cytoplasmic membrane. This requires efficient targeting systems that recognize N-terminal signal sequences in client proteins and deliver them to protein transport complexes in the membrane. While the importance of these protein transport machineries for the spatial organization of the bacterial cell is well documented in multiple studies, the contribution of mRNA targeting and localized translation to protein transport is only beginning to emerge. mRNAs can exhibit diverse subcellular localizations in the bacterial cell and can accumulate at sites where new protein is required. This is frequently observed for mRNAs encoding membrane proteins, but the physiological importance of membrane enrichment of mRNAs and the consequences it has for the insertion of the encoded protein have not been explored in detail. Here, we briefly highlight some basic concepts of signal sequence-based protein targeting and describe in more detail strategies that enable the monitoring of mRNA localization in bacterial cells and potential mechanisms that route mRNAs to particular positions within the cell. Finally, we summarize some recent developments that demonstrate that mRNA targeting and localized translation can sustain membrane protein insertion under stress conditions when the protein-targeting machinery is compromised. Thus, mRNA targeting likely acts as a back-up strategy and complements the canonical signal sequence-based protein targeting., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The universally conserved YchF/Ola1 ATPases regulate stress response pathways in prokaryotes and eukaryotes. Deletion of YchF/Ola1 leads to increased resistance against environmental stressors, such as reactive oxygen species, while their upregulation is associated with tumorigenesis in humans. The current study shows that in E. coli , the absence of YchF stimulates the synthesis of the alternative sigma factor RpoS by a transcription-independent mechanism. Elevated levels of RpoS then enhance the transcription of major stress-responsive genes. In addition, the deletion of ychF increases the levels of polyphosphate kinase, which in turn boosts the production of the evolutionary conserved and ancient chemical chaperone polyphosphate. This potentially provides a unifying concept for the increased stress resistance in bacteria and eukaryotes upon YchF/Ola1 deletion. Intriguingly, the simultaneous deletion of ychF and the polyphosphate-degrading enzyme exopolyphosphatase causes synthetic lethality in E. coli , demonstrating that polyphosphate production needs to be fine-tuned to prevent toxicity., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

The universally conserved protein YidC aids in the insertion and folding of transmembrane polypeptides. Supposedly, a charged arginine faces its hydrophobic lipid core, facilitating polypeptide sliding along YidC's surface. How the membrane barrier to other molecules may be maintained is unclear. Here, we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon ribosome or ribosome-nascent chain complex (RNC) binding. In contrast to monomeric YidC structures, an AlphaFold parallel YidC dimer model harbors a pore. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, single-molecule fluorescence photobleaching observations, and crosslinking experiments. In the dimeric model, the conserved arginine and other residues interacting with nascent chains point into the putative pore. This result suggests the possibility of a YidC-assisted insertion mode alternative to the insertase mechanism.

Maintaining a functional proteome under different environmental conditions is challenging for every organism, in particular for unicellular organisms, such as bacteria. In order to cope with changing environments and stress conditions, bacteria depend on strictly coordinated proteostasis networks that control protein production, folding, trafficking, and degradation. Regulation of ribosome biogenesis and protein synthesis are cornerstones of this cellular adaptation in all domains of life, which is rationalized by the high energy demand of both processes and the increased resistance of translationally silent cells against internal or external poisons. Reduced protein synthesis ultimately also reduces the substrate load for protein transport systems, which are required for maintaining the periplasmic, inner, and outer membrane subproteomes. Consequences of impaired protein transport have been analyzed in several studies and generally induce a multifaceted response that includes the upregulation of chaperones and proteases and the simultaneous downregulation of protein synthesis. In contrast, generally less is known on how bacteria adjust the protein targeting and transport machineries to reduced protein synthesis, e.g., when cells encounter stress conditions or face nutrient deprivation. In the current review, which is mainly focused on studies using Escherichia coli as a model organism, we summarize basic concepts on how ribosome biogenesis and activity are regulated under stress conditions. In addition, we highlight some recent developments on how stress conditions directly impair protein targeting to the bacterial membrane. Finally, we describe mechanisms that allow bacteria to maintain the transport of stress-responsive proteins under conditions when the canonical protein targeting pathways are impaired., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The presequence translocase of the mitochondrial inner membrane (TIM23) represents the major route for the import of nuclear-encoded proteins into mitochondria 1,2 . About 60% of more than 1,000 different mitochondrial proteins are synthesized with amino-terminal targeting signals, termed presequences, which form positively charged amphiphilic α-helices 3,4 . TIM23 sorts the presequence proteins into the inner membrane or matrix. Various views, including regulatory and coupling functions, have been reported on the essential TIM23 subunit Tim17 (refs. 5-7 ). Here we mapped the interaction of Tim17 with matrix-targeted and inner membrane-sorted preproteins during translocation in the native membrane environment. We show that Tim17 contains conserved negative charges close to the intermembrane space side of the bilayer, which are essential to initiate presequence protein translocation along a distinct transmembrane cavity of Tim17 for both classes of preproteins. The amphiphilic character of mitochondrial presequences directly matches this Tim17-dependent translocation mechanism. This mechanism permits direct lateral release of transmembrane segments of inner membrane-sorted precursors into the inner membrane., (© 2023. The Author(s).)

Signal-sequence-dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and is facilitated by dedicated protein targeting factors such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins but can also be present within mRNAs. By in vivo and in vitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors but is dependent on the SecYEG translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metabolic sensing is a crucial prerequisite for cells to adjust their physiology to rapidly changing environments. In bacteria, the response to intra- and extracellular ligands is primarily controlled by transcriptional regulators, which activate or repress gene expression to ensure metabolic acclimation. Translational control, such as ribosomal stalling, can also contribute to cellular acclimation and has been shown to mediate responses to changing intracellular molecules. In the current study, we demonstrate that the cotranslational export of the Rhodobacter capsulatus protein CutF regulates the translation of the downstream cutO -encoded multicopper oxidase CutO in response to extracellular copper (Cu). Our data show that CutF, acting as a Cu sensor, is cotranslationally exported by the signal recognition particle pathway. The binding of Cu to the periplasmically exposed Cu-binding motif of CutF delays its cotranslational export via its C-terminal ribosome stalling-like motif. This allows for the unfolding of an mRNA stem-loop sequence that shields the ribosome-binding site of cutO , which favors its subsequent translation. Bioinformatic analyses reveal that CutF-like proteins are widely distributed in bacteria and are often located upstream of genes involved in transition metal homeostasis. Our overall findings illustrate a highly conserved control mechanism using the cotranslational export of a protein acting as a sensor to integrate the changing availability of extracellular nutrients into metabolic acclimation. IMPORTANCE Metabolite sensing is a fundamental biological process, and the perception of dynamic changes in the extracellular environment is of paramount importance for the survival of organisms. Bacteria usually adjust their metabolisms to changing environments via transcriptional regulation. Here, using Rhodobacter capsulatus, we describe an alternative translational mechanism that controls the bacterial response to the presence of copper, a toxic micronutrient. This mechanism involves a cotranslationally secreted protein that, in the presence of copper, undergoes a process resembling ribosomal stalling. This allows for the unfolding of a downstream mRNA stem-loop and enables the translation of the adjacent Cu-detoxifying multicopper oxidase. Bioinformatic analyses reveal that such proteins are widespread, suggesting that metabolic sensing using ribosome-arrested nascent secreted proteins acting as sensors may be a common strategy for the integration of environmental signals into metabolic adaptations.

Membrane proteins require protein machineries to insert their hydrophobic transmembrane domains (TMDs) into the lipid bilayer. A functional analysis of protein insertases in this issue of PLOS Biology reveals that the fundamental mechanism of membrane protein insertion is universally conserved., Competing Interests: The authors declare that there are no conflicts of interest.

The stringent response enables bacteria to respond to nutrient limitation and other stress conditions through production of the nucleotide-based second messengers ppGpp and pppGpp, collectively known as (p)ppGpp. Here, we report that (p)ppGpp inhibits the signal recognition particle (SRP)-dependent protein targeting pathway, which is essential for membrane protein biogenesis and protein secretion. More specifically, (p)ppGpp binds to the SRP GTPases Ffh and FtsY, and inhibits the formation of the SRP receptor-targeting complex, which is central for the coordinated binding of the translating ribosome to the SecYEG translocon. Cryo-EM analysis of SRP bound to translating ribosomes suggests that (p)ppGpp may induce a distinct conformational stabilization of the NG domain of Ffh and FtsY in Bacillus subtilis but not in E. coli., (© 2022. The Author(s).)

Acute respiratory infections (ARIs) are the most common childhood illnesses worldwide whereby the reported frequency varies widely, often depending on type of assessment. Symptom diaries are a powerful tool to counteract possible under-reporting, particularly of milder infections, and thus offer the possibility to assess the full burden of ARIs. The following analyses are based on symptom diaries from participants of the German birth cohort study LoewenKIDS. Primary analyses included frequencies of ARIs and specific symptoms. Factors, which might be associated with an increased number of ARIs, were identified using the Poisson regression. A subsample of two hundred eighty-eight participants were included. On average, 13.7 ARIs (SD: 5.2 median: 14.0 IQR: 10-17) were reported in the first two years of life with an average duration of 11 days per episode (SD: 5.8, median: 9.7, IQR: 7-14). The median age for the first ARI episode was 91 days (IQR: 57-128, mean: 107, SD: 84.5). Childcare attendance and having siblings were associated with an increased frequency of ARIs, while exclusive breastfeeding for the first three months was associated with less ARIs, compared to exclusive breastfeeding for a longer period. This study provides detailed insight into the symptom burden of ARIs in German infants.

The ability to respond to metabolic or environmental changes is an essential feature in all cells and involves both transcriptional and translational regulators that adjust the metabolic activity to fluctuating conditions. While transcriptional regulation has been studied in detail, the important role of the ribosome as an additional player in regulating gene expression is only beginning to emerge. Ribosome-interacting proteins are central to this translational regulation and include universally conserved ribosome interacting proteins, such as the ATPase YchF (Ola1 in eukaryotes). In both eukaryotes and bacteria, the cellular concentrations of YchF/Ola1 determine the ability to cope with different stress conditions and are linked to several pathologies in humans. The available data indicate that YchF/Ola1 regulates the stress response via controlling non-canonical translation initiation and via protein degradation. Although the molecular mechanisms appear to be different between bacteria and eukaryotes, increased non-canonical translation initiation is a common consequence of YchF/Ola1 regulated translational control in E. coli and H. sapiens . In this review, we summarize recent insights into the role of the universally conserved ATPase YchF/Ola1 in adapting translation to unfavourable conditions.

The large abundance of small open reading frames (smORFs) in prokaryotic and eukaryotic genomes and the plethora of smORF-encoded small proteins became only apparent with the constant advancements in bioinformatic, genomic, proteomic, and biochemical tools. Small proteins are typically defined as proteins of < 50 amino acids in prokaryotes and of less than 100 amino acids in eukaryotes, and their importance for cell physiology and cellular adaptation is only beginning to emerge. In contrast to antimicrobial peptides, which are secreted by prokaryotic and eukaryotic cells for combatting pathogens and competitors, small proteins act within the producing cell mainly by stabilizing protein assemblies and by modifying the activity of larger proteins. Production of small proteins is frequently linked to stress conditions or environmental changes, and therefore, cells seem to use small proteins as intracellular modifiers for adjusting cell metabolism to different intra- and extracellular cues. However, the size of small proteins imposes a major challenge for the cellular machinery required for protein folding and intracellular trafficking and recent data indicate that small proteins can engage distinct trafficking pathways. In the current review, we describe the diversity of small proteins in prokaryotes and eukaryotes, highlight distinct and common features, and illustrate how they are handled by the protein trafficking machineries in prokaryotic and eukaryotic cells. Finally, we also discuss future topics of research on this fascinating but largely unexplored group of proteins., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The universally conserved P-loop ATPase Ola1 is implicated in various cellular stress response pathways, as well as in cancer and tumor progression. However, Ola1p functions are divergent between species, and the involved mechanisms are only poorly understood. Here, we studied the role of Ola1p in the heat shock response of the yeast Saccharomyces cerevisiae using a combination of quantitative and pulse labeling-based proteomics approaches, in vitro studies, and cell-based assays. Our data show that when heat stress is applied to cells lacking Ola1p, the expression of stress-protective proteins is enhanced. During heat stress Ola1p associates with detergent-resistant protein aggregates and rapidly forms assemblies that localize to stress granules. The assembly of Ola1p was also observed in vitro using purified protein and conditions, which resembled those in living cells. We show that loss of Ola1p results in increased protein ubiquitination of detergent-insoluble aggregates recovered from heat-shocked cells. When cells lacking Ola1p were subsequently relieved from heat stress, reinitiation of translation was delayed, whereas, at the same time, de novo synthesis of central factors required for protein refolding and the clearance of aggregates was enhanced when compared with wild-type cells. The combined data suggest that upon acute heat stress, Ola1p is involved in the stabilization of misfolded proteins, which become sequestered in cytoplasmic stress granules. This function of Ola1p enables cells to resume translation in a timely manner as soon as heat stress is relieved., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H 2 , CO 2 , or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO 2 to methane by electrons derived from β-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop-driven reduction of CO 2 by acyl-coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO 2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of β-oxidation in (methyl)menaquinone-containing organisms., Competing Interests: The authors declare no competing interest.

Copper (Cu)-transporting P 1B -type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P 1B -type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb 3 -type cytochrome oxidase ( cbb 3 -Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb 3 -Cox. CcoI and cbb 3 -Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb 3 -Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb 3 -Cox assembly., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Andrei, Di Renzo, Öztürk, Meisner, Daum, Frank, Rauch, Daldal, Andrade and Koch.)

Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu 1+ to the less toxic Cu 2+ . Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb 3 -type cytochrome oxidase ( cbb 3 -Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb 3 -Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Öztürk, Blaby-Haas, Daum, Andrei, Rauch, Daldal and Koch.)

CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT ( C co A - l ike T ransporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb 3 -type cytochrome c oxidase ( cbb 3 -Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met 233 xxxMet 237 and His 261 xxxMet 265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met 233 and His 261 are essential for Cu uptake and cbb 3 -Cox production, whereas Met 237 and Met 265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met 237 and Met 265 completely abolishes Cu uptake and cbb 3 -Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys 49 located at the end of TMH2, the Cys 247 on a surface loop between TMH7 and THM8, and the C 367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo , possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb 3 -Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function. IMPORTANCE Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.

The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, and intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli . By western blot and promoter fusion experiments, we demonstrate that YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress, or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor 3 (IF3) and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs, and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Landwehr, Milanov, Angebauer, Hong, Jüngert, Hiersemenzel, Siebler, Schmit, Öztürk, Dannenmaier, Drepper, Warscheid and Koch.)

The spatial and temporal coordination of protein transport is an essential cornerstone of the bacterial adaptation to different environmental conditions. By adjusting the protein composition of extra-cytosolic compartments, like the inner and outer membranes or the periplasmic space, protein transport mechanisms help shaping protein homeostasis in response to various metabolic cues. The universally conserved SecYEG translocon acts at the center of bacterial protein transport and mediates the translocation of newly synthesized proteins into and across the cytoplasmic membrane. The ability of the SecYEG translocon to transport an enormous variety of different substrates is in part determined by its ability to interact with multiple targeting factors, chaperones and accessory proteins. These interactions are crucial for the assisted passage of newly synthesized proteins from the cytosol into the different bacterial compartments. In this review, we summarize the current knowledge about SecYEG-mediated protein transport, primarily in the model organism Escherichia coli , and describe the dynamic interaction of the SecYEG translocon with its multiple partner proteins. We furthermore highlight how protein transport is regulated and explore recent developments in using the SecYEG translocon as an antimicrobial target., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Oswald, Njenga, Natriashvili, Sarmah and Koch.)

Streptomycetes are well-known producers of numerous bioactive secondary metabolites widely used in medicine, agriculture, and veterinary. Usually, their genomes encode 20-30 clusters for the biosynthesis of natural products. Generally, the onset and production of these compounds are tightly coordinated at multiple regulatory levels, including cluster-situated transcriptional factors. Rishirilides are biologically active type II polyketides produced by Streptomyces bottropensis . The complex regulation of rishirilides biosynthesis includes the interplay of four regulatory proteins encoded by the rsl -gene cluster: three SARP family regulators (RslR1-R3) and one MarR-type transcriptional factor (RslR4). In this work, employing gene deletion and overexpression experiments we revealed RslR1-R3 to be positive regulators of the biosynthetic pathway. Additionally, transcriptional analysis indicated that rslR2 is regulated by RslR1 and RslR3. Furthermore, RslR3 directly activates the transcription of rslR2 , which stems from binding of RslR3 to the rslR2 promoter. Genetic and biochemical analyses demonstrated that RslR4 represses the transcription of the MFS transporter rslT4 and of its own gene. Moreover, DNA-binding affinity of RslR4 is strictly controlled by specific interaction with rishirilides and some of their biosynthetic precursors. Altogether, our findings revealed the intricate regulatory network of teamworking cluster-situated regulators governing the biosynthesis of rishirilides and strain self-immunity.

Eeyarestatin 1 (ES1) is an inhibitor of endoplasmic reticulum (ER) associated protein degradation, Sec61-dependent Ca 2+ homeostasis and protein translocation into the ER. Recently, evidence was presented showing that a smaller analog of ES1, ES24, targets the Sec61-translocon, and captures it in an open conformation that is translocation-incompetent. We now show that ES24 impairs protein secretion and membrane protein insertion in Escherichia coli via the homologous SecYEG-translocon. Transcriptomic analysis suggested that ES24 has a complex mode of action, probably involving multiple targets. Interestingly, ES24 shows antibacterial activity toward clinically relevant strains. Furthermore, the antibacterial activity of ES24 is equivalent to or better than that of nitrofurantoin, a known antibiotic that, although structurally similar to ES24, does not interfere with SecYEG-dependent protein trafficking. Like nitrofurantoin, we find that ES24 requires activation by the NfsA and NfsB nitroreductases, suggesting that the formation of highly reactive nitroso intermediates is essential for target inactivation in vivo., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The complex phosphorylation pattern of natural and modified pentaphosphorylated magic spot nucleotides is generated in a highly efficient way. A cyclic pyrophosphoryl phosphoramidite (cPyPA) reagent is used to introduce four phosphates on nucleosides regioselectively in a one-flask key transformation. The obtained magic spot nucleotides are used to develop a capillary electrophoresis UV detection method, enabling nucleotide assignment in complex bacterial extracts.

OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, which is critical to regulate pore selectivity, is also required for SecY-mediated transport of OmpC and OmpF into the periplasm. Variants with a deleted or mutated motif were expressed in the cytosol and translocation was initiated. However, after signal peptide cleavage, import into the periplasm was aborted and the mutated proteins were redirected to the cytosol. Strikingly, reducing the proof-reading capacity of SecY by introducing the PrlA4 substitutions restored transport of OmpC with a mutated channel domain into the periplasm. Our study identified a SecY-mediated quality control pathway to restrict transport of outer membrane porin proteins with a deregulated channel activity into the periplasm.

Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro- and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27-amino-acid-long model protein YohP is recognized by the signal recognition particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries., Competing Interests: The authors have declared that no competing interests exist.

Copper (Cu) is an essential trace element for all living organisms and used as cofactor in key enzymes of important biological processes, such as aerobic respiration or superoxide dismutation. However, due to its toxicity, cells have developed elaborate mechanisms for Cu homeostasis, which balance Cu supply for cuproprotein biogenesis with the need to remove excess Cu. This review summarizes our current knowledge on bacterial Cu homeostasis with a focus on Gram-negative bacteria and describes the multiple strategies that bacteria use for uptake, storage and export of Cu. We furthermore describe general mechanistic principles that aid the bacterial response to toxic Cu concentrations and illustrate dedicated Cu relay systems that facilitate Cu delivery for cuproenzyme biogenesis. Progress in understanding how bacteria avoid Cu poisoning while maintaining a certain Cu quota for cell proliferation is of particular importance for microbial pathogens because Cu is utilized by the host immune system for attenuating pathogen survival in host cells.

Context: Spinal cord injury (SCI) is a complex chronic condition with multiple self-management requirements and a high prevalence of complications. Pressure injuries (PIs) are among the most common ones and represent a frequent reason for re-hospitalization. This study aimed to identify styles of prevention that individuals with SCI adopt to deal with the risk of developing PIs. Design: Qualitative explorative interview study. Data was collected through semi-structured interviews, which were transcribed verbatim and analyzed following the principles of thematic analysis. Setting: Switzerland. Participants: The participants were a purposive sample of community-dwelling Swiss residents with SCI for at least five years. Interventions: Not applicable. Outcome measures: Not applicable. Results: Although all participants (N = 20) showed at least a basic knowledge of prevention of PIs by describing some preventive measures, they had different prevention styles characterized by different behavioral patterns (i.e. complying with all recommended measures, performing only a selection of them or delegating them to others) and different beliefs and attitudes towards prevention. Conclusion: By identifying the style of prevention of an individual, it is possible to develop tailored interventions that have an impact on the factors which seem to play a role in determining the adoption of preventive behaviors (i.e. perceived susceptibility to PIs, attitudes towards prevention, and self-efficacy). Such interventions would constitute a concrete effort to support individuals with SCI during their self-management. Besides alleviating a frequent and disabling medical complication and contributing to an enhanced quality of life, these interventions might also help decrease healthcare costs.

Objective: To identify barriers to access healthcare services and reveal determinants of satisfaction with healthcare services in people with chronic spinal cord injury (SCI). Design: Cross-sectional survey. Setting : Community setting in Switzerland. Participants : People with chronic SCI. Interventions : Non-applicable. Outcome Measures : Questionnaire-based evaluation of availability and quality of healthcare services for secondary health conditions, satisfaction with fulfillment of healthcare needs, and preference for care from a hypothetical service provider with limited specialized SCI care expertise but in close proximity over comprehensive care from an existing specialized SCI center located at a greater distance. Results: Close to three-quarter of participants (70%) indicated satisfaction with healthcare services received for SCI related health conditions. Elderly individuals (61+ years old) rated the availability and quality of healthcare 6% to 11% higher than younger individuals. The perceived fulfillment of healthcare needs was lower in people with incomplete paraplegia (odds ratio (OR) 2.11, 95%-credibility interval (CI) 1.18-3.84), chronic pain (OR 1.85, CI 1.12-3.08), insufficient access to long distance transportation (OR 5.81, CI 2.74-12.82), and longer travel distances to specialized SCI centers. Conclusion: Perceived inadequateness of access to healthcare services was partly related to transportation barriers, suggesting that outreach services or support with transportation are possible solutions. People with incomplete paralysis and pain consistently rated the fulfillment of care needs associated with SCI less favorably, pointing to the need for enhanced advocacy for this vulnerable groups.

The SecYEG translocon constitutes the major protein transport channel in bacteria and transfers an enormous variety of different secretory and inner-membrane proteins. The minimal core of the SecYEG translocon consists of three inner-membrane proteins, SecY, SecE, and SecG, which, together with appropriate targeting factors, are sufficient for protein transport in vitro However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, likely allowing the SecYEG translocon to process its diverse substrates. To obtain a global view on SecYEG plasticity in Escherichia coli , here we performed a quantitative interaction proteomic analysis, which identified several known SecYEG-interacting proteins, verified the interaction of SecYEG with quality-control proteins, and revealed several previously unknown putative SecYEG-interacting proteins. Surprisingly, we found that the chaperone complex PpiD/YfgM is the most prominent interaction partner of SecYEG. Detailed analyses of the PpiD-SecY interaction by site-directed cross-linking revealed that PpiD and the established SecY partner protein YidC use almost completely-overlapping binding sites on SecY. Both PpiD and YidC contacted the lateral gate, the plug domain, and the periplasmic cavity of SecY. However, quantitative MS and cross-linking analyses revealed that despite having almost identical binding sites, their binding to SecY is noncompetitive. This observation suggests that the SecYEG translocon forms different substrate-independent subassemblies in which SecYEG either associates with YidC or with the PpiD/YfgM complex. In summary, the results of this study indicate that the PpiD/YfgM chaperone complex is a primary interaction partner of the SecYEG translocon., (© 2019 Jauss et al.)

The ribosome is a sophisticated cellular machine, composed of RNA and protein, which translates the mRNA-encoded genetic information into protein and thus acts at the center of gene expression. Still, the ribosome not only decodes the genetic information, it also coordinates many ribosome-associated processes like protein folding and targeting. The ribosomal protein uL23 is crucial for this coordination and is located at the ribosomal tunnel exit where it serves as binding platform for targeting factors, chaperones and modifying enzymes. This includes the signal recognition particle (SRP), which facilitates co-translational protein targeting in pro- and eukaryotes, the chaperone Trigger Factor and methionine aminopeptidase, which removes the start methionine in many bacterial proteins. A recent report revealed the intricate interaction of uL23 with yet another essential player in bacteria, the ATPase SecA, which is best known for its role during post-translational secretion of proteins across the bacterial SecYEG translocon., Competing Interests: Conflict of interest: The authors declare no conflict of interests.

Copper (Cu)-containing proteins execute essential functions in prokaryotic and eukaryotic cells, but their biogenesis is challenged by high Cu toxicity and the preferential presence of Cu(II) under aerobic conditions, while Cu(I) is the preferred substrate for Cu chaperones and Cu-transport proteins. These proteins form a coordinated network that prevents Cu accumulation, which would lead to toxic effects such as Fenton-like reactions and mismetalation of other metalloproteins. Simultaneously, Cu-transport proteins and Cu chaperones sustain Cu(I) supply for cuproprotein biogenesis and are therefore essential for the biogenesis of Cu-containing proteins. In eukaryotes, Cu(I) is supplied for import and trafficking by cell-surface exposed metalloreductases, but specific cupric reductases have not been identified in bacteria. It was generally assumed that the reducing environment of the bacterial cytoplasm would suffice to provide sufficient Cu(I) for detoxification and cuproprotein synthesis. Here, we identify the proposed cbb 3 -type cytochrome c oxidase ( cbb 3 -Cox) assembly factor CcoG as a cupric reductase that binds Cu via conserved cysteine motifs and contains 2 low-potential [4Fe-4S] clusters required for Cu(II) reduction. Deletion of ccoG or mutation of the cysteine residues results in defective cbb 3 -Cox assembly and Cu sensitivity. Furthermore, anaerobically purified CcoG catalyzes Cu(II) but not Fe(III) reduction in vitro using an artificial electron donor. Thus, CcoG is a bacterial cupric reductase and a founding member of a widespread class of enzymes that generate Cu(I) in the bacterial cytosol by using [4Fe-4S] clusters., Competing Interests: The authors declare no competing interest.

Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome. IMPORTANCE Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time., (Copyright © 2019 Knüpffer et al.)

Aims of the Study: The effective management of spinal cord injury (SCI) requires partnership between people with SCI and health professionals (HPs). This paper identifies HPs&rsquo; perceived challenges in building and maintaining this partnership, with a specific focus on how people with SCI and HPs collaborate in the prevention and treatment of pressure injuries (PIs) in SCI., Design: This study has a qualitative and explorative design. Data were collected through semi-structured interviews and analysed following the principles of thematic analysis., Population and Setting: The study population consisted of a purposive sample of HPs (n = 26) working in inpatient and outpatient care in Switzerland with experience in the care of people with SCI., Results: The analysis identified three main challenges: defining responsibilities and expectations, negotiating priorities and establishing and strengthening trust and respect. The HPs argue that the prevention of PIs and self-management are mainly the responsibility of the person with SCI. The HPs have, however, the responsibility to empower, guide, and support persons with SCI in self-management by educating and motivating them., Conclusion: Building and maintaining a partnership with individuals with SCI to prevent and treat PIs is crucial, but it is not an easy task for HPs. Specific communication skills can help HPs and patients find personalised solutions that take into account the patients&rsquo; expertise and preferences. Additionally, the healthcare system must develop solutions that go beyond personal partnership to better integrate the prevention and treatment of PIs into the lifelong self-management of SCI. Assistive technologies, such as mobile technology, might help in this endeavour.

Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu-detoxification and Cu delivery for cytochrome c oxidase (cbb 3 -Cox) assembly depend on two distinct Cu-exporting P 1B -type ATPases. The low-affinity CopA is suggested to export excess Cu and the high-affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb 3 -Cox biogenesis. In most organisms, CopA-like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI-like ATPases is unknown. Here we identified a CopZ-like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox-sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu-sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb 3 -Cox activity is reduced, suggesting that CopZ not only supplies Cu to P 1B -type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI-CopZ protein complex in native R. capsulatus membranes., (© 2018 John Wiley & Sons Ltd.)

Comparative genomic studies of the bacterial MFS-type copper importer CcoA, required for cbb 3 -type cytochrome c oxidase (cbb 3 -Cox) biogenesis, revealed a widespread CcoA-like transporters (CalT) family, containing the conserved CcoA Cu-binding MxxxM and HxxxM motifs. Surprisingly, this family also included the RfnT-like proteins, earlier suggested to transport riboflavin. However, presence of the Cu-binding motifs in these proteins raised the possibility that they might be Cu transporters. To test this hypothesis, the genomic context of the corresponding genes was examined, and three of such genes from Ochrobactrum anthropi, Rhodopseudomonas palustris and Agrobacterium tumefaciens were expressed in Escherichia coli (ΔribB) and Rhodobacter capsulatus (ΔccoA) mutants. Copper and riboflavin uptake abilities of these strains were compared with those expressing R. capsulatus CcoA and Rhizobium leguminosarum RibN as bona fide copper and riboflavin importers, respectively. Overall data demonstrated that the "RfnT-like" CalT proteins are unable to efficiently transport riboflavin, but they import copper like CcoA. Nevertheless, even though expressed and membrane-localized in a R. capsulatus mutant lacking CcoA, these transporters were unable to accumulate Cu or complement for cbb 3 -Cox defect. This lack of functional exchangeability between the different subfamilies of CalT homologs suggests that MFS-type bacterial copper importers might be species-specific.

Background: People with spinal cord injury (SCI) suffer from complex secondary health conditions and rely on specialized health care services, which are often centralized and difficult to reach for individuals living in remote areas. As a consequence, they might move to regions where they expect better access to care. The aims of this study were: 1) to identify regions where people with SCI live compared with the general population, 2) to examine whether their choice of residence is related to the availability of local health care infrastructure, and 3) to ascertain determinants of their consideration to change residence when aging., Methods: This study used information from a nationwide Swiss SCI cohort and inpatient hospital discharge data. To detect clusters in the distribution of people with chronic SCI in Switzerland, a spatial cluster detection test was conducted using the normative population of a region as offset. To identify associations between the residential location of people with SCI and infrastructure variables, a negative binomial model was set up at a regional level with the frequency of people with SCI as outcome, geographical indicators as explanatory variables, and the normative population as offset. Determinants of the consideration to change residence when aging were investigated using logistic regression models., Results: People with SCI were not living equally distributed among the normative population, but clustered in specific areas. They were more likely than the general population to reside close to specialized SCI centers, in areas with a high density of outpatient physicians, and in urban regions. People with SCI living in rural areas were more likely to consider relocating when aging than those living in urban areas. However, only a few people with SCI considered moving closer to specialized centers when such a move required crossing language barriers., Conclusions: Good access to appropriate health care services and amenities of daily life seems to play such an important role in the lives of people with SCI that they are willing to choose their residential location based on local availability of appropriate health care services.

Cytochrome c oxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (Cu B ) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the Cu B atom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacterium Rhodobacter capsulatus indicated that the copper atom needed for the Cu B site of cbb 3 -type cytochrome c oxidase ( cbb 3 -Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis of CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochrome c oxidases. To define the specificity of CcoA activity, we investigated its function in Rhodobacter sphaeroides , a close relative of R. capsulatus that contains both cbb 3 - and aa 3 -Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production of cbb 3 -Cox and not that of aa 3 -Cox was affected. We therefore concluded that CcoA is dedicated solely to cbb 3 -Cox biogenesis, establishing that distinct copper uptake systems provide the Cu B atoms to the catalytic sites of these two similar cytochrome c oxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions. IMPORTANCE The cbb 3 - and aa 3 -type cytochrome c oxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and cofactor composition, yet they all share a conserved heme-copper binuclear site within their catalytic subunit. In this study, we show that the copper atoms of the catalytic center of two similar cytochrome c oxidases from this superfamily are provided by different copper uptake systems during their biogenesis. This finding illustrates different strategies by which organisms fine-tune the trafficking of copper, which is an essential but toxic micronutrient., (Copyright © 2018 Khalfaoui-Hassani et al.)

YidC/Oxa1/Alb3 are essential proteins that operate independently or cooperatively with the Sec machinery during membrane protein insertion in bacteria, archaea and eukaryotic organelles. Although the interaction between the bacterial SecYEG translocon and YidC has been observed in multiple studies, it is still unknown which domains of YidC are in contact with the SecYEG translocon. By in vivo and in vitro site-directed and para-formaldehyde cross-linking we identified the auxiliary transmembrane domain 1 of E. coli YidC as a major contact site for SecY and SecG. Additional SecY contacts were observed for the tightly packed globular domain and the C1 loop of YidC, which reveals that the hydrophilic cavity of YidC faces the lateral gate of SecY. Surprisingly, YidC-SecYEG contacts were only observed when YidC and SecYEG were present at about stoichiometric concentrations, suggesting that the YidC-SecYEG contact in vivo is either very transient or only observed for a very small SecYEG sub-population. This is different for the YidC-SRP and YidC-FtsY interaction, which involves the C1 loop of YidC and is efficiently observed even at sub-stoichiometric concentrations of SRP/FtsY. In summary, our data provide a first detailed view on how YidC interacts with the SecYEG translocon and the SRP-targeting machinery.

About one-quarter to nearly one-third of the proteins synthesized in the cytosol of eukaryotic cells are integrated into the plasma membrane or are secreted. Translocation of secretory proteins into the lumen of the endoplasmic reticulum or the periplasm of bacteria is mediated by a highly conserved heterotrimeric membrane protein complex denoted Sec61 in eukaryotes and SecYEG in bacteria. To evaluate a possible modulation of the translocation efficiency by secondary structures of the nascent peptide chain, we performed a comparative analysis in bacteria, yeast, and mammalian cells. Strikingly, neither the bacterial SecY nor the eukaryotic Sec61 translocon was able to efficiently transport proteins entirely composed of intrinsically disordered domains (IDDs) or β-strands. However, translocation could be restored by α-helical domains in a position- and organism-dependent manner. In bacteria, we found that the α-helical domains have to precede the IDD or β-strands, whereas in mammalian cells, C-terminally located α-helical domains are sufficient to promote translocation. Our study reveals an evolutionarily conserved deficiency of the Sec61/SecY complex to translocate IDDs and β-strands in the absence of α-helical domains. Moreover, our results may suggest that adaptive pathways co-evolved with the expansion of IDDs in the proteome of eukaryotic cells to increase the transport capacity of the Sec61 translocon., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic proteins by catalyzing the formation of intramolecular disulfide bonds. It has an important role in various cellular functions, including cell division. The purple non-sulfur bacterium Rhodobacter capsulatus mutants lacking DsbA show severe temperature-sensitive and medium-dependent respiratory growth defects. In the presence of oxygen, at normal growth temperature (35°C), DsbA - mutants form colonies on minimal medium, but they do not grow on enriched medium where cells elongate and lyse. At lower temperatures (i.e., 25°C), cells lacking DsbA grow normally in both minimum and enriched media, however, they do not produce the cbb 3 -type cytochrome c oxidase ( cbb 3 -Cox) on enriched medium. Availability of chemical oxidants (e.g., Cu 2+ or a mixture of cysteine and cystine) in the medium becomes critical for growth and cbb 3 -Cox production in the absence of DsbA. Indeed, addition of Cu 2+ to the enriched medium suppresses, and conversely, omission of Cu 2+ from the minimal medium induces, growth and cbb 3 -Cox defects. Alleviation of these defects by addition of redox-active chemicals indicates that absence of DsbA perturbs cellular redox homeostasis required for the production of an active cbb 3 -Cox, especially in enriched medium where bioavailable Cu 2+ is scarce. This is the first report describing that DsbA activity is required for full respiratory capability of R. capsulatus , and in particular, for proper biogenesis of its cbb 3 -Cox. We propose that absence of DsbA, besides impairing the maturation of the c -type cytochrome subunits, also affects the incorporation of Cu into the catalytic subunit of cbb 3 -Cox. Defective high affinity Cu acquisition pathway, which includes the MFS-type Cu importer CcoA, and lower production of the c -type cytochrome subunits lead together to improper assembly and degradation of cbb 3 -Cox.

Study Design: Cross-sectional survey., Objectives: To investigate annual rates and geographic variation of health care utilization in persons with spinal cord injury (SCI), and to identify factors associated with health care utilization., Setting: Community setting, entire country of Switzerland., Methods: Annual rates of planned and emergency visits to the general practitioner (GP), planned and emergency outpatient clinic visits and in-patient hospitalizations were compared between individuals with chronic SCI, over 16 years of age residing in Switzerland between late 2011 and early 2013 and a population sample (2012) of the Swiss general population. Risk factors for increased health service utilization were identified by means of regression models adjusted for spatial variation., Results: Of 492 participants (86.2% response rate), 94.1% visited a health care provider in the preceding year, with most persons visiting GPs (88.4%) followed by outpatient clinics (53.1%) and in-patient hospitals (35.9%). The increase in utilization as compared with the general population was 1.3-, 4.0- and 2.9-fold for GP, outpatient clinic and in-patient hospital visit, respectively. GP utilization was highest in persons with low income (incidence rate ratio (IRR) 1.85) and old age (IRR 2.62). In the first 2 years post injury, health service visits were 1.7 (GP visits) to 5.8 times (emergency outpatient clinic visits) more likely compared with those later post injury., Conclusions: People with SCI more frequently use health services as compared with the general population, across all types of medical service institutions. GP services were used most often in areas where availability of specialized outpatient clinic services was low.

Study Design: This was a cross-sectional questionnaire survey., Objectives: The objective of this study was to identify the care-seeking behavior of persons with spinal cord injury (SCI) with respect to the various health care providers and ascertain circumstances that lead to situations where required care was not received., Setting: This study was conducted in the entire country of Switzerland., Methods: Statistical analysis of frequency of annual visits to health care providers by 17 specialties, and description of situations where health care was required but not received, in persons with chronic SCI living in the community., Results: Main medical contact person was the general practitioner (GP; visited by 88% during last 12 months). The physiotherapist (visited by 72%) was the health care provider with the most visits (average of 30 visits in 12 months). GPs, physiotherapists, urologists and spinal medicine specialists were often contacted in combination, by many participants, often for check-up visits. A situation where care was required but not received was reported by 53 (11%) of participants, with a substantially higher rate in migrants (29%). Main problems why care was not received were bladder and bowel problems and main reasons of care not received were regional or temporal unavailability., Conclusions: Individuals with SCI are frequent users of medical services. There is no group of medical specialists that covers all needs of persons with SCI, what emphasizes health care provision from a comprehensive perspective including a wide array of services. Instances with care required but not received appeared to be rare and more likely in participants with migration background.