Biochemical, biophysical and functional analysis of the DsrMKJOP transmembrane complex from <em>Allochromatium vinosum</em>

In the present study the DsrMKJOP transmembrane complex was investigated and characterized by biochemical, biophysical and functional analyses. Each of the proteins was successfully produced in E. coli as recombinant proteins and purified. The complex was furthermore enriched from A. vinosum and analysed. It has been shown that DsrJ is a triheme cytochrome c and EPR spectroscopy provided evidence for a possible, but only partial, His/Cys heme ligation in one of the hemes. This heme shows heterogeneous coordination with methionine being another candidate ligand. Cysteine 46 was replaced by serine using site-directed mutagenesis, with the mutant protein showing a significant decrease in the EPR signal attributed to His/Cys coordination, but identical UV-Vis and resonance Raman spectra. The redox potentials of the hemes in the wild type protein were determined to be -20,-200 and -220 mV and were found to be virtually identical in the mutant protein. However, in vivo the same ligand exchange led to a dramatically altered phenotype highlighting the importance of Cys46. The results suggest that Cys46 may be involved in catalytic sulfur chemistry rather than electron transfer. It was furthermore shown that the signal peptide of DsrJ is not cleaved off in A. vinosum and that it may anchor the protein into the membrane. DsrM was identified as a diheme cytochrome b and the two hemes were found to be in low spin state. Their midpoint redox potentials were determined to be +60 and +110 mV. Although no hemes were predicted for DsrP, it was also clearly identified as a b-type cytochrome. As obvious from the literature, this is the first time that heme binding has been experimentally proven for a member of the NrfD protein family. Both cytochromes were partly reduced after addition of a menaquinol analogue suggesting interaction with quinones in vivo. DsrO and DsrK were both experimentally proven to be Fe-S-containing proteins. In addition, DsrK was shown to be membrane-associated most likely via an in-plane membrane anchor. Coelution assays provide support for the proposed interaction of DsrK with the soluble cytoplasmic protein DsrC, which might be its substrate. The results obtained in this study were combined in a model for the function of the DsrMKJOP complex in A. vinosum

The Lantibiotic Nisin Induces Lipid II Aggregation, Causing Membrane Instability and Vesicle Budding

AbstractThe antimicrobial peptide nisin exerts its activity by a unique dual mechanism. It permeates the cell membranes of Gram-positive bacteria by binding to the cell wall precursor Lipid II and inhibits cell wall synthesis. Binding of nisin to Lipid II induces the formation of large nisin-Lipid II aggregates in the membrane of bacteria as well as in Lipid II-doped model membranes. Mechanistic details of the aggregation process and its impact on membrane permeation are still unresolved. In our experiments, we found that fluorescently labeled nisin bound very inhomogeneously to bacterial membranes as a consequence of the strong aggregation due to Lipid II binding. A correlation between cell membrane damage and nisin aggregation was observed in vivo. To further investigate the aggregation process of Lipid II and nisin, we assessed its dynamics by single-molecule microscopy of fluorescently labeled Lipid II molecules in giant unilamellar vesicles using light-sheet illumination. We observed a continuous reduction of Lipid II mobility due to a steady growth of nisin-Lipid II aggregates as a function of time and nisin concentration. From the measured diffusion constants of Lipid II, we estimated that the largest aggregates contained tens of thousands of Lipid II molecules. Furthermore, we observed that the formation of large nisin-Lipid II aggregates induced vesicle budding in giant unilamellar vesicles. Thus, we propose a membrane permeation mechanism that is dependent on the continuous growth of nisin-Lipid II aggregation and probably involves curvature effects on the membrane

A Comparative Genomic Analysis of Energy Metabolism in Sulfate Reducing Bacteria and Archaea

The number of sequenced genomes of sulfate reducing organisms (SRO) has increased significantly in the recent years, providing an opportunity for a broader perspective into their energy metabolism. In this work we carried out a comparative survey of energy metabolism genes found in 25 available genomes of SRO. This analysis revealed a higher diversity of possible energy conserving pathways than classically considered to be present in these organisms, and permitted the identification of new proteins not known to be present in this group. The Deltaproteobacteria (and Thermodesulfovibrio yellowstonii) are characterized by a large number of cytochromes c and cytochrome c-associated membrane redox complexes, indicating that periplasmic electron transfer pathways are important in these bacteria. The Archaea and Clostridia groups contain practically no cytochromes c or associated membrane complexes. However, despite the absence of a periplasmic space, a few extracytoplasmic membrane redox proteins were detected in the Gram-positive bacteria. Several ion-translocating complexes were detected in SRO including H+-pyrophosphatases, complex I homologs, Rnf, and Ech/Coo hydrogenases. Furthermore, we found evidence that cytoplasmic electron bifurcating mechanisms, recently described for other anaerobes, are also likely to play an important role in energy metabolism of SRO. A number of cytoplasmic [NiFe] and [FeFe] hydrogenases, formate dehydrogenases, and heterodisulfide reductase-related proteins are likely candidates to be involved in energy coupling through electron bifurcation, from diverse electron donors such as H2, formate, pyruvate, NAD(P)H, β-oxidation, and others. In conclusion, this analysis indicates that energy metabolism of SRO is far more versatile than previously considered, and that both chemiosmotic and flavin-based electron bifurcating mechanisms provide alternative strategies for energy conservation

pyPESTO: A modular and scalable tool for parameter estimation for dynamic models

Mechanistic models are important tools to describe and understand biological processes. However, they typically rely on unknown parameters, the estimation of which can be challenging for large and complex systems. We present pyPESTO, a modular framework for systematic parameter estimation, with scalable algorithms for optimization and uncertainty quantification. While tailored to ordinary differential equation problems, pyPESTO is broadly applicable to black-box parameter estimation problems. Besides own implementations, it provides a unified interface to various popular simulation and inference methods. pyPESTO is implemented in Python, open-source under a 3-Clause BSD license. Code and documentation are available on GitHub (https://github.com/icb-dcm/pypesto)

Antimicrobial Potential of Bacteria Associated with Marine Sea Slugs from North Sulawesi, Indonesia

Nudibranchia, marine soft-bodied organisms, developed, due to the absence of a protective shell, different strategies to protect themselves against putative predators and fouling organisms. One strategy is to use chemical weapons to distract predators, as well as pathogenic microorganisms. Hence, these gastropods take advantage of the incorporation of chemical molecules. Thereby the original source of these natural products varies; it might be the food source, de novo synthesis from the sea slug, or biosynthesis by associated bacteria. These bioactive molecules applied by the slugs can become important drug leads for future medicinal drugs. To test the potential of the associated bacteria, the latter were isolated from their hosts, brought into culture and extracts were prepared and tested for antimicrobial activities. From 49 isolated bacterial strains 35 showed antibiotic activity. The most promising extracts were chosen for further testing against relevant pathogens. In that way three strains showing activity against methicillin resistant Staphylococcus aureus and one strain with activity against enterohemorrhagic Escherichia coli, respectively, were identified. The obtained results indicate that the sea slug associated microbiome is a promising source for bacterial strains, which hold the potential for the biotechnological production of antibiotics

Complications and Short-Term Explantation Rate Following Artificial Urinary Sphincter Implantation: Results from a Large Middle European Multi-Institutional Case Series

Background/Aims/Objectives: To analyze perioperative complication and short-term explantation rates after perineal or penoscrotal single-cuff and double-cuff artificial urinary sphincter (AUS) implantation in a large middle European multi-institutional patient cohort. Methods: 467 male patients with stress urinary incontinence underwent implantation of a perineal single-cuff (n = 152), penoscrotal single-cuff (n = 99), or perinea! double-cuff (n = 216) AUS between 2010 and 2012. Postoperative complications and 6-month explantation rates were assessed. For statistical analysis, Fisher's exact test and Kruskal Wallis rank sum test, and a multiple logistic regression model were used (p < 0.05). Results: Compared to perineal single-cuff AUS, penoscrotal single-cuff implantation led to significantly increased short-term explantation rates (8.6% (perinea)) vs. 19.2% (penoscrotal), p = 0.019). The postoperative infection rate was significantly higher after double-cuff compared to single cuff implantation (6.0% (single-cuff) vs. 13.9% (double-cuff), p = 0.019). The short-term explantation rate after primary double-cuff placement was 6.5% (p = 0.543 vs. perineal single -cuff). In multivariate analysis, the penoscrotal approach (p = 0.004), intraoperative complications (p = 0.005), postoperative bleeding (p = 0.011), and perioperative infection (p < 0.001) were independent risk factors for short-term explantation. Conclusions: Providing data from a large contemporary multi-institutional patient cohortfrom high-volume and low-volume institutions, our results reflect the current standard of care in middle Europe. We indicate that the penoscrotal approach is an independent risk factor for increased short-term explantation rates. (C) 2016 S. Karger AG, Base

An antibiotic from an uncultured bacterium binds to an immutable target

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55PP, lipid II, and lipid III WTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development. </p

A new antibiotic from an uncultured bacterium binds to an immutable target

Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55 PP, Lipid II, Lipid WTA ). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline

An antibiotic from an uncultured bacterium binds to an immutable target

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55PP, lipid II, and lipid III WTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development

Biochemical Characterization of Individual Components of the Allochromatium vinosum DsrMKJOP Transmembrane Complex Aids Understanding of Complex Function In Vivo▿ †

The DsrMKJOP transmembrane complex has a most important function in dissimilatory sulfur metabolism and consists of cytoplasmic, periplasmic, and membrane integral proteins carrying FeS centers and b- and c-type cytochromes as cofactors. In this study, the complex was isolated from the purple sulfur bacterium Allochromatium vinosum and individual components were characterized as recombinant proteins. The two integral membrane proteins DsrM and DsrP were successfully produced in Escherichia coli C43(DE3) and C41(DE3), respectively. DsrM was identified as a diheme cytochrome b, and the two hemes were found to be in low-spin state. Their midpoint redox potentials were determined to be +60 and +110 mV. Although no hemes were predicted for DsrP, it was also clearly identified as a b-type cytochrome. To the best of our knowledge, this is the first time that heme binding has been experimentally proven for a member of the NrfD protein family. Both cytochromes were partly reduced after addition of a menaquinol analogue, suggesting interaction with quinones in vivo. DsrO and DsrK were both experimentally proven to be FeS-containing proteins. In addition, DsrK was shown to be membrane associated, and we propose a monotopic membrane anchoring for this protein. Coelution assays provide support for the proposed interaction of DsrK with the soluble cytoplasmic protein DsrC, which might be its substrate. A model for the function of DsrMKJOP in the purple sulfur bacterium A. vinosum is presented