Abstract
The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant
Staphylococcus aureus
(MRSA) and drug-resistant ...enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca
2+
-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.
The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo. Here we report the results of our efforts to elucidate the mechanism of ...regulation of PknB activity. The specific residues in the PknB extracytoplasmic domain that are essential for ligand interaction and survival of the bacterium are identified. The extracytoplasmic domain interacts with mDAP-containing LipidII, and this is abolished upon mutation of the ligand-interacting residues. Abrogation of ligand-binding or sequestration of the ligand leads to aberrant localization of PknB. Contrary to the prevailing hypothesis, abrogation of ligand-binding is linked to activation loop hyperphosphorylation, and indiscriminate hyperphosphorylation of PknB substrates as well as other proteins, ultimately causing loss of homeostasis and cell death. We propose that the ligand-kinase interaction directs the appropriate localization of the kinase, coupled to stringently controlled activation of PknB, and consequently the downstream processes thereof.
Microbial sulfate reduction has governed Earth's biogeochemical sulfur cycle for at least 2.5 billion years. However, the enzymatic mechanisms behind this pathway are incompletely understood, ...particulary for the reduction of sulfite—a key intermediate in the pathway. This critical reaction is performed by DsrAB, a widespread enzyme also involved in other dissimilatory sulfur metabolisms. Using in vitro assays with an archaeal DsrAB, supported with genetic experiments in a bacterial system, we show that the product of sulfite reduction by DsrAB is a protein-based trisulfide, in which a sulfite-derived sulfur is bridging two conserved cysteines of DsrC. Physiological studies also reveal that sulfate reduction rates are determined by cellular levels of DsrC. Dissimilatory sulfate reduction couples the four-electron reduction of the DsrC trisulfide to energy conservation.
Behind the versatile nature of prokaryotic energy metabolism is a set of redox proteins having a highly modular character. It has become increasingly recognized that a limited number of redox modules ...or building blocks appear grouped in different arrangements, giving rise to different proteins and functionalities. This modularity most likely reveals a common and ancient origin for these redox modules, and is obviously reflected in similar energy conservation mechanisms. The dissimilation of sulfur compounds was probably one of the earliest biological strategies used by primitive organisms to obtain energy. Here, we review some of the redox proteins involved in dissimilatory sulfur metabolism, focusing on sulfate reducing organisms, and highlight links between these proteins and others involved in different processes of anaerobic respiration. Noteworthy are links to the complex iron–sulfur molybdoenzyme family, and heterodisulfide reductases of methanogenic archaea. We discuss how chemiosmotic and electron bifurcation/confurcation may be involved in energy conservation during sulfate reduction, and how introduction of an additional module, multiheme cytochromes c, opens an alternative bioenergetic strategy that seems to increase metabolic versatility. Finally, we highlight new families of heterodisulfide reductase-related proteins from non-methanogenic organisms, which indicate a widespread distribution for these protein modules and may indicate a more general involvement of thiol/disulfide conversions in energy metabolism. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.
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► Dissimilation of sulfur compounds is an ancient metabolism. ► We present proteins involved in dissimilatory sulfur metabolism. ► Their modular character and links to other respiratory systems are discussed. ► Unique complexes show links to CISM family and heterodisulfide reductases. ► Electron bifurcation linked to chemiosmotic mechanisms are proposed.
The bacterial cell wall is essential for viability, but despite its ability to withstand internal turgor must remain dynamic to permit growth and division. Peptidoglycan is the major cell wall ...structural polymer, whose synthesis requires multiple interacting components. The human pathogen
is a prolate spheroid that divides in three orthogonal planes. Here, we have integrated cellular morphology during division with molecular level resolution imaging of peptidoglycan synthesis and the components responsible. Synthesis occurs across the developing septal surface in a diffuse pattern, a necessity of the observed septal geometry, that is matched by variegated division component distribution. Synthesis continues after septal annulus completion, where the core division component FtsZ remains. The novel molecular level information requires re-evaluation of the growth and division processes leading to a new conceptual model, whereby the cell cycle is expedited by a set of functionally connected but not regularly distributed components.
The 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.
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 H(2), 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.
When oxidizing reduced sulfur compounds, the phototrophic sulfur bacterium
forms spectacular sulfur globules as obligatory intracellular-but extracytoplasmic-intermediates. The globule envelope ...consists of three extremely hydrophobic proteins: SgpA and SgpB, which are very similar and can functionally replace each other, and SgpC which is involved in the expansion of the sulfur globules. The presence of a fourth protein, SgpD, was suggested by comparative transcriptomics and proteomics of purified sulfur globules. Here, we investigated the in vivo function of SgpD by coupling its carboxy-terminus to mCherry. This fluorescent protein requires oxygen for chromophore maturation, but we were able to use it in anaerobically growing
provided the cells were exposed to oxygen for one hour prior to imaging. While mCherry lacking a signal peptide resulted in low fluorescence evenly distributed throughout the cell, fusion with SgpD carrying its original Sec-dependent signal peptide targeted mCherry to the periplasm and co-localized it exactly with the highly light-refractive sulfur deposits seen in sulfide-fed
cells. Insertional inactivation of the
gene showed that the protein is not essential for the formation and degradation of sulfur globules.
Sufficient access to transition metals such as iron is essential for bacterial proliferation and their active limitation within host tissues effectively restricts infection. To overcome iron ...limitation, the invasive pathogen
uses the iron-regulated surface determinant (Isd) system to acquire hemoglobin-derived heme. While heme transport over the cell wall is well understood, its transport over the membrane is hardly investigated. In this study, we show the heme-specific permease IsdF to be energized by the general ATPase FhuC. Additionally, we show that IsdF needs appropriate location within the membrane for functionality. The membrane of
possesses special compartments (functional membrane microdomains FMMs) to organize membrane complexes. We show IsdF to be associated with FMMs, to directly interact with the FMM scaffolding protein flotillin A (FloA) and to co-localize with the latter on intact bacterial cells. Additionally, Isd-dependent bacterial growth required FMMs and FloA. Our study shows that Isd-dependent heme acquisition requires a highly structured cell envelope to allow coordinated transport over the cell wall and membrane and it gives the first example of a bacterial nutrient acquisition system that depends on FMMs.
Summary
In this work we identified the gene for the tetrathionate‐forming thiosulfate dehydrogenase (TsdA) from the purple sulfur bacterium Allochromatium vinosum by sequence analysis and reverse ...genetics. The recombinant protein produced in Escherichia coli is a periplasmic, monomeric 25.8 kDa dihaem cytochrome c with an enzyme activity optimum at pH 4. UV‐visible and electron paramagnetic resonance spectroscopy indicate methionine (strictly conserved M222 or M236) and cysteine (C123) as probable sixth distal axial ligands of the two haem irons in TsdA. These results place TsdA in the group of c‐type cytochromes with an unusual axial histidine‐cysteine coordination of the haem iron. These proteins appear to play a pivotal role in sulfur‐based energy metabolism. Exchange of C123 to glycine rendered thiosulfate dehydrogenase inactive, proving the importance of this residue for catalysis.
TsdA homologues are present in α‐, β‐, δ‐, γ‐ and ε‐Proteobacteria. Three of these were produced in E. coli and exhibited the expected enzymatic activity. The widespread occurrence of tsdA agrees with reports of tetrathionate formation not only by specialized sulfur oxidizers but also by many chemoorganoheterotrophs that use thiosulfate as a supplemental but not as the sole energy source.
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