Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics.antibiotics | daptomycin | membrane potential | cell wall biosynthesis | Bacillus subtilis

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.

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, particularly 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.

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with grampositive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drugresistant 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.

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.

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.

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 Staphylococcus aureus 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.

Flavin-based electron bifurcation (FBEB) is an important mechanism for the energy metabolism of anaerobes. A new family of NADH dehydrogenases, the flavin oxidoreductase (FlxABCD, previously called FloxABCD), was proposed to perform FBEB in sulphate-reducing organisms coupled with heterodisulfide reductase (HdrABC). We found that the hdrABC-flxABCD gene cluster is widespread among anaerobic bacteria, pointing to a general and important role in their bioenergetics. In this work, we studied FlxABCD of Desulfovibrio vulgaris Hildenborough. The hdr-flx genes are part of the same transcriptional unit and are increased in transcription during growth in ethanol-sulfate, and to a less extent during pyruvate fermentation. Two mutant strains were generated: one where expression of the hdr-flx genes was interrupted and another lacking the flxA gene. Both strains were unable to grow with ethanol-sulfate, whereas growth was restored in a flxA-complemented strain. The mutant strains also produced very reduced amounts of ethanol compared with the wild type during pyruvate fermentation. Our results show that in D. vulgaris, the FlxABCD-HdrABC proteins are essential for NADH oxidation during growth on ethanol, probably involving a FBEB mechanism that leads to reduction of ferredoxin and the small protein DsrC, while in fermentation they operate in reverse, reducing NAD(+) for ethanol production.