Total synthesis of himastatin.

The natural product himastatin has an unusual homodimeric structure that presents a substantial synthetic challenge. We report the concise total synthesis of himastatin from readily accessible precursors, incorporating a final-stage dimerization strategy that was inspired by a detailed consideration of the compound's biogenesis. Combining this approach with a modular synthesis enabled expedient access to more than a dozen designed derivatives of himastatin, including synthetic probes that provide insight into its antibiotic activity.

Bacterial developmental checkpoint that directly monitors cell surface morphogenesis.

Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous "coat" and an inner peptidoglycan "cortex," separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.

Single molecule/particle tracking analysis program SMTracker 2.0 reveals different dynamics of proteins within the RNA degradosome complex in Bacillus subtilis.

Single-molecule (particle) tracking is a powerful method to study dynamic processes in cells at highest possible spatial and temporal resolution. We have developed SMTracker, a graphical user interface for automatic quantifying, visualizing and managing of data. Version 2.0 determines distributions of positional displacements in x- and y-direction using multi-state diffusion models, discriminates between Brownian, sub- or superdiffusive behaviour, and locates slow or fast diffusing populations in a standardized cell. Using SMTracker, we show that the Bacillus subtilis RNA degradosome consists of a highly dynamic complex of RNase Y and binding partners. We found similar changes in molecule dynamics for RNase Y, CshA, PNPase and enolase, but not for phosphofructokinase, RNase J1 and J2, to inhibition of transcription. However, the absence of PfkA or of RNase J2 affected molecule dynamics of RNase Y-mVenus, indicating that these two proteins are indeed part of the degradosome. Molecule counting suggests that RNase Y is present as a dimer in cells, at an average copy number of about 500, of which 46% are present in a slow-diffusive state and thus likely engaged within degradosomes. Thus, RNase Y, CshA, PNPase and enolase likely play central roles, and RNase J1, J2 and PfkA more peripheral roles, in degradosome architecture.

Temporal compartmentalization of viral infection in bacterial cells.

Virus infection causes major rearrangements in the subcellular architecture of eukaryotes, but its impact in prokaryotic cells was much less characterized. Here, we show that infection of the bacterium Bacillus subtilis by bacteriophage SPP1 leads to a hijacking of host replication proteins to assemble hybrid viral-bacterial replisomes for SPP1 genome replication. Their biosynthetic activity doubles the cell total DNA content within 15 min. Replisomes operate at several independent locations within a single viral DNA focus positioned asymmetrically in the cell. This large nucleoprotein complex is a self-contained compartment whose boundaries are delimited neither by a membrane nor by a protein cage. Later during infection, SPP1 procapsids localize at the periphery of the viral DNA compartment for genome packaging. The resulting DNA-filled capsids do not remain associated to the DNA transactions compartment. They bind to phage tails to build infectious particles that are stored in warehouse compartments spatially independent from the viral DNA. Free SPP1 structural proteins are recruited to the dynamic phage-induced compartments following an order that recapitulates the viral particle assembly pathway. These findings show that bacteriophages restructure the crowded host cytoplasm to confine at different cellular locations the sequential processes that are essential for their multiplication.

Oxalate decarboxylase uses electron hole hopping for catalysis.

The hexameric low-pH stress response enzyme oxalate decarboxylase catalyzes the decarboxylation of the oxalate mono-anion in the soil bacterium Bacillus subtilis. A single protein subunit contains two Mn-binding cupin domains, and catalysis depends on Mn(III) at the N-terminal site. The present study suggests a mechanistic function for the C-terminal Mn as an electron hole donor for the N-terminal Mn. The resulting spatial separation of the radical intermediates directs the chemistry toward decarboxylation of the substrate. A π-stacked tryptophan pair (W96/W274) links two neighboring protein subunits together, thus reducing the Mn-to-Mn distance from 25.9 Å (intrasubunit) to 21.5 Å (intersubunit). Here, we used theoretical analysis of electron hole-hopping paths through redox-active sites in the enzyme combined with site-directed mutagenesis and X-ray crystallography to demonstrate that this tryptophan pair supports effective electron hole hopping between the C-terminal Mn of one subunit and the N-terminal Mn of the other subunit through two short hops of ∼8.5 Å. Replacement of W96, W274, or both with phenylalanine led to a large reduction in catalytic efficiency, whereas replacement with tyrosine led to recovery of most of this activity. W96F and W96Y mutants share the wildtype tertiary structure. Two additional hole-hopping networks were identified leading from the Mn ions to the protein surface, potentially protecting the enzyme from high Mn oxidation states during turnover. Our findings strongly suggest that multistep hole-hopping transport between the two Mn ions is required for enzymatic function, adding to the growing examples of proteins that employ aromatic residues as hopping stations.

Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation.

The Gram-positive bacterium Bacillus subtilis can divide via two modes. During vegetative growth, the division septum is formed at the midcell to produce two equal daughter cells. However, during sporulation, the division septum is formed closer to one pole to yield a smaller forespore and a larger mother cell. Using cryo-electron tomography, genetics and fluorescence microscopy, we found that the organization of the division machinery is different in the two septa. While FtsAZ filaments, the major orchestrators of bacterial cell division, are present uniformly around the leading edge of the invaginating vegetative septa, they are only present on the mother cell side of the invaginating sporulation septa. We provide evidence suggesting that the different distribution and number of FtsAZ filaments impact septal thickness, causing vegetative septa to be thicker than sporulation septa already during constriction. Finally, we show that a sporulation-specific protein, SpoIIE, regulates asymmetric divisome localization and septal thickness during sporulation.

Ultraviolet-C inactivation and hydrophobicity of Bacillus subtilis and Bacillus velezensis spores isolated from extended shelf-life milk.

Bacterial spores are important in food processing due to their ubiquity, resistance to high temperature and chemical inactivation. This work aims to study the effect of ultraviolet C (UVC) on the spores of Bacillus subtilis and Bacillus velezensis at a molecular and individual level to guide in deciding on the right parameters that must be applied during the processing of liquid foods. The spores were treated with UVC using phosphate buffer saline (PBS) as a suspension medium and their lethality rate was determined for each sample. Purified spore samples of B. velezensis and B. subtilis were treated under one pass in a UVC reactor to inactivate the spores. The resistance pattern of the spores to UVC treatment was determined using dipicolinic acid (Ca-DPA) band of spectral analysis obtained from Raman spectroscopy. Flow cytometry analysis was also done to determine the effect of the UVC treatment on the spore samples at the molecular level. Samples were processed for SEM and the percentage spore surface hydrophobicity was also determined using the Microbial Adhesion to Hydrocarbon (MATH) assay to predict the adhesion strength to a stainless-steel surface. The result shows the maximum lethality rate to be 6.5 for B. subtilis strain SRCM103689 (B47) and highest percentage hydrophobicity was 54.9% from the sample B. velezensis strain LPL-K103 (B44). The difference in surface hydrophobicity for all isolates was statistically significant (P < 0.05). Flow cytometry analysis of UVC treated spore suspensions clarifies them further into sub-populations unaccounted for by plate counting on growth media. The Raman spectroscopy identified B4002 as the isolate possessing the highest concentration of Ca-DPA. The study justifies the critical role of Ca-DPA in spore resistance and the possible sub-populations after UVC treatment that may affect product shelf-life and safety. UVC shows a promising application in the inactivation of resistant spores though there is a need to understand the effects at the molecular level to design the best parameters during processing.

A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline.

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.

CwlQ Is Required for Swarming Motility but Not Flagellar Assembly in Bacillus subtilis.

Lytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan must be unfailingly stable to preserve cell integrity, but must also be dynamically remodeled for the cell to grow, divide, and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG, and flagellar insertion is aided by localized activity of a dedicated PG lyase in Gram-negative bacteria. To date, there is no known dedicated lyase in Gram-positive bacteria for the insertion of flagella. Here, we take a reverse-genetic candidate-gene approach and find that cells mutated for the lytic transglycosylase CwlQ exhibit a severe defect in flagellum-dependent swarming motility. We further show that CwlQ is expressed by the motility sigma factor SigD and is secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells with mutations of CwlQ remain proficient for flagellar biosynthesis even when mutated in combination with four other lyases related to motility (LytC, LytD, LytF, and CwlO). The PG lyase (or lyases) essential for flagellar synthesis in B. subtilis, if any, remains unknown.IMPORTANCE Bacteria are surrounded by a wall of peptidoglycan and early work in Bacillus subtilis was the first to suggest that bacteria needed to enzymatically remodel the wall to permit insertion of the flagellum. No PG remodeling enzyme alone or in combination, however, has been found to be essential for flagellar assembly in B. subtilis Here, we take a reverse-genetic candidate-gene approach and find that the PG lytic transglycosylase CwlQ is required for swarming motility. Subsequent characterization determined that while CwlQ was coexpressed with motility genes and is secreted by the flagellar secretion apparatus, it was not required for flagellar synthesis. The PG lyase needed for flagellar assembly in B. subtilis remains unknown.

Transcriptional coupling and repair of 8-OxoG activate a RecA-dependent checkpoint that controls the onset of sporulation in Bacillus subtilis.

During sporulation Bacillus subtilis Mfd couples transcription to nucleotide excision repair (NER) to eliminate DNA distorting lesions. Here, we report a significant decline in sporulation following Mfd disruption, which was manifested in the absence of external DNA-damage suggesting that spontaneous lesions activate the function of Mfd for an efficient sporogenesis. Accordingly, a dramatic decline in sporulation efficiency took place in a B. subtilis strain lacking Mfd and the repair/prevention guanine oxidized (GO) system (hereafter, the ∆GO system), composed by YtkD, MutM and MutY. Furthermore, the simultaneous absence of Mfd and the GO system, (i) sensitized sporulating cells to H2O2, and (ii) elicited spontaneous and oxygen radical-induced rifampin-resistance (Rifr) mutagenesis. Epifluorescence (EF), confocal and transmission electron (TEM) microscopy analyses, showed a decreased ability of ∆GO ∆mfd strain to sporulate and to develop the typical morphologies of sporulating cells. Remarkably, disruption of sda, sirA and disA partially, restored the sporulation efficiency of the strain deficient for Mfd and the ∆GO system; complete restoration occurred in the RecA- background. Overall, our results unveil a novel Mfd mechanism of transcription-coupled-repair (TCR) elicited by 8-OxoG which converges in the activation of a RecA-dependent checkpoint event that control the onset of sporulation in B. subtilis.

Homology modeling and heterologous expression of highly alkaline subtilisin-like serine protease from Bacillus halodurans C-125.

Here we report heterologous expression, enzymatic characterization and structure homology modeling of a subtilisin-like alkaline serine protease (ASP) from Bacillus halodurans C-125. Encoding gene was successfully obtained by PCR and cloned into pMA0911 shuttle vector under the control of strong HpaII promoter and expressed extracellularly. ASP enzyme was successfully expressed in B. subtilis WB800 cell line lacking eight extracellular proteases and produced extracellularly in the culture medium. Km, Vmax and specific activity parameters of the recombinantly produced ASP were identified as 0.2899 mg/ml, 76.12 U/ml and 9500 U/mg, respectively. The purified enzyme revealed remarkable proteolytic activity at highly alkaline conditions with a pH optimum 12.0 and notable thermostability with temperature optimum at 60 °C. Furthermore, substrate-free enzyme revealed remarkable pH stability at pH 12.0 and maintained 93% of its initial activity when incubated at 37 °C for 24 h and 60% of its initial activity upon incubation at 60 °C for 1 h. Theoretically calculated molecular mass of ASP protein was confirmed through SDS-PAGE and western blot analysis (Mw: 28.3 kDa). The secondary and tertiary structures of ASP protein were also identified through homology modeling and further examined in detail. ASP harbors a typical S8/S53 peptidase domain comprising 17 ß-sheets and 9 α-helixes within its secondary structure. The structure dynamics analysis of modeled 3D structure further revealed that transient inactivating propeptide chain is the most dynamic region of ASP enzyme with 8.52 Å2 ß-Factor value. Additional residue-dependent fluctuation plot analysis also confirmed the elevated structure dynamics patterning of ASP N-terminus which could be the potential prerequisite for the autonomous propeptide removal of alkaline serine peptidases. Yet the functional domain of ASP becomes quite stable after autonomous exclusion of its propeptide. Although the sequence homology between ASP and commercial detergent additive B. lentus protease (PDB ID:1GCI) was moderate (65.4% sequence similarity), their overlaid 3D structures revealed much higher similarity (98.14%) within 0.80 Å RMSD. In conclusions, with remarkable pH stability, notable thermostability and particularly high specific activity at extreme alkaline conditions, the unveiled ASP protein stands out as a novel protease candidate for various industrial sectors such as textile, detergent, leather, feed, waste, pharmaceutical and others.

Structural Basis for Bacterial Ribosome-Associated Quality Control by RqcH and RqcP.

In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails." How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.

Mimicry of Canonical Translation Elongation Underlies Alanine Tail Synthesis in RQC.

Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation.

Multistep optimization of a cell-penetrating peptide towards its antimicrobial activity.

Multidrug resistant (MDR) bacteria have adapted to most clinical antibiotics and are a growing threat to human health. One promising type of candidates for the everlasting demand of new antibiotic compounds constitute antimicrobial peptides (AMPs). These peptides act against different types of microbes by permeabilizing pathogen cell membranes, whereas being harmless to mammalian cells. Contrarily, another class of membrane-active peptides, namely cell-penetrating peptides (CPPs), is known to translocate in eukaryotic cells without substantially affecting the cell membrane. Since CPPs and AMPs share several physicochemical characteristics, we hypothesized if we can rationally direct the activity of a CPP towards antimicrobial activity. Herein, we describe the screening of a synthetic library, based on the CPP sC18, including structure-based design to identify the active residues within a CPP sequence and to discover novel AMPs with high activity. Peptides with increased hydrophobicity were tested against various bacterial strains, and hits were further optimized leading to four generations of peptides, with the last also comprising fluorinated amino acid building blocks. Interestingly, beside strong antibacterial activities, we also detected activity in cancer cells, while non-cancerous cells remained unharmed. The results highlight our new candidates, particularly those from generation 4, as a valuable and promising source for the development of future therapeutics with antibacterial activity and beyond.

Impact of an Engineered Copper-Titanium Dioxide Nanocomposite and Parent Substrates on the Bacteria Viability, Antioxidant Enzymes and Fatty Acid Profiling.

Due to the systematic increase in the production of nanomaterials (NMs) and their applications in many areas of life, issues associated with their toxicity are inevitable. In particular, the performance of heterogeneous NMs, such as nanocomposites (NCs), is unpredictable as they may inherit the properties of their individual components. Therefore, the purpose of this work was to assess the biological activity of newly synthesized Cu/TiO2-NC and the parent nanoparticle substrates Cu-NPs and TiO2-NPs on the bacterial viability, antioxidant potential and fatty acid composition of the reference Escherichia coli and Bacillus subtilis strains. Based on the toxicological parameters, it was found that B. subtilis was more sensitive to NMs than E. coli. Furthermore, Cu/TiO2-NC and Cu-NPs had an opposite effect on both strains, while TiO2-NPs had a comparable mode of action. Simultaneously, the tested strains exhibited varied responses of the antioxidant enzymes after exposure to the NMs, with Cu-NPs having the strongest impact on their activity. The most considerable alternations in the fatty acid profiles were found after the bacteria were exposed to Cu/TiO2-NC and Cu-NPs. Microscopic images indicated distinct interactions of the NMs with the bacterial outer layers, especially in regard to B. subtilis. Cu/TiO2-NC generally proved to have less distinctive antimicrobial properties on B. subtilis than E. coli compared to its parent components. Presumably, the biocidal effects of the tested NMs can be attributed to the induction of oxidative stress, the release of metal ions and specific electrochemical interactions with the bacterial cells.

High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action.

The increase in speed of the high-speed atomic force microscopy (HS-AFM) compared to that of the conventional AFM made possible the first-ever visualisation at the molecular-level of the activity of an antimicrobial peptide on a membrane. We investigated the medically prescribed but poorly understood lipopeptide Daptomycin under infection-like conditions (37 °C, bacterial lipid composition and antibiotic concentrations). We confirmed so far hypothetical models: Dap oligomerization and the existence of half pores. Moreover, we detected unknown molecular mechanisms: new mechanisms to form toroidal pores or to resist Dap action, and to unprecedently quantify the energy profile of interacting oligomers. Finally, the biological and medical relevance of the findings was ensured by a multi-scale multi-nativeness-from the molecule to the cell-correlation of molecular-level information from living bacteria (Bacillus subtilis strains) to liquid-suspended vesicles and supported-membranes using electron and optical microscopies and the lipid tension probe FliptR, where we found that the cells with a healthier state of their cell wall show smaller membrane deformations.

Morphological Changes in Spores during Germination in Bacillus cereus and Bacillus subtilis.

Processes from spore germination to outgrowth were observed in detail using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for Bacillus cereus and Bacillus subtilis. At 15 and 30 min after germination induction, SEM observation and SEM-EDX analysis of Bacillus spores prepared by freeze substitution showed that spherical structures including compounds having the same elemental ratio as that of the spore were observed on the surface of the spores. The results suggested the leakages of the cellular materials from the spores. At 360 min, B. cereus spores in outgrowth phase elongated with hemispherical structures at the end of the long side of the cells. The discoid structures with a hole (20-30 nm diameter) in the center was observed at 360 min. Confocal laser scanning microscopy after staining with fluorescence-labeled anti-spore antibodies showed that the hemispherical and discoid structures originated from the spore coat. These structures broke down after detached from the cells in outgrowth phase.

Bacterial nanotubes as a manifestation of cell death.

Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells' genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.

Structures of B. subtilis Maturation RNases Captured on 50S Ribosome with Pre-rRNAs.

The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.

The Effect of Chlorogenic Acid on Bacillus subtilis Based on Metabolomics.

Chlorogenic acid (CGA), a natural phenolic compound, is an important bioactive compound, and its antibacterial activity has been widely concerned, but its antibacterial mechanism remains largely unknown. Protein leakage and the solution exosmosis conductivity of Bacillus subtilis 24434 (B. subtilis) reportedly display no noticeable differences before and after CGA treatment. The bacterial cells treated with CGA displayed a consistently smooth surface under the electron microscope, indicating that CGA cannot directly disrupt bacterial membranes. However, CGA induced a significant decrease in the intracellular adenosine triphosphate (ATP) concentration, possibly by affecting the material and energy metabolism or cell-signaling transduction. Furthermore, metabolomic results indicated that CGA stress had a bacteriostatic effect by inducing the intracellular metabolic imbalance of the tricarboxylic acid (TCA) cycle and glycolysis, leading to metabolic disorder and death of B. subtilis. These findings improve the understanding of the complex action mechanisms of CGA antimicrobial activity and provide theoretical support for the application of CGA as a natural antibacterial agent.