Two broadly conserved families of polyprenyl-phosphate transporters.

Peptidoglycan and almost all surface glycopolymers in bacteria are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP)1-4. These UndP-linked precursors are transported across the membrane and polymerized or directly transferred to surface polymers, lipids or proteins. UndP is then flipped to regenerate the pool of cytoplasmic-facing UndP. The identity of the flippase that catalyses transport has remained unknown. Here, using the antibiotic amphomycin that targets UndP5-7, we identified two broadly conserved protein families that affect UndP recycling. One (UptA) is a member of the DedA superfamily8; the other (PopT) contains the domain DUF368. Genetic, cytological and syntenic analyses indicate that these proteins are UndP transporters. Notably, homologues from Gram-positive and Gram-negative bacteria promote UndP transport in Bacillus subtilis, indicating that recycling activity is broadly conserved among family members. Inhibitors of these flippases could potentiate the activity of antibiotics targeting the cell envelope.

Aflatoxin B1-degrading activity from Bacillus subtilis BCC 42005 isolated from fermented cereal products.

Aflatoxin B1 is a naturally occurring mycotoxin that is produced as secondary metabolite by Aspergillus spp., especially A. flavus and A. parasiticus. This is the most severe toxin due to its carcinogenic, mutagenic, and teratogenic properties. Hence, methods for toxin degradation have been received increasing interest from both scientific communities and industries. In this study, 32 isolates of Bacillus spp. from various fermented cereal products were screened for their aflatoxin B1 degradation ability. The results indicated the extracellular fraction of Bacillus subtilis BCC 42005 isolated from Iru (African locust bean) potentially possessed aflatoxin B1-degrading ability. The maximum activity of the active fraction was at 50°C and pH 8.0. The activity was stable in a wide range of pH (5.0-8.0) and temperature (25-60°C). The aflatoxin B1-degrading mechanisms of this strain may be possibly involved by enzyme(s). This extracellular fraction was not toxic at IC50 4 mg/ml and it can be combined with water as a soaking agent for maize, which results in 54% of aflatoxin B1 reduction after contact time 120 min. Hence, the extracellular fraction of Bacillus subtilis BCC 42005 can be further applied as an effective soaking agent in a pretreatment process with a practical and easy-to-implement condition and also probably used to reduce the aflatoxin B1 contamination in other foods and feeds commodities.

A protein phosphorylation module patterns the Bacillus subtilis spore outer coat.

Assembly of the Bacillus subtilis spore coat involves over 80 proteins which self-organize into a basal layer, a lamellar inner coat, a striated electrodense outer coat and a more external crust. CotB is an abundant component of the outer coat. The C-terminal moiety of CotB, SKRB , formed by serine-rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH. We show that another coat protein, CotG, with a central serine-repeat region, SKRG , interacts with the C-terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a strain producing an inactive form of CotH, like those formed by a cotG deletion mutant, lack the pattern of electrondense outer coat striations, but retain the crust. In contrast, deletion of the SKRB region, has no major impact on outer coat structure. Thus, phosphorylation of CotG by CotH is a key factor establishing the structure of the outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis hints at the importance of this protein phosphorylation module in the morphogenesis of the spore surface layers.

Identification of an amphipathic peptide sensor of the Bacillus subtilis fluid membrane microdomains.

Regions of increased fluidity are newly found bacterial membrane microdomains that are composed of short, unsaturated and branched fatty acyl chains in a fluid and disordered state. Currently, little is known about how proteins are recruited and localized to these membrane domains. Here, we identify a short amphipathic α-peptide in a previously unreported crystal structure and show that it is responsible for peripheral localization of the phosphate acyltransferase PlsX to the fluid microdomains in Bacillus subtilis. Mutations disrupting the amphipathic interaction or increasing the nonpolar interaction are found to redistribute the protein to the cytosol or other part of the plasma membrane, causing growth defects. These results reveal a mechanism of peripheral membrane sensing through optimizing nonpolar interaction with the special lipids in the microdomains. This finding shows that the fluid membrane microdomains may take advantage of their unique lipid environment as a means of recruiting and organizing proteins.

Mammalian antimicrobial peptide protegrin-4 self assembles and forms amyloid-like aggregates: Assessment of its functional relevance.

Protegrin-4 (PG-4) is a member of the porcine leukocyte protegrins family of cysteine-rich antimicrobial peptides (AMPs) isolated from Sus scrofa. It consists of 18 amino acid residues and works as a part of innate immune system. In this study, we examined the intrinsic aggregation propensity of this AMP using multiple computational algorithms, namely, TANGO, AGGRESCAN, FOLDAMYLOID, AMYLPRED, and ZYGGREGATOR, and found that the peptide is predicted to have a high propensity for the ß sheet formation that disposes this peptide to be amyloidogenic. Under in vitro conditions, PG-4 formed visible aggregates and displayed the hallmark properties of typical amyloids such as enhanced binding of Congo red, increased fluorescence with Thioflavin-T, and fibrillar morphology under transmission electron microscopy. Then we examined its antimicrobial activity against Bacillus subtilis and found that the aggregated peptide retained its antimicrobial activity. Additionally, the aggregates remain non-toxic to the HEK293 and Caco2 cells. Our study suggests that the inherent aggregation properties of AMP can rationally be explored as a potential source of peptide-based antimicrobials with enhanced stability.

Structure-Activity Relationships of Photoswitchable Diarylethene-Based ß-Hairpin Peptides as Membranolytic Antimicrobial and Anticancer Agents.

Five series (28 structures) of photoswitchable ß-hairpin peptides were synthesized based on the cyclic scaffold of the natural antibiotic gramicidin S. Cell-type selectivity was compared for all activated (diarylethene "ring-open") and deactivated ("ring-closed") forms in terms of antibacterial activity (MIC against Escherichia coli and Bacillus subtilis), anticancer activity (IC50 against HeLa cell line), and hemolytic cytotoxicity (HC50 against human erythrocytes). Correlations between the conformational plasticity of the peptides, their hydrophobicity, and their bioactivity were also analyzed. Considerable improvements in selectivity were achieved compared to the reference compound. We found a dissociation of the anticancer activity from hemolysis. Phototherapeutic indices (PTI), HC50(closed)/MIC(open) and HC50(closed)/IC50(open), were introduced for the peptides as safety criteria. The highest PTI for HeLa-selective toxicity were observed among analogues containing hydroxyleucine on the hydrophobic face. For one compound, high PTIs were demonstrated across a range of different cancer cell lines, including a doxorubicin-resistant one.

Structural and functional insights into the regulation of the lysis-lysogeny decision in viral communities.

Communication is vital for all organisms including microorganisms, which is clearly demonstrated by the bacterial quorum-sensing system. However, the molecular mechanisms underlying communication among viruses (phages) via the quorum-sensing-like 'arbitrium' system remain unclear. Viral or host densities are known to be related to an increased prevalence of lysogeny; however, how the switch from the lytic to the lysogenic pathway occurs is unknown. Thus, we sought to reveal mechanisms of communication among viruses and determine the lysogenic dynamics involved. Structural and functional analyses of the phage-derived SAIRGA and GMPRGA peptides and their corresponding receptors, phAimR and spAimR, indicated that SAIRGA directs the lysis-lysogeny decision of phi3T by modulating conformational changes in phAimR, whereas GMPRGA regulates the lysis-lysogeny pathway by stabilizing spAimR in the dimeric state. Although temperate viruses are thought to share a similar lytic-lysogenic cycle switch model, our study suggests the existence of alternative strain-specific mechanisms that regulate the lysis-lysogeny decision. Collectively, these findings provide insights into the molecular mechanisms underlying communication among viruses, offering theoretical applications for the treatment of infectious viral diseases.

Structural basis of the arbitrium peptide-AimR communication system in the phage lysis-lysogeny decision.

A bacteriophage can replicate and release virions from a host cell in the lytic cycle or switch to a lysogenic process in which the phage integrates itself into the host genome as a prophage. In Bacillus cells, some types of phages employ the arbitrium communication system, which contains an arbitrium hexapeptide, the cellular receptor AimR and the lysogenic negative regulator AimX. This system controls the decision between the lytic and lysogenic cycles. However, both the mechanism of molecular recognition between the arbitrium peptide and AimR and how downstream gene expression is regulated remain unknown. Here, we report crystal structures for AimR from the SPbeta phage in the apo form and the arbitrium peptide-bound form at 2.20 Å and 1.92 Å, respectively. With or without the peptide, AimR dimerizes through the C-terminal capping helix. AimR assembles a superhelical fold and accommodates the peptide encircled by its tetratricopeptide repeats, which is reminiscent of RRNPP family members from the quorum-sensing system. In the absence of the arbitrium peptide, AimR targets the upstream sequence of the aimX gene; its DNA binding activity is prevented following peptide binding. In summary, our findings provide a structural basis for peptide recognition in the phage lysis-lysogeny decision communication system.

Real-Time Evaluation of Bacterial Viability Using Gold Nanoparticles.

Real-time evaluation of bacterial viability is important for various purposes such as hygiene management, development of antibacterial agents, and effective utilization of bacterial resources. Here, we demonstrate a simple procedure for evaluating bacterial viability using gold nanoparticles (Au NPs). The color of bacterial suspensions containing Au NPs strongly depended on the bacterial viability. We found that the dispersion state of Au NPs affected the color of the suspension, based on the interaction of Au NPs with substances secreted by the bacteria. This color change was easily recognized with the naked eye, and viability was accurately determined by measuring the absorbance at a specific wavelength. This method was applicable to various bacterial species, regardless of whether they were Gram-positive or Gram-negative.

Arylthiazole antibiotics targeting intracellular methicillin-resistant Staphylococcus aureus (MRSA) that interfere with bacterial cell wall synthesis.

The promising antibacterial potency of arylthiazole antibiotics is offset by their limited activity against intracellular bacteria (namely methicillin-resistant Staphylococcus aureus (MRSA)), similar to many clinically-approved antibiotics. The failure to target these hidden pathogens is due to the compounds' lack of proper characteristics to accumulate intracellularly. Fine tuning of the size and polar-surface-area of the linking heteroaromatic ring provided a new series of 5-thiazolylarylthiazoles with balanced properties that allow them to sufficiently cross and accumulate inside macrophages infected with MRSA. The most promising compound 4i exhibited rapid bactericidal activity, good metabolic stability and produced over 80% reduction of intracellular MRSA in infected macrophages.

Septal membrane localization by C-terminal amphipathic α-helices of MinD in Bacillus subtilis mutant cells lacking MinJ or DivIVA.

The Min system, which inhibits assembly of the cytokinetic protein FtsZ, is largely responsible for positioning the division site in rod-shaped bacteria. It has been reported that MinJ, which bridges DivIVA and MinD, is targeted to the cell poles by an interaction with DivIVA, and that MinJ in turn recruits MinCD to the cell poles. MinC, however, is located primarily at active division sites at mid-cell when expressed from its native promoter. Surprisingly, we found that Bacillus subtilis MinD is located at nascent septal membranes and at an asymmetric site on lateral membranes between nascent septal membranes in filamentous cells lacking MinJ or DivIVA. Bacillus subtilis MinD has two amphipathic α-helices rich in basic amino acid residues at its C-terminus; one of these, named MTS1 here, is the counterpart of the membrane targeting sequence (MTS) in Escherichia coli MinD while the other, named MTS-like sequence (MTSL), is the nearest helix to MTS1. These amphipathic helices were located independently at nascent septal membranes in cells lacking MinJ or DivIVA, whereas elimination of the helices from the wild type protein reduced its localization considerably. MinD variants with altered MTS1 and MTSL, in which basic amino acid residues were replaced with proline or acidic residues, were not located at nascent septal membranes, indicating that the binding to the nascent septal membranes requires basic residues and a helical structure. The septal localization of MTSL, but not of MTS1, was dependent on host cell MinD. These results suggest that MinD is targeted to nascent septal membranes via its C-terminal amphipathic α-helices in B. subtilis cells lacking MinJ or DivIVA. Moreover, the diffuse distribution of MinD lacking both MTSs suggests that only a small fraction of MinD depends on MinJ for its localization to nascent septal membranes.

Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR.

Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ΔspeD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.

[Metabolism and cell cycle, two interconnected processes in bacteria]. / Métabolisme et cycle cellulaire, deux processus interconnectés chez les bactéries.

To increase chances for a cell to survive in its natural environment, metabolism and cell cycle necessarily have to be interconnected. Indeed, cells need, on the one hand, to check their metabolic status before initiating a cell cycle step often energy-consuming, and, on the other hand, to complete some cell cycle steps before modifying their metabolism. Because bacteria do not differ from the rules, an increasing number of examples of connection between metabolism and cell cycle emerged these last years. Identifying metabolic enzymes as messengers coordinating metabolism and cell cycle allows the addition of another dimension to metabolic maps. Likewise, the very high conservation of these metabolic maps, from bacteria to human, allows inspiring research on tumor cells that are known to have an unrestrained cell cycle and a voracious appetite.

Enhanced poly(γ-glutamic acid) production by H2 O2 -induced reactive oxygen species in the fermentation of Bacillus subtilis NX-2.

Effects of reactive oxygen species (ROS) on cell growth and poly(γ-glutamic acid) (γ-PGA) synthesis were studied by adding hydrogen peroxide to a medium of Bacillus subtilis NX-2. After optimizing the addition concentration and time of H2 O2 , a maximum concentration of 33.9 g/L γ-PGA was obtained by adding 100 µM H2 O2 to the medium after 24 H. This concentration was 20.6% higher than that of the control. The addition of diphenyleneiodonium chloride (ROS inhibitor) can interdict the effect of H2 O2 -induced ROS. Transcriptional levels of the cofactors and relevant genes were also determined under ROS stress to illustrate the possible metabolic mechanism contributing to the improve γ-PGA production. The transcriptional levels of genes belonging to the tricarboxylic acid cycle and electron transfer chain system were significantly increased by ROS, which decreased the NADH/NAD+ ratio and increased the ATP levels, thereby providing more reducing power and energy for γ-PGA biosynthesis. The enhanced γ-PGA synthetic genes also directly promoted the formation of γ-PGA. This study was the first to use the ROS control strategy for γ-PGA fermentation and provided valuable information on the possible mechanism by which ROS regulated γ-PGA biosynthesis in B. subtilis NX-2.

Honey glycoproteins containing antimicrobial peptides, Jelleins of the Major Royal Jelly Protein 1, are responsible for the cell wall lytic and bactericidal activities of honey.

We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A-affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log10 reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey's active principle responsible for this activity.

Silk macromolecules with amino acid-poly(ethylene glycol) grafts for controlling layer-by-layer encapsulation and aggregation of recombinant bacterial cells.

This study introduces double-brush designs of functionalized silk polyelectrolytes based upon regenerated silk fibroin (SF), which is modified with poly-L-lysine (SF-PLL), poly-L-glutamic acid (SF-PGA), and poly(ethylene glycol) (PEG) side chains with different grafting architecture and variable amino acid-PEG graft composition for cell encapsulation. The molecular weight of poly amino acids (length of side chains), molecular weight and degree of PEG grafting (D) were varied in order to assess the formation of cytocompatible and robust layer-by-layer (LbL) shells on two types of bacterial cells (Gram-negative and Gram-positive bacteria). We observed that shells assembled with charged polycationic amino acids adversely effected the properties of microbial cells while promoting the formation of large cell aggregates. In contrast, hydrogen-bonded shells with high PEG grafting density were the most cytocompatible, while promoting formation of stable colloidal suspensions of individual cell encapsulates. The stability to degradation of silk shells (under standard cell incubation procedure) was related to the intrinsic properties of thermodynamic bonding forces, with shells based on electrostatic interactions having stronger resistance to deterioration compared to pure hydrogen-bonded silk shells. By optimizing the charge density of silk polyelectrolytes brushes, as well as the length and the degree of PEG side grafts, robust and cytocompatible cell coatings were engineered that can control aggregation of cells for biosensor devices and other potential biomedical applications.

Adaptor-mediated Lon proteolysis restricts Bacillus subtilis hyperflagellation.

The Lon AAA+ protease is a highly conserved intracellular protease that is considered an anticancer target in eukaryotic cells and a crucial virulence regulator in bacteria. Lon degrades both damaged, misfolded proteins and specific native regulators, but how Lon discriminates among a large pool of candidate targets remains unclear. Here we report that Bacillus subtilis LonA specifically degrades the master regulator of flagellar biosynthesis SwrA governed by the adaptor protein swarming motility inhibitor A (SmiA). SmiA-dependent LonA proteolysis is abrogated upon microbe-substrate contact causing SwrA protein levels to increase and elevate flagellar density above a critical threshold for swarming motility atop solid surfaces. Surface contact-dependent cellular differentiation in bacteria is rapid, and regulated proteolysis may be a general mechanism of transducing surface stimuli.

Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution.

Tea is one of the most widely consumed beverages in the world and known for its antimicrobial activity against many microorganisms. Preliminary studies have shown that tea polyphenols can inhibit the growth of a wide range of Gram-positive bacteria. However, the effect of these compounds on germination and outgrowth of bacterial spores is unclear. Spore-forming bacteria are an aggravating problem for the food industry due to spore formation and their subsequent returning to vegetative state during food storage, thus posing spoilage and food safety challenges. Here we analysed the effect of tea compounds: gallic acid, gallocatechin gallate, Teavigo (>90% epigallocatechin gallate), and theaflavin 3,3'-digallate on spore germination and outgrowth and subsequent growth of vegetative cells of Bacillus subtilis. To quantitatively analyse the effect of these compounds, live cell images were tracked from single phase-bright spores up to microcolony formation and analysed with the automated image analysis tool "SporeTracker". In general, the tested compounds had a significant effect on most stages of germination and outgrowth. However, germination efficiency (ability of spores to become phase-dark) was not affected. Gallic acid most strongly reduced the ability to grow out. Additionally, all compounds, in particular theaflavin 3,3'-digallate, clearly affected the growth of emerging vegetative cells.

A conserved cysteine residue of Bacillus subtilis SpoIIIJ is important for endospore development.

During sporulation in Bacillus subtilis, the onset of activity of the late forespore-specific sigma factor σG coincides with completion of forespore engulfment by the mother cell. At this stage, the forespore becomes a free protoplast, surrounded by the mother cell cytoplasm and separated from it by two membranes that derive from the asymmetric division septum. Continued gene expression in the forespore, isolated from the surrounding medium, relies on the SpoIIIA-SpoIIQ secretion system assembled from proteins synthesised both in the mother cell and in the forespore. The membrane protein insertase SpoIIIJ, of the YidC/Oxa1/Alb3 family, is involved in the assembly of the SpoIIIA-SpoIIQ complex. Here we show that SpoIIIJ exists as a mixture of monomers and dimers stabilised by a disulphide bond. We show that residue Cys134 within transmembrane segment 2 (TM2) of SpoIIIJ is important to stabilise the protein in the dimeric form. Labelling of Cys134 with a Cys-reactive reagent could only be achieved under stringent conditions, suggesting a tight association at least in part through TM2, between monomers in the membrane. Substitution of Cys134 by an Ala results in accumulation of the monomer, and reduces SpoIIIJ function in vivo. Therefore, SpoIIIJ activity in vivo appears to require dimer formation.

Optimization of Bacillus subtilis cell growth effecting jiean-peptide production in fed batch fermentation using central composite design

Background Optimization of nutrient feeding was developed to improve the growth of Bacillus subtilis in fed batch fermentation to increase the production of jiean-peptide (JAA). A central composite design (CCD) was used to obtain a model describing the relationship between glucose, total nitrogen, and the maximum cell dry weight in the culture broth with fed batch fermentation in a 5 L fermentor. Results The results were analyzed using response surface methodology (RSM), and the optimized values of glucose and total nitrogen concentration were 30.70 g/L and 1.68 g/L in the culture, respectively. The highest cell dry weight was improved to 77.50 g/L in fed batch fermentation, which is 280% higher than the batch fermentation concentration (20.37 g/L). This led to a 44% increase of JAA production in fed batch fermentation as compared to the production of batch fermentation. Conclusion The results of this work improve the present production of JAA and may be adopted for other objective products' production.