Siderophore-Mediated Iron Uptake Promotes Yeast–Bacterial Symbiosis

In the present study, siderophore produced by the marine yeast Aureobasidium pullulans was characterized as hydroxamate by chemical and bioassays. The hydroxamate assignment was supported by the appearance of peaks at 1,647.21?C1,625.99?cm^?1 and at 1,435.04?cm^?1 in the infrared spectrum. The purified siderophore exhibited specific growth-promoting activity under iron-limited conditions for siderophore auxotrophic probiotic bacteria. Cross-utilization of siderophore indicates a symbiotic relationship between the yeast A. pullulans and the selected probiotic bacterial strains. Statistical optimization of medium components for improved siderophore production in A. pullulans was depicted by response surface methodology. The shift in UV?CVis spectroscopy indicates the photoreactive property and subsequent oxidative cleavage of purified siderophore on exposure to sunlight.     

An In Vitro Study of the Effect of Viburnum opulus Extracts on Key Processes in the Development of Staphylococcal Infections

Staphylococcus aureus is still one of the leading causes of both hospital- and community-acquired infections. Due to the very high percentage of drug-resistant strains, the participation of drug-tolerant biofilms in pathological changes, and thus the limited number of effective antibiotics, there is an urgent need to search for alternative methods of prevention or treatment for S. aureus infections. In the present study, biochemically characterized (HPLC/UPLC–QTOF–MS) acetonic, ethanolic, and water extracts from fruits and bark of Viburnum opulus L. were tested in vitro as diet additives that potentially prevent staphylococcal infections. The impacts of V. opulus extracts on sortase A (SrtA) activity (Fluorimetric Assay), staphylococcal protein A (SpA) expression (FITC-labelled specific antibodies), the lipid composition of bacterial cell membranes (LC-MS/MS, GC/MS), and biofilm formation (LIVE/DEAD BacLight) were assessed. The cytotoxicity of V. opulus extracts to the human fibroblast line HFF-1 was also tested (MTT reduction). V. opulus extracts strongly inhibited SrtA activity and SpA expression, caused modifications of S. aureus cell membrane, limited biofilm formation by staphylococci, and were non-cytotoxic. Therefore, they have pro-health potential. Nevertheless, their usefulness as diet supplements that are beneficial for the prevention of staphylococcal infections should be confirmed in animal models in the future.     

Legonoxamines A-B,two new hydroxamate siderophores from the soil bacterium, Streptomyces sp. MA37

Two new siderophores belonging to the hydroxamate class, Legonoxamine A (1) and B (2) have been isolated from the soil bacterium, Streptomyces sp. MA37, together with one known compound, desferrioxamine B (3). Their structures were elucidated based on spectroscopic methods including 1D, 2D NMR, MS, as well as by comparison with the relevant literatures. To our knowledge, this is the first report describing a siderophore containing the N-hydroxyl phenylacetyl cadaverine (HPAC) moiety in the structure. Based on bioinformatics analysis and previous knowledge of the biosynthesis of the hydroxamate-type siderophore, the biosynthetic gene cluster (lgo) responsible for the production of 1–3 was identified in the annotated genome of the producing strain. The supplementation of phenylacetate and benzoate analogues with meta substitution into the cultures of Streptomyces sp. MA37 resulted in the production of new legonoxamine A derivatives as observed in LC-HR-ESIMS, suggesting that the legonoxamine biosynthetic pathway has a good degree of natural flexibility of accepting unnatural precursors with different functional groups.     

Solid phase microextraction/gas chromatography/mass spectrometry integrated with chemometrics for detection of Salmonella typhimurium contamination in a packaged fresh vegetable

A rapid method for detection of Salmonella typhimurium contamination in packaged alfalfa sprouts using solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) integrated with chemometrics was investigated. Alfalfa sprouts were inoculated with S. typhimurium, packed into commercial LDPE bags and stored at 10 + 2 °C for 0, 1, 2 and 3 days. Uninoculated sprouts were used as control samples. A SPME device was used to collect the volatiles from the headspace above the samples and the volatiles were identified using GC/MS. Chemometric techniques including linear discriminant analysis (LDA) and artificial neural network (ANN) were used as data processing tools. Numbers of Salmonella were followed using a colony counting method. From LDA, it was able to differentiate control samples from sprouts contaminated with S. typhimurium. The potential to predict the number of contaminated S. typhimurium from the SPME/GC/MS data was investigated using multilayer perceptron (MLP) neural network with back propagation training. The MLP comprised an input layer, one hidden layer, and an output layer, with a hyperbolic tangent sigmoidal transfer function in the hidden layer and a linear transfer function in the output layer. The MLP neural network with a back propagation algorithm could predict number of S. typhimurium in unknown samples using the volatile fingerprints. Good prediction was found as measured by a regression coefficient (R² = 0.99) between actual and predicted data.     

Siderophore Production by Bacillus megaterium: Effect of Growth Phase and Cultural Conditions

Siderophore production by Bacillus megaterium was detected, in an iron-deficient culture medium, during the exponential growth phase, prior to the sporulation, in the presence of glucose; these results suggested that the onset of siderophore production did not require glucose depletion and was not related with the sporulation. The siderophore production by B. megaterium was affected by the carbon source used. The growth on glycerol promoted the very high siderophore production (1,182 μmol g^?1 dry weight biomass); the opposite effect was observed in the presence of mannose (251 μmol g^?1 dry weight biomass). The growth in the presence of fructose, galactose, glucose, lactose, maltose or sucrose, originated similar concentrations of siderophore (546–842 μmol g^?1 dry weight biomass). Aeration had a positive effect on the production of siderophore. Incubation of B. megaterium under static conditions delayed and reduced the growth and the production of siderophore, compared with the incubation in stirred conditions.     

A spectrophotometric method to determine the siderophore production by strains of fluorescent Pseudomonas in the presence of copper and iron

A simple procedure to determine levels of siderophore production by strains of Pseudomonas, particularly the Avm strain is described. Bacterial cells were incubated for 24 h in iron-rich (RM) and iron-limiting conditions (RM-Fe) with and without 6 and 60 μM of CuSO₄. Cells grown under iron-limiting conditions developed a green color even in the presence of Cu. The spent media supernatants from the Avm cells grown in RM-Fe medium showed a maximum peak of absorbance at 400 nm, which suggest that this strain produced a single type of siderophore. The presence of 60 μM of CuSO₄ in the cultures did not interfere with the detection of siderophores in the spent media. Clear supernatants obtained from cultures of 10 fluorescent Pseudomonas were diluted 1 to 10 in deionized water and the absorption at 400 nm was determined. The results demonstrated the clear discriminating capacity of this highly practical procedure to categorize a great number of fluorescent Pseudomonas strains by the range of siderophore production.     

Enantioselective Hydrolysis of dl-Menthyl Benzoate by Cell-Free Extract of Newly Isolated Acinetobacter sp. ECU2040

Production of l-menthol by bioprocesses attracts increasing attention nowadays. Herein, we attempted to develop a bioresolution process for production of l-menthol through enantioselective hydrolysis of dl-menthyl benzoate using a newly isolated bacterium from soil samples. Among 129 active soil isolates screened rapidly by thin-layer chromatography, an outstanding bacterial strain numbered ECU2040, which was subsequently identified as Acinetobacter species, was finally selected as our target enzyme producer due to its highest activity and the best enantioselectivity toward l-substrate as confirmed by chiral gas chromatography. The catalytic performance of the cell-free extract from Acinetobacter sp. ECU2040 was preliminarily examined, indicating that its optimal pH and temperature for the reaction were 7.5 and 37 °C, respectively. Under the optimal conditions, the enzymatic reaction was performed on a 1-L scale, affording l-menthol in 48 % yield and 71 % ee.     

Siderophore as a Potential Plant Growth-Promoting Agent Produced by Pseudomonas aeruginosa JAS-25

Siderophores scavenges Fe⁺³ from the vicinity of the roots of plants, and thus limit the amount of iron required for the growth of pathogens such as Fusarium oxysporum, Pythium ultimum, and Fusarium udum, which cause wilt and root rot disease in crops. The ability of Pseudomonas to grow and to produce siderophore depends upon the iron content, pH, and temperature. Maximum yield of siderophore of 130 μM was observed at pH 7.0?±?0.2 and temperature of 30 °C at 30 h. The threshold level of iron was 50 μM, which increases up to 150 μM, favoring growth but drastically affecting the production of siderophore by Pseudomonas aeruginosa JAS-25. The seeds of agricultural crops like Cicer arietinum (chick pea), Cajanus cajan (pigeon pea), and Arachis hypogaea (ground nut) were treated with P. aeruginosa JAS-25, which enhanced the seed germination, root length, shoot length, and dry weight of chick pea, pigeon pea, and ground nut plants under pot studies. The efficient growth of the plants was not only due to the biocontrol activity of the siderophore produced by P. aeruginosa JAS-25 but also may be by the production of indole acetic acid (IAA), which influences the growth of the plants as phytohormones.     

Mass spectrometry profiling analysis enables the identification of new modifications in ribosomal RNA

Ribosomal RNAs (rRNAs) provide the structural framework of ribosomes and play critical roles in protein translation. In ribosome biogenesis, rRNAs acquire various modifications that can influence the structure and catalytic activity of ribosomes. However, rRNA modifications in plants have yet to be fully defined. Herein, we proposed a method to purify rRNAs by a successive isolation with different strategies, including polyA-based mRNA depletion and agarose gel electrophoresis-based purification, with which highly pure rRNAs could be obtained. In addition, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to systematically profile and characterize modifications from the isolated highly pure plant 18S rRNA and 25S rRNA. LC-ESI-MS/MS analysis showed that 10 and 12 kinds of modifications were present in plant 18S rRNA and 25S rRNA, respectively. Notably, among these identified modifications, 2 kinds of modifications of N²,N²-dimethylguanosine (m^2,2G) and N⁶,N⁶-dimethyladenosine (m^6,6A) in 18S rRNA, and 4 kinds of modifications of m^2,2G, m^6,6A, N7-methylguanosine (m⁷G) and 3-methyluridin (m³U) in 25S rRNA, were first reported to be present in plants. Moreover, exposure of Arabidopsis thaliana to cadmium (Cd) led to significant changes of modifications in both 18S rRNA and 25S rRNA of plants, indicating that rRNA modifications play important roles in response to environmental stress. The discovery of new modifications in plant rRNAs improves the spectra of plant rRNA modifications and may promote the investigation of the functional roles of plant ribosomes in regulating gene expression.     

Intrinsic Characteristics of Cr6+-Resistant Bacteria Isolated from an Electroplating Industry Polluted Soils for Plant Growth-Promoting Activities

The Cr⁶⁺-resistant plant growth-promoting bacteria was isolated from soil samples that were collected from an electroplating industry at Coimbatore, India, that had tolerated chromium concentrations up to 500?mg Cr⁶⁺/L in Luria-Bertani medium. Based on morphology, physiology, and biochemical characteristics, the strain was identified as Bacillus sp. following the Bergey's manual of determinative bacteriology. Evaluation of plant growth-promoting parameters has revealed the intrinsic ability of the strain for the production of indole-3-acetic acid (IAA), siderophore, and solubilization of insoluble phosphate. Bacillus sp. have utilized tryptophan as a precursor for their growth and produced IAA (122???g/mL). Bacillus sp. also exhibited the production of siderophore that was tested qualitatively using Chrome Azurol S (CAS) assay solution and utilized the insoluble tricalcium phosphate as the sole source of phosphate exhibiting higher rate of phosphate solubilization after 72?h of incubation (1.45???g/mL). Extent of Cr⁶⁺ uptake and accumulation of Cr⁶⁺ in the cell wall of Bacillus sp. was investigated using atomic absorption spectrophotometer and scanning electron microscope-energy dispersive spectroscopy, respectively. The congenital capability of this Cr⁶⁺-resistant plant growth-promoting Bacillus sp. could be employed as bacterial inoculum for the improvement of phytoremediation in heavy metal contaminated soils.     

Determination of carbonyl compounds in the atmosphere by DNPH derivatization and LC-ESI-MS/MS detection

A method of determination of 32 carbonyl compounds by high performance liquid chromatography (HPLC) and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) after derivatization with 2,4-dinitrophenylhydrazine (DNPH) was developed and successfully applied to the atmosphere sample of a residential area of Liwan District (S1) and a research institute of Tianhe District (S2) in Guangzhou, China. Some operation conditions of ESI-MS/MS in the negative ion mode including selection of parent and daughter ions, declustering potential (DP), entrance potential (EP), collision energy (CE), collision cell exit potential (CXP) and effect of buffer in ESI-MS/MS process were optimized. The regression coefficient of the calibration curves (R²), recovery, reproducibility (R.S.D., n = 5) and limit of detection (LOD) were in the range of 0.9938-0.9999, 90-104%, 1.7-11% and 0.4-9.4 ng/m³, respectively. Among most of the samples, acetone was the most abundant carbonyl in two sampling sites and formaldehyde, acetaldehyde and butyraldehyde/2-butanone were also abundant carbonyls. In contrast to LC-UV method, the LOD, the separation of some co-eluting compounds and the precision (mainly to higher molecular weight carbonyls) are all improved by LC-ESI-MS/MS.     

<Emphasis Type="Italic">Bacillus</Emphasis> spp. of Human Origin: A Potential Siderophoregenic Probiotic Bacteria

Bacillus spp. ST13, isolated from human stool, was evaluated for siderophoregenic and probiotic qualities prior to its possible application for iron nutrition in humans and animals. It was tested for siderophore production in iron-limiting conditions and found to produce catecholate type of siderophore on the basis of high-performance liquid chromatography (HPLC), FT-IR, NMR, and mass spectra analysis. The isolate was screened for probiotic properties as per WHO and FAO guidelines. The strain ST13 can survive stomach acidity, bile salt and partially simulated gastrointestinal tract conditions. It was susceptible to most of the antibiotic tested and showed antimicrobial activity against enteric pathogens like Salmonella typhimurium, Streptococcus pyogenes, and Staphylococcus aureus. Strain ST13 showed close similarity with Bacillus subtilis using 16S r-RNA gene sequence analysis and biochemical characterization. The methanolic extract of ST13 siderophore was evaluated for DPPH radical scavenging activity, which showed 94.55 ± 0.9% of radical scavenging effect.     

Purification and Biochemical Characterization of a Novel Thermo-stable Carboxymethyl Cellulase from Azorean Isolate Bacillus mycoides S122C

Bacillus mycoides S122C was identified as carboxymethyl cellulase (CMcellulase)-producing bacteria from the Azorean Bacillus collection (Lab collection), which was isolated from local soil samples. The bacteria was identified by 16S rRNA sequence and designated as B. mycoides S122C. NCBI blast analysis showed that the B. mycoides S122C 16S rRNA sequence has high identity compared to other B. mycoides strains. CMcellulase was purified from the culture filtrates using anion-exchange chromatography. After mono-Q purification, the protein folds and recovery were 13.7 and 0.76?%, respectively. SDS-PAGE analysis showed that the molecular weight of the purified CMcellulase protein was estimated to be about 62?kDa and that it was composed of a single subunit. MALDI-MS/MS analysis yielded each four peptides of the purified protein; it has identity to other cellulases. The purified CMcellulase showed high activity with CMcellulose followed by ??-glucan as a substrate. Optimum temperature and pH for the purified CMcellulase activity were found to be at 50?°C and pH?7.0, respectively. The purified CMcellulase was stable with about 60?% activity in broad pH ranges from 5 to 10 and temperature of 40 to 60?°C. However, purified CMcellulase was stable at about 70?% at 70?°C and also stable overall at 78?% for surfactants. CMcellulase activity was inhibited by ions such as HgCl₂, followed by CuSo₄, FeCl₂, and MnCl₂, while CoCl₂ activated CMcellulase activity. The purified CMcellulase activity was strongly inhibited by EDTA.     

Synthesis,Antibacterial and Pharmacokinetic Evaluation of Novel Derivatives of Harmine N9-Cinnamic Acid

A series of 16 new derivatives of harmine N⁹-Cinnamic acid were synthesized and fully characterized using NMR and MS. The in vitro antibacterial evaluation revealed that most of the synthesized harmine derivatives displayed better antibacterial activities against Gram-positive strains (S. aureus, S. albus and MRSA) than Gram-negative strains (E. coli and PA). In particular, compound 3c showed the strongest bactericidal activity with a minimum inhibitory concentration of 13.67 μg/mL. MTT assay showed that compound 3c displayed weaker cytotoxicity than harmine with IC₅₀ of 340.30, 94.86 and 161.67 μmol/L against WI-38, MCF-7 and HepG2 cell lines, respectively. The pharmacokinetic study revealed that the distribution and elimination of 3c in vivo were rapid in rats with an oral bioavailability of 6.9%.     

Resolution-Enhanced Kendrick Mass Defect Analysis of Polycyclic Aromatic Hydrocarbons and Fullerenes in the Diffusion Flame from a Butane Torch

A modified Kendrick Mass Defect (KMD) analysis was applied to the analysis of polycyclic aromatic hydrocarbons (PAHs) and fullerenes in the diffusion flame from a handheld butane torch.

Open image in new window

Graphical Abstract ?

     

Spectrometric analysis,phenolics isolation and cytotoxic activity of <Emphasis Type="Italic">Stipagrostis plumosa</Emphasis> (Family Poaceae)

Grasses (family Poaceae) are economically important plants; they are used as crops and animal foods. Stipagrostis plumosa (L.) Munro ex T. Anderson is a member of this family and subjected to chemical and biological studies. The chromatographic techniques,  LC–ESI–MS and GC/MS were used for identification of polar and non-polar compounds in its extract. Ten compounds, including one new flavone glycoside; tricin 7-O-galactoside, three known flavones, three C-glycosyl flavones and three phenolic acids, were isolated from S. plumosa for the first time except tricin. Their structures were elucidated on the basis of extensive spectroscopic interpretation. In addition to the isolated compounds, eleven compounds were tentatively identified using LC–ESI–MS, five of them were detected for the first time from this species. 29 non polar compounds were identified using GC–MS analysis, representing 83.13% of S. plumosa diethyl ether extract. In addition to the DPPH activity evaluation, the crude extract and the isolated compounds were investigated against five human carcinoma cell lines; A549, HCT-116, HepG2, MCF-7 and PC3 at a concentration of 100 μg/ml. From the isolated compounds tricin and luteolin 6,8-di-C-glucoside could be considered as natural-free radical scavenging agents.     

Isolation and structure determination of a new lasso peptide subterisin from Sphingomonas subterranea

A new lasso peptide named subterisin was isolated from the culture broth of Sphingomonas subterranea NBRC 16086^T. The molecular formula of subterisin was established as C₇₈H₁₂₁O₂₂N₂₁ based on accurate mass analysis. The chemical structure of subterisin was determined by 2D NMR experiments. The presence of macrolactam ring of Gly1–Glu8 was indicated by NOESY experiment and MS/MS analysis. The three-dimensional structure of subterisin in solution was established by calculation based on NMR data. The proposed biosynthetic gene cluster of subterisin was found on the genome of S. subterranea.     

Galbofloxacin: a xenometal-antibiotic with potent in vitro and in vivo efficacy against S. aureus

Siderophore-antibiotic drug conjugates are considered potent tools to deliver and potentiate the antibacterial activity of antibiotics, but only few have seen preclinical and clinical success. Here, we introduce the gallium(iii) complex of a ciprofloxacin-functionalized linear desferrichrome, Galbofloxacin, with a cleavable serine linker as a potent therapeutic for S. aureus bacterial infections. We employed characterization using in vitro inhibitory assays, radiochemical, tracer-based uptake and pharmacokinetic assessment of our lead compound, culminating in in vivo efficacy studies in a soft tissue model of infection. Galbofloxacin exhibits a minimum inhibitory concentration of (MIC₉₈) 93 nM in wt S. aureus, exceeding the potency of the parent antibiotic ciprofloxacin (0.9 μM). Galbofloxacin is a protease substrate that can release the antibiotic payload in the bacterial cytoplasm. Radiochemical experiments with wt bacterial strains reveal that ⁶⁷Galbofloxacin is taken up efficiently using siderophore mediated, active uptake. Biodistribution of ⁶⁷Galbofloxacin in a mouse model of intramuscular S. aureus infection revealed renal clearance and enhanced uptake in infected muscle when compared to ⁶⁷Ga-citrate, which showed no selectivity. A subsequent in vivo drug therapy study reveals efficient reduction in S. aureus infection burden and sustained survival with Galbofloxacin for 7 days. Ciprofloxacin had no treatment efficacy at identical molecular dose (9.3 μmol kg⁻¹) and resulted in death of all study animals in <24 hours. Taken together, the favorable bacterial growth inhibitory, pharmacokinetic and in vivo efficacy properties qualify Galbofloxacin as the first rationally designed Ga-coordination complex for the management of S. aureus bacterial infections.

Galbofloxacin, a novel theranostic xenosiderophore antibiotic, exhibits unparalleled potency in combating S. aureus infections in vivo.     

Salvia officinalis L.: Antitrypanosomal Activity and Active Constituents against Trypanosoma brucei rhodesiense

As part of our studies on antiprotozoal activity of approved herbal medicinal products, we previously found that a commercial tincture from Salvia officinalis L. (common Sage, Lamiaceae) possesses high activity against Trypanosoma brucei rhodesiense (Tbr), causative agent of East African Human Trypanosomiasis. We have now investigated in detail the antitrypanosomal constituents of this preparation. A variety of fractions were tested for antitrypanosomal activity and analyzed by UHPLC/+ESI QqTOF MS. The resulting data were used to generate a partial least squares (PLS) regression model that highlighted eight particular constituents that were likely to account for the major part of the bioactivity. These compounds were then purified and identified and their activity against the pathogen tested. All identified compounds (one flavonoid and eight diterpenes) displayed significant activity against Tbr, in some cases higher than that of the total tincture. From the overall results, it can be concluded that the antitrypanosomal activity of S. officinalis L. is, for the major part, caused by abietane-type diterpenes of the rosmanol/rosmaquinone group.     

Leaves of Chenopodium album as source of natural fungicides against Sclertium rolfsii

Series of experiments were conducted to identify the possible antifungal components of Chenopodium album leaves for the management of a highly destructive soil-borne fungal pathogen Sclerotium rolfsii. A 4% methanolic extract caused up to 82% reduction in biomass of the target organism. This extract was partitioned using solvents of variable polarities, and the obtained subfractions were evaluated for their activity against S. rolfsii. The best antifungal activity was detected for the ethyl acetate subfraction (60–74%) followed by n-hexane (51–69%), n-butanol (50–60%), chloroform (20–40%) while the aqueous sub-fraction had the lowest activity (9–35%) as detected by the decrease in biomass of the pathogen. Ethyl acetate and n-hexane sub-fractions were analyzed for their chemical constituents by GC–MS technique. Literature survey showed that among the identified compounds kitazin P, imidazole-4-carboxylic acid, 2-fluoro-1-methoxymethyl-, ethyl ester and 9,12,15-octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)- had antifungal activities against other fungal species and could be responsible for control of S. rolfsii by methanolic extract in the present study.