Effect of a phthalate derivative purified from Bacillus zhangzhouensis SK4 on quorum sensing regulated virulence factors of Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes life-threatening diseases and is resistant to almost all conventional antibiotics. The quorum sensing (QS) system of P. aeruginosa contributes to many pathogenic factors some of which are pigment production, motility, and biofilm. The disruption of quorum sensing system may be an impactful strategy to deal with infections. The present study investigates the anti-quorum sensing property of a bioactive molecule extracted from marine epibiotic bacteria present on the surface of seaweeds. Among all the isolates tested against monitor strain Chromobacterium violaceum (MTCC 2656), the one with the highest activity was identified as Bacillus zhangzhouensis SK4. The culture supernatant was extracted with chloroform which was then partially purified by TLC and column chromatography. The probable anti-QS compound was identified as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl ester) by GC-MS and NMR analysis. The treatment of P. aeruginosa MCC 3457 with the lead compound resulted in the reduced production of pyocyanin, rhamnolipids, exopolysaccharide, biofilm, and motility. The observations of light and scanning electron microscopy also supported the biofilm inhibition. The lead compound showed synergism with the meropenem antibiotic and significantly reduced MIC. The molecular docking and pharmacokinetics study predicted 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl ester), a phthalate derivative as a good drug candidate. The molecular dynamics study was also performed to check the stability of the lead compound and LasR complex. Further, lead compounds did not exhibit any cytotoxicity when tested on human embryonic kidney cells. As per our knowledge, this is the first report on the anti-QS activity of B. zhangzhouensis SK4, indicating that epibiotic bacteria can be a possible source of novel compounds to deal with the multidrug resistance phenomenon.

Effect of Different Selenium Species on Indole-3-Acetic Acid Activity of Selenium Nanoparticles Producing Strain Bacillus altitudinis LH18.

Acting as a growth regulator, Indole-3-acetic acid (IAA) is an important phytohormone that can be produced by several Bacillus species. However, few studies have been published on the comprehensive evaluation of the strains for practical applications and the effects of selenium species on their IAA-producing ability. The present study showed the selenite reduction strain Bacillus altitudinis LH18, which is capable of producing selenium nanoparticles (SeNPs) at a high yield in a cost-effective manner. Bio-SeNPs were systematically characterized by using DLS, zeta potential, SEM, and FTIR. The results showed that these bio-SeNPs were small in particle size, homogeneously dispersed, and highly stable. Significantly, the IAA-producing ability of strain was differently affected under different selenium species. The addition of SeNPs and sodium selenite resulted in IAA contents of 221.7 µg/mL and 91.01 µg/mL, respectively, which were 3.23 and 1.33 times higher than that of the control. This study is the first to examine the influence of various selenium species on the IAA-producing capacity of Bacillus spp., providing a theoretical foundation for the enhancement of the IAA-production potential of microorganisms.

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes.

Bacillus flexus strain SSAI1 isolated from agro-industry waste, Tuem, Goa, India displayed high arsenite resistance as minimal inhibitory concentration was 25 mM in mineral salts medium. This bacterial strain exposed to 10 mM arsenite demonstrated rapid arsenite oxidation and internalization of 7 mM arsenate within 24 h. The Fourier transformed infrared (FTIR) spectroscopy of cells exposed to arsenite revealed important functional groups on the cell surface interacting with arsenite. Furthermore, scanning electron microscopy combined with electron dispersive X-ray spectroscopy (SEM-EDAX) of cells exposed to arsenite revealed clumping of cells with no surface adsorption of arsenite. Transmission electron microscopy coupled with electron dispersive X-ray spectroscopic (TEM-EDAX) analysis of arsenite exposed cells clearly demonstrated ultra-structural changes and intracellular accumulation of arsenic. Whole-genome sequence analysis of this bacterial strain interestingly revealed the presence of large number of metal(loid) resistance genes, including aioAB genes encoding arsenite oxidase responsible for the oxidation of highly toxic arsenite to less toxic arsenate. Enzyme assay further confirmed that arsenite oxidase is a periplasmic enzyme. The genome of strain SSAI1 also carried glpF, aioS and aioE genes conferring resistance to arsenite. Therefore, multi-metal(loid) resistant arsenite oxidizing Bacillus flexus strain SSAI1 has potential to bioremediate arsenite contaminated environmental sites and is the first report of its kind.

Synthesis, antimicrobial, anti-cancer and in silico studies of new urea derivatives.

The reaction of an alkyl or aryl isocyanates with some primary amines in acetonitrile at room temperature afforded the corresponding alkyl- and aryl-urea derivatives. All the prepared urea compounds have been elucidated by FTIR, NMR, and elemental analysis. The compounds 1 and 3 were confirmed by single-crystal X-ray diffraction. The 4-tolylsulfonyl isocyanate reacted with the aryl amines 1, 2, 3, and 2,4-dichloroaniline to afford the corresponding sulfonylurea derivatives 5-8. Likewise, the reaction of the isocyanates with 2,4-dichloroaniline, 5-methyl isoxazole-3-amine, and 2-aminothiazole derivatives gave the corresponding urea derivatives 9-17. All the prepared compounds 5-17 were tested in vitro as anti-microbial and anti-HepG2 agents. Moreover, analyzing gene expression of TP53-exon4 and TP53-exon7, DNA damage values, and DNA fragmentation percentages have been discussed. The compounds 5 and 8 recorded the highest activity against the tested microbial strains with maximum activity against C. albicans (50 mm) and B. mycoides (40 mm), respectively. The compounds 5 inhibited the growth of E. coli, S. aureus, and C. Albicans at the MIC level of 0.0489 µM, while the compound 8 was able to inhibit the visible growth of E. coli and C. albicans at MIC value of 3.13 µM and S. aureus at 0.3912 µM. In the same line, compound 5 showed the best cytotoxic activity against the HepG2 cell line (IC50 = 4.25 µM) compared to 5 fluorouracil with IC50 = 316.25 µM. Expression analysis of liver cancer related to a gene including TP53-exon4 and TP53-exon7 was used in HepG2 Liver cancer cell lines using RT-qPCR. The expression values of TP53-exon4 and TP53-exon7 genes were decreased. The DNA damage values and DNA fragmentation percentages were increased significantly (P < 0.01) in the treated HepG2 (5) sample compared with the negative control. Docking studies were performed for the synthetic compounds against 2 bacterial proteins (DNA gyrase subunit B, and penicillin binding protein 1a) that are known targets for some antibiotics, and one cell division protein kinase 2 (CDK2) as target for anticancer drugs.

Lead metal biosorption and isotherms studies by metal-resistant Bacillus strain MRS-2 bacterium.

In this study, lead (Pb) biosorption studies in aqueous solution were performed with metal-resistant Bacillus strain MRS-2 (ATCC 55674) bacterium which was previously isolated from wastewater plant. It showed minimum inhibition concentration of 300 ppm Pb on the nutrient agar plates. Pb biosorption using MRS-2 bacteria was investigated under different parameters such as pH, temperature, biomass dosage, initial Pb concentration, contact time, and type of biomass by batch experiments. Pb concentration was analyzed through Inductively coupled plasma-optical emission spectrometry. The rate of biosorption (Q) and Pb biosorption capacity (qe ) were calculated for above mentioned parameters. It was observed that Pb precipitates by itself from the solution at pH 2 and 8 or above without bacteria and precipitation did not increase even in the presence of bacteria. The results showed that the highest biosorption rate and biosorption capacity (mg/g) were observed at pH 7, 25°C, 2-h contact time with live bacteria. The highest biosorption rate was observed at 1.5 g/L biomass dose and 5 ppm initial Pb concentration, whereas the highest Pb biosorption capacity was observed at 0.25 g/L biomass dose and 12.5 ppm initial Pb concentration. It was observed that Pb biosorption by live bacteria occurred through adsorption on cell surface. In this study, the biosorption isotherm analysis favored the Langmuir isotherm model indicating monolayer biosorption. This Bacillus strain showed higher Pb biosorption capacity than most of the previously reported Bacillus strains. In conclusion, this study indicates that the Bacillus MRS-2 strain can be used to remove Pb from industrial wastewaters in an ecofriendly approach.

Theoretical Evaluation of Novel Thermolysin Inhibitors from Bacillus thermoproteolyticus. Possible Antibacterial Agents.

The search for new antibacterial agents that could decrease bacterial resistance is a subject in continuous development. Gram-negative and Gram-positive bacteria possess a group of metalloproteins belonging to the MEROPS peptidase (M4) family, which is the main virulence factor of these bacteria. In this work, we used the previous results of a computational biochemistry protocol of a series of ligands designed in silico using thermolysin as a model for the search of antihypertensive agents. Here, thermolysin from Bacillus thermoproteolyticus, a metalloprotein of the M4 family, was used to determine the most promising candidate as an antibacterial agent. Our results from docking, molecular dynamics simulation, molecular mechanics Poisson-Boltzmann (MM-PBSA) method, ligand efficiency, and ADME-Tox properties (Absorption, Distribution, Metabolism, Excretion, and Toxicity) indicate that the designed ligands were adequately oriented in the thermolysin active site. The Lig783, Lig2177, and Lig3444 compounds showed the best dynamic behavior; however, from the ADME-Tox calculated properties, Lig783 was selected as the unique antibacterial agent candidate amongst the designed ligands.

Statistical Based Bioprocess Design for Improved Production of Amylase from Halophilic Bacillus sp. H7 Isolated from Marine Water.

Amylase (EC 3.2.1.1) enzyme has gained tremendous demand in various industries, including wastewater treatment, bioremediation and nano-biotechnology. This compels the availability of enzyme in greater yields that can be achieved by employing potential amylase-producing cultures and statistical optimization. The use of Plackett-Burman design (PBD) that evaluates various medium components and having two-level factorial designs help to determine the factor and its level to increase the yield of product. In the present work, we are reporting the screening of amylase-producing marine bacterial strain identified as Bacillus sp. H7 by 16S rRNA. The use of two-stage statistical optimization, i.e., PBD and response surface methodology (RSM), using central composite design (CCD) further improved the production of amylase. A 1.31-fold increase in amylase production was evident using a 5.0 L laboratory-scale bioreactor. Statistical optimization gives the exact idea of variables that influence the production of enzymes, and hence, the statistical approach offers the best way to optimize the bioprocess. The high catalytic efficiency (kcat/Km) of amylase from Bacillus sp. H7 on soluble starch was estimated to be 13.73 mL/s/mg.

Silicea terra and Zincum metallicum Modulate the Activity of Macrophages Challenged with BCG In Vitro.

BACKGROUND: The homeopathic medicines Silicea terra (Sil) and Zincum metallicum (Zinc) modulate macrophage activity and were assessed in an experimental study in-vitro for their effects on macrophage-BCG (Bacillus Calmette-Guérin) interaction. METHODS: RAW 264.7 macrophages were infected with BCG, treated with different potencies of Sil and Zinc (6cH, 30cH and 200cH) or vehicle, and assessed 24 and 48 h later for bacilli internalization, hydrogen peroxide (H2O2) and cytokine production, and lysosomal activity. RESULTS: Treatment with vehicle was associated with non-specific inhibition of H2O2 production to the levels exhibited by uninfected macrophages. Sil 200cH induced significant reduction of H2O2 production (p < 0.001) compared with the vehicle and all other treatments, as well as higher lysosomal activity (p ≤ 0.001) and increased IL-10 production (p ≤ 0.05). Such effects were considered specific for this remedy and potency. The number of internalized bacilli was inversely proportional to Zinc potencies, with statistically significant interaction between dilution and treatment (p = 0.003). Such linear-like behavior was not observed for Sil dilutions: peak internalization occurred with the 30cH dilution, accompanied by cellular degeneration, and IL-6 and IL-10 increased (p ≤ 0.05) only in the cells treated with Sil 6cH. CONCLUSION: Sil and Zinc presented different patterns of potency-dependent effect on macrophage activity. Bacterial digestion and a balanced IL-6/IL-10 production were related to Sil 6cH, though reduced oxidative stress with increased lysosomal activity was related to Sil 200cH. Degenerative effects were exclusively related to Sil 30cH, and potency-dependent phagocytosis was related only to Zinc.

Evidences for antioxidant response and biosorption potential of Bacillus simplex strain 115 against lead.

In this study, we investigated effects of lead on growth response and antioxidant defense protection in a new identified strain isolated from a soil, in the rhizosphere of Sainfoin Hedysarum coronarium L. Different concentrations of lead (0, 0.2, 1.5 and 3 g L-1) added to Bacillus simplex strain 115 cultures surprisingly did not inhibit its growth. However, a resulting oxidative stress as attested by overproduction of H2O2 (+ 6.2 fold) and malondialdehyde (+ 2.3 fold) concomitantly to the enhancement of proteins carbonylation (+ 221%) and lipoxygenase activity (+ 59%) was observed in presence of 3 g L-1 of lead. Intrinsic antioxidant defenses were revealed by the coupled up-regulation of catalase (+ 416%) and superoxide dismutase (+ 4 fold) activities, with a more important Fe-SOD increase in comparison to the other isoforms. Bioaccumulation assays showed both intracellular and extracellular lead accumulation. Biosorption was confirmed as a particularly lead resistance mechanism for Bacillus simplex strain 115 as the metal sequestration in cell wall accounted for 88.5% to 98.5% of the total endogenous metal accumulation. Potentiality of this new isolated microorganism as a biotechnological tool for agricultural soil lead bioremediation was thus proposed.

Antibacterial and antioxidant metabolites from the insect-associated fungus Aspergillus fumigatus.

The research on bioactive secondary metabolites from Aspergillus fumigatus afforded six compounds, which were identified by mass spectrometer (MS) and nuclear magnetic resonance (NMR) spectroscopic analysis as cyclopyazonic acid (1), trypacidin A (2), asterric acid (3), methyl asterrate (4), demethylcitreoviranol (5), as well as (5-hydroxy-2-oxo-2H-pyran-4-yl) methyl acetate (6). Cyclopyazonic acid (1) was found to have potent antibacterial effects, especially against Bacillus licheniformis with minimal inhibitory concentration (MIC) value of 3.7µg/mL. Its antibacterial effects were possibly related to the olefinic acid group in the structure. Phenyl ether derivatives 3 and 4, and trypacidin A (2) also exhibited antimicrobial effects. In addition, compound 6 showed significant antioxidant effects with half maximal effective concentration (EC50) value of 10.2µM in the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assay, which was better than the positive control.

Antimicrobial peptide CAMA-syn expressed in pulmonary epithelium by recombination adenovirus inhibited the growth of intracellular bacteria.

BACKGROUND: Intracellular bacteria, especially Mycobacterium tuberculosis, are important pathogenic microorganisms that endanger human health. Purified and synthesized cecropin A-magainin 2 (CAMA-syn) can exhibit a higher antibacterial activity and lower cytotoxicity. To enhance such antimicrobial potential, it would be desirable to deliver CAMA-syn expressed in lung epithelial cells by an adenovirus vector using gene therapy. METHODS: A549 cells in vitro and lung epithelial cells in vivo were used to express CAMA-syn by transducing recombinant adenovirus Ad-SPC-CAMA/GFP, and the expression of CAMA-syn was determined by a reverse transcriptase-polymerase reaction (RT-PCR) and immunofluorescence. The antimicrobial activity in cells was investigated by colony-forming rate and growth curve. Forty Kunming mice of a Bacillus Calmette-Guerin (BCG) infection animal model were randomly divided into three groups: adenoviruses delivery of Ad-SPC-CAMA/GFP, Ad-CMV-CAMA/GFP and empty-virus Ad-CMV-GFP. The expression of CAMA-syn in mice was confirmed by RT-PCR and immunofluorescence. After tracheal injection of adenoviral vector for 3 days, lungs from the mouse model were extracted and homogenized for detection of colony-forming efficiency. RESULTS: CAMA-syn expressed in lung epithelial cells A549 conferred antimicrobial activity against a series of bacteria, including Salmonella abortusovis and BCG. The results obtained in vivo showed that the colony-forming rate of Ad-SPC-CAMA/GFP (74.54%) and Ad-CMV-CAMA/GFP (62.31%) transduced into mice was significantly lower than that of the control group. CONCLUSIONS: Lung epithelial-specific expression of antimicrobial peptide CAMA-syn mediated by adenovirus suppressed the growth of intracellular bacteria, providing a promising approach for the control of refractory intracellular infection.

An operon consisting of a P-type ATPase gene and a transcriptional regulator gene responsible for cadmium resistances in Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25.

BACKGROUND: Cadmium (Cd) is a severely toxic heavy metal to most microorganisms. Many bacteria have developed Cd2+ resistance. RESULTS: In this study, we isolated two different Cd2+ resistance Bacillus sp. strains, Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25, which could be grown in the presence of Cd2+ at concentration up to 0.3 mM and 0.8 mM, respectively. According to the genomic sequencing, transcriptome analysis under cadmium stress, and other related experiments, a gene cluster in plasmid p25 was found to be a major contributor to Cd2+ resistance in B. marisflavi 151-25. The cluster in p25 contained orf4802 and orf4803 which encodes an ATPase transporter and a transcriptional regulator protein, respectively. Although 151-6 has much lower Cd2+ resistance than 151-25, they contained similar gene cluster, but in different locations. A gene cluster on the chromosome containing orf4111, orf4112 and orf4113, which encodes an ATPase transporter, a cadmium efflux system accessory protein and a cadmium resistance protein, respectively, was found to play a major role on the Cd2+ resistance for B. vietamensis 151-6. CONCLUSIONS: This work described cadmium resistance mechanisms in newly isolated Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25. Based on homologies to the cad system (CadA-CadC) in Staphylococcus aureus and analysis of transcriptome under Cd2+ induction, we inferred that the mechanisms of cadmium resistance in B. marisflavi 151-25 was as same as the cad system in S. aureus. Although Bacillus vietamensis 151-6 also had the similar gene cluster to B. marisflavi 151-25 and S. aureus, its transcriptional regulatory mechanism of cadmium resistance was not same. This study explored the cadmium resistance mechanism for B. vietamensis 151-6 and B. marisflavi 151-25 and has expanded our understanding of the biological effects of cadmium.

Effects of process conditions and yeast extract on the synthesis of selenium nanoparticles by a novel indigenous isolate Bacillus sp. EKT1 and characterization of nanoparticles.

Selenium nanoparticles (SeNPs) are attractive nanomaterials for application in medical diagnosis, because their toxicities are lower than the elemental selenium which is a functional element and essential for human. In the current study, SeNPs synthesis capability of a novel soil originated indigenous Bacillus isolate was investigated. In this context, effects of processing conditions (SeO2 concentration, pH, temperature, and time), and yeast extract supplementation on the synthesis of SeNPs have been tested. In addition, nanoparticles were characterized and antioxidant capacity was determined. The cell-free supernatant of the bacterium, which was obtained after the cultivation of the isolate in nutrient broth at 33 °C for 24 h, was used for the synthesis. During the synthesis color change from light yellow to red-orange was an indication of the formation of SeNPs. Effect of SeO2 concentration was tested on the formation of nanoparticles and at concentrations higher than 10 mM, there was no formation of nanoparticles. The best production was achieved at 6.4 mM concentration, at pH 9 and 33 °C in 72 h. Field emission scanning electron microscopy (FESEM) images revealed that SeNPs were spherical in shape having the diameters between 31 and 335 nm, and the average diameter was determined to be 126 nm. Energy dispersive X-ray spectroscopy analysis confirmed the presence of elemental selenium. SeNPs possessed significant antioxidant activity that the scavenging capacity was up to 56.5 ± 5% (IC50 322.8 µg/mL).

Determination of bioremediation properties of soil-borne Bacillus sp. 5O5Y11 and its effect on the development of Zea mays in the presence of copper.

Today, industrial activities lead to the accumulation of heavy metals in the soil, water, and air due to mine deposits and operations, fertilizers, and drugs used in agriculture, and urban wastes. Using microorganism bioremediation of metals is an important technique in solving these problems. Herein, a rhizoid bacterium isolated from orchids that grow in Ovit plateau was defined as Bacillus sp. 5O5Y11 by conventional and molecular methods and the bioremediation properties of strain were investigated. It was capable of growth at high salt (10-15%) concentration, wide temperature (10-45 °C) and pH range (pH 4.5-8.0), and was observed to have strong lecithinase, gelatinase activity, and nitrate reduction. When the plant growth-promoting properties of this strain were examined, strong siderophore and ammonium production were observed in in vitro conditions. Bacillus sp. 5O5Y11 was found to have high tolerance to a group of heavy metals [iron (Fe), copper (Cu), lead (Pb), silver (Ag), zinc (Zn)]. Minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) values of copper metal on Bacillus sp. 5O5Y11 were determined as 12.5 mM and 50 mM, respectively. The effectiveness of this bacterium on the germination and growth of maize plant in the presence and absence of copper were investigated. These results suggest that Bacillus sp. 5O5Y11 is a microorganism, which has potential in metal bioremediation and plant growth promotion.

Marine bacteria associated with shallow hydrothermal systems in the Gulf of California with the capacity to produce biofilm inhibiting compounds.

Shallow hydrothermal systems are extreme environments. The sediments and fluids emitted from the vents present unusual physical and chemical conditions compared to other marine areas, which promotes unique biodiversity that has been of great interest for biotechnology for some years. In this work, a bioprospective study was carried out to evaluate the capacity of bacteria associated with shallow hydrothermal vents to produce biofilm-inhibiting compounds. Degradation assays of N-acyl homoserine lactone (AHL) autoinducers (C6HSL) involved in the quorum sensing process were carried out on 161 strains of bacteria isolated from three shallow hydrothermal systems located in Baja California Sur (BCS), Mexico. The biosensor Chromobacterium violaceum CV026 was used. Twenty-three strains showed activity, and organic extracts were obtained with ethyl acetate. The potential of the extracts to inhibit the formation of biofilms was tested against two human pathogenic strains (Pseudomonas aeruginosa PAO1 and Aeromonas caviae ScH3), a shrimp pathogen (Vibrio parahaemolyticus M8), and two marine strains identified as producing biofilms on submerged surfaces (Virgibacillus sp C29 and Vibrio alginolyticus C96). The results showed that Vibrio alginolyticus and Brevibacillus thermoruber, as well as some thermotolerant strains (mostly Bacillus), produce compounds that inhibit bacterial biofilms (B. licheniformis, B. paralicheniformis, B. firmus, B. oceanizedimenis, B. aerius and B. sonorensis).

Inactivation of bacterial endospores on surfaces by plasma processed air.

AIMS: In case of biological hazards and pandemics, personal protective equipment of rescue forces is currently manually decontaminated with harmful disinfectants, primarily peracetic acid. To overcome current drawbacks regarding supply, handling and disposal of chemicals, the use of plasma processed air (PPA) represents a promising alternative for surface decontamination on site. In this study, the sporicidal efficiency of a portable plasma system, designed for field applications, was evaluated. METHODS AND RESULTS: The developed plasma device is based on a dielectric barrier discharge (DBD) and operated with ambient air as process gas. PPA from the plasma nozzle was flushed into a treatment chamber (volume: 300 l) and bacterial endospores (Bacillus subtilis and Bacillus atrophaeus) dried on different surfaces were treated under variable conditions. Reductions in spores by more than 4 log10 were found within 3 min of PPA exposure. However, the presence of endospores in agglomerates or in an organic matrix as well as the complexity of the respective surface microstructure negatively affected the inactivation efficiency. When endospores were embedded in a dried protein matrix, mechanical wiping with swabs during exposure to PPA increased the inactivation effect significantly. Gaseous ozone alone did not provide a sporicidal effect. Significant spore inactivation was only obtained when water vapour was injected into the PPA stream. CONCLUSION: The results show that endospores dried on surfaces can be reduced by several orders of magnitude within few minutes in a treatment chamber which is flushed with PPA from of a DBD plasma nozzle. SIGNIFICANCE AND IMPACT OF THE STUDY: Plasma processed air generated on site by DBD plasma nozzles could be a suitable alternative for the disinfection of various surfaces in closed rooms.

Induced root-secreted D-galactose functions as a chemoattractant and enhances the biofilm formation of Bacillus velezensis SQR9 in an McpA-dependent manner.

Chemotaxis towards root exudates and subsequent biofilm formation are very important for root colonization and for providing the beneficial functions of plant growth-promoting rhizobacteria (PGPRs). In this study, in comparison with other root-secreted compounds, D-galactose in the root exudates of cucumber was found to be a strong chemoattractant at the concentration of 1 µM for Bacillus velezensis SQR9. Chemotaxis assays with methyl-accepting chemotaxis proteins (MCPs) deletion strains demonstrated that McpA was solely responsible for chemotaxis towards D-galactose. Interestingly, D-galactose significantly enhanced the biofilm formation of SQR9 in an McpA-dependent manner. Further experiment showed that D-galactose also enhanced root colonization by SQR9. In addition, the secretion of D-galactose by cucumber roots could be induced by inoculation with SQR9, indicating that D-galactose may be an important signal in the interaction between plant and SQR9. These findings suggested that the root-secreted D-galactose was a signal, the secretion of which was induced by the beneficial bacteria, and which in turn induced colonization of the bacteria.

Expression and purification of the 5'-nucleotidase YitU from Bacillus species: its enzymatic properties and possible applications in biotechnology.

5'-Nucleotidases (EC 3.1.3.5) are enzymes that catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides to their corresponding nucleosides plus phosphate. In the present study, to search for new genes encoding 5'-nucleotidases specific for purine nucleotides in industrially important Bacillus species, "shotgun" cloning and the direct selection of recombinant clones grown in purine nucleosides at inhibitory concentrations were performed in the Escherichia coli GS72 strain, which is sensitive to these compounds. As a result, orthologous yitU genes from Bacillus subtilis and Bacillus amyloliquefaciens, whose products belong to the ubiquitous haloacid dehalogenase superfamily (HADSF), were selected and found to have a high sequence similarity of 87%. B. subtilis YitU was produced in E. coli as an N-terminal hexahistidine-tagged protein, purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity with respect to various deoxyribo- and ribonucleoside monophosphates: dAMP, GMP, dGMP, CMP, AMP, XMP, IMP and 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranosyl 5'-monophosphate (AICAR-P). However, the preferred substrate for recombinant YitU was shown to be flavin mononucleotide (FMN). B. subtilis and B. amyloliquefaciens yitU overexpression increased riboflavin (RF) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) accumulation and can be applied to breed highly performing RF- and AICAR-producing strains.

Characterization of a Cu2+, SDS, alcohol and glucose tolerant GH1 ß-glucosidase from Bacillus sp. CGMCC 1.16541.

A ß-glucosidase gene (bsbgl1a) from Bacillus sp. CGMCC 1.16541 was expressed in Escherichia coli BL21 and subsequently characterized. The amino acid sequence shared 83.64% identity with ß-glucosidase (WP_066390903.1) from Fictibacillus phosphorivorans. The recombinant ß-glucosidase (BsBgl1A) had a molecular weight of 52.2 kDa and could hydrolyze cellobiose, cellotriose, cellotetrose, p-nitrophenyl-ß-D-glucopyranoside (pNPG), and p-nitrophenyl-ß-D-xylopyranoside (pNPX). Optimal activity for BsBgl1A was recorded at 45 °C with a pH between 5.6 and 7.6, and 100% of its activity was maintained after a 24 h incubation between pH 4 and 9. Kinetic characterization revealed an enzymatic turnover (Kcat) of 616 ± 2 s-1 (with cellobiose) and 3.5 ± 0.1 s-1 (with p-nitrophenyl-ß-D-glucopyranoside). Interestingly, the recombinant enzyme showed cupric ion (Cu2+), sodium dodecyl sulfate (SDS) and alcohol tolerance at 10 mM for Cu2+ and 10% for both SDS and alcohol. Additionally, BsBgl1A had high tolerance for glucose (Ki = 2095 mM), which is an extremely desirable feature for industrial applications. Following the addition of BsBgl1A (0.05 mg/ml) to a commercial cellulase reaction system, glucose yields from sugarcane bagasse increased 100% after 1 day at 45 °C. This work identifies a Cu2+, SDS, alcohol, and glucose tolerant GH1 ß-glucosidase with potential applications in the hydrolysis of cellulose for the bioenergy industry.

Litoralimycins A and B, New Cytotoxic Thiopeptides from Streptomonospora sp. M2.

Streptomonospora sp. M2 has been isolated from a soil sample collected at the Wadden Sea beach in our ongoing program aimed at the isolation of rare Actinobacteria, ultimately targeting the discovery of new antibiotics. Because crude extracts derived from cultures of this strain showed inhibitory activity against the indicator organism Bacillus subtilis, it was selected for further analysis. HPLC-MS analysis of its culture broth revealed the presence of lipophilic metabolites. The two major metabolites of those were isolated by preparative reversed-phase HPLC and preparative TLC. Their planar structures were elucidated using high-resolution electrospray ionization mass spectrometry (HRESIMS), 1D and 2D NMR data as new thiopeptide antibiotics and named litoralimycin A (1) and B (2). Although rotating frame nuclear Overhauser effect spectroscopy (ROESY) data established a Z configuration of the Δ21,26 double bond, the stereochemistry of C-5 and C-15 were assigned as S by Marfey's method after ozonolysis. The biological activity spectrum of 1 and 2 is highly uncommon for thiopeptide antibiotics, since they showed only insignificant antibacterial activity, but 1 showed strong cytotoxic effects.