Comparison of Growth Performance, Pigment Synthesis, and Esterase Activity of Synechococcus sp. HS01 and Limnothrix sp. KO01 in Response to Cadmium Toxicity.

Various studies have been conducted to understand the impact of environmental pollutants on cyanobacteria due to their abundant presence in aquatic and terrestrial environments, specific morphological and physiological characteristics, and high ecological flexibility in response to environmental changes. Here, the effect of different concentrations of cadmium on two native strains of cyanobacteria, namely Synechococcus sp. HS01 and Limnothrix sp. KO01 was studied and compared with each other. In this regard, the cyanobacterial growth, pigment contents, and esterase enzyme activity were evaluated after exposure of the cells to different concentrations of cadmium (II). The toxic effects of Cd(II) on the growth rate of Limnothrix sp. KO01, even at low concentrations, tended to be higher than those for Synechococcus sp. HS01. The content of pigments decreased by an increase in Cd(II) concentration. In compliance with the cell growth, the changes occurred in pigment contents of Limnothrix sp. KO01 was more sensitive than Synechococcus sp. HS01 in the presence of different concentrations of cadmium. Flow cytometry analysis of Cd(II) effects on esterase activity of both strains after 6, 24, 48, and 72 h of exposure to Cd(II) concentrations of 9, 27, 63, and 90 µM showed that tolerance to Cd(II) toxicity in Limnothrix sp. KO01 is less than Synechococcus sp. HS01. The results obtained in this study suggest high potentials of Synechococcus sp. HS01 for heavy metal bioaccumulation due to its considerable tolerance to cadmium.

Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation.

A longstanding awareness in generating resistance to common antimicrobial therapies by Gram-negative bacteria has made them a major threat to global health. The application of antimicrobial peptides as a therapeutic agent would be a great opportunity to combat bacterial diseases. Here, we introduce a new antimicrobial peptide (∼8.3 kDa) from probiotic strain Lactobacillus acidophilus ATCC 4356, designated acidocin 4356 (ACD). This multifunctional peptide exerts its anti-infective ability against Pseudomonas aeruginosa through an inhibitory action on virulence factors, bacterial killing, and biofilm degradation. Reliable performance over tough physiological conditions and low hemolytic activity confirmed a new hope for the therapeutic setting. Antibacterial kinetic studies using flow cytometry technique showed that the ACD activity is related to the change in permeability of the membrane. The results obtained from molecular dynamic (MD) simulation were perfectly suited to the experimental data of ACD behavior. The structure-function relationship of this natural compound, along with the results of transmission electron microscopy analysis and MD simulation, confirmed the ability of the ACD aimed at enhancing bacterial membrane perturbation. The peptide was effective in the treatment of P. aeruginosa infection in mouse model. The results support the therapeutic potential of ACD for the treatment of Pseudomonas infections.IMPORTANCE Multidrug-resistant bacteria are a major threat to global health, and the Pseudomonas bacterium with the ability to form biofilms is considered one of the main causative agents of nosocomial infections. Traditional antibiotics have failed because of increased resistance. Thus, finding new biocompatible antibacterial drugs is essential. Antimicrobial peptides are produced by various organisms as a natural defense mechanism against pathogens, inspiring the possible design of the next generation of antibiotics. In this study, a new antimicrobial peptide was isolated from Lactobacillus acidophilus ATCC 4356, counteracting both biofilm and planktonic cells of Pseudomonas aeruginosa A detailed investigation was then conducted concerning the functional mechanism of this peptide by using fluorescence techniques, electron microscopy, and in silico methods. The antibacterial and antibiofilm properties of this peptide may be important in the treatment of Pseudomonas infections.

Nostoc entophytum cell response to cadmium exposure: A possible role of chaperon proteins GroEl and HtpG in cadmium-induced stress.

The present study is pursuing our previous research, focused on some aspects of Nostoc entophytum ISC32 cell response to the stress caused by exposure to cadmium at the cellular and molecular levels. Variations in the antioxidant system (catalase and ascorbate peroxidase activity) of N. entophytum ISC32 exposed to varying concentrations of Cd (2, and 5 mg/L) resulted in a significant increase in the activity of both catalase and peroxidase. Activity of these enzymes was, however, not significantly changed in the presence of Cd concentrations of 10 and 20 mg/L. Levels of lipid peroxidation, as measured by malondialdehyde (MDA) assay, were observed in response to exposure to Cd (20 mg/L). There was, however, a sharp drop in both antioxidant and lipid peroxidation activities of Cd treated cells after 5 days exposure, likely in consequence of cellular damage. The content of chlorophyll a and phycobiliproteins of living cells were altered under Cd-induced conditions. TEM images of cyanobacterial cells treated with Cd showed cell surface alteration and modification along with altered cellular microcompartments. Cyanobacterial cells treated with Cd at concentrations below the minimum inhibitory concentration (MIC) remained with no apparent structural changes. However, at a higher concentration of Cd (30 mg/L), a clear detachment effect was observed between the mucilage external layer and cell membrane which may be attributed to cell plasmolysis due to toxic effects of Cd. Subsequently, the thickness of the ring-shaped mucilage external layer increased likely as a result of the cell defense mechanisms against toxic concentrations of Cd. Characterization of cells treated with Cd (30 and 150 mg/L) by scanning electron microscopy (SEM) indicated cell shrinkage with varying degrees of distortion and surface wrinkling. Energy-dispersive X-ray spectrometry (EDS) analysis suggested that Cd was not present as nanoparticles within the cell, but in the form of salt or other molecular structures. The up-regulation of chaperons was confirmed for GroEL and HtpG using real-time PCR and northern blot analyses. Interestingly, the expression of GroEL was markedly increased at lower Cd concentration (5 mg/L). However, the ISC32 strain accrued higher levels of HtpG transcript in response to an elevated concentration of Cd (15 mg/L). This pattern seems to be related to the fast and early induction of GroEL, which may be necessary for induction of other factors and heat shock proteins such as HtpG in Cd-treated Nostoc cells. The result of this study paves the way for a more detailed exploration of Cd effects on the defense mechanisms of cyanobacteria. Our research also shed some light on how cyanobacterial cells have evolved to respond to the heavy metal toxicity at the cellular, molecular and ultrastructural levels.

High phenol degradation capacity of a newly characterized Acinetobacter sp. SA01: Bacterial cell viability and membrane impairment in respect to the phenol toxicity.

An efficient phenol-degrading bacterial strain, belonging to Acinetobacter genus, was isolated and selected to study the impact of different environmentally relevant phenol concentrations on the degradation process. The bacterial isolate, labeled as Acinetobacter sp. SA01 was able to degrade the maximum phenol concentration of 1 g/l during 60 h at optimum condition of pH 7, 30 °C and 180 rpm. Aeration and initial cell density, the two important factors, were carefully examined in the optimal growth conditions. The results showed that these two variables related proportionally with phenol degradation rate. Further investigations showed no effect of inoculum size on the enhancement of degradation of phenol at over 1 g/l. Flow cytometry (FCM) study was performed to find out the relationship between phenol-induced damages and phenol degradation process. Single staining using propidium iodide (PI) showed increased cell membrane permeability with an increase of phenol concentration, while single staining with carboxyfluorescein diacetate (cFDA) demonstrated a considerable reduction in esterase activity of the cells treated with phenol at more than 1 g/l. A detailed investigation of cellular viability using concurrent double staining of cFDA/PI revealed that the cell death increases in cells exposed to phenol at more than 1 g/l. The rate of cell death was low but noticeable in the presence of phenol concentration of 2 g/l, over time. Phenol at concentrations of 3 and 4 g/l caused strong toxicity in living cells of Acinetobacter sp. SA01. The plate count method and microscopy analysis of the cells treated with phenol at 1.5 and 2 g/l confirmed an apparent reduction in cell number over time. It was assumed that the phenol concentrations higher than 1 g/l have destructive effects on membrane integrity of Acinetobacter sp. SA01. Our results also revealed that the toxicity did not reduce by increasing initial cell density. Scanning electron microscopy (SEM) examination of bacterial cells revealed the surface morphological changes following exposure to phenol. The bacterial cells, with wizened appearance and wrinkled surface, were observed by exposing to phenol (1 g/l) at lag phase. A morphological change occurred in the mid-logarithmic phase as the bacterial cells demonstrated coccobacilli form as well as elongated filamentous shape. The wrinkled cell surface were totally disappeared in mid-stationary phase, suggesting that the complete degradation of phenol relieve the stress and direct bacterial cells toward possessing smoother cell membrane.

Biochemical properties of Glu-SH3 as a family 13 glycoside hydrolase with remarkable substrate specificity for trehalose: Implications to sequence-based classification of CAZymes.

A novel glycoside hydrolase from Exiguobacterium sp. SH3 was characterized. The enzyme, designated as Glu-SH3, was predicted by in silico analysis to have structural similarity with members of oligo-1,6-glucosidase and trehalose-6-phosphate hydrolase subfamilies in the GH-13 family of glycoside hydrolases. The gene was expressed in Escherichia coli and the recombinant enzyme was purified as a His-tagged protein of about 60 kDa. The enzyme was shown to have remarkable substrate specificity for trehalose. The characteristic ability of Glu-SH3 to hydrolyze trehalose was ascertained by zymography, thin layer chromatography, and NMR spectroscopy. The maximum activity of Glu-SH3 was obtained at 35 °C and pH 7, but it was able to exhibit more than 90% of the activity within the pH range of 5-8. The Vmax and Km values were estimated to be 170 U and 4.5 mg ml(-1), respectively. By comparison with trehalases, Glu-SH3 with Kcat and Kcat/Km values of 1552 s(-1) and 119.4 mM(-1) s(-1) can be recognized as a very efficient trehalose-hydrolyzing glycosidase. Given the phylogeny and the substrate specificity of Glu-SH3, it may be assumed that the enzyme shares a common ancestor with oligo-1,6-glucosidases but have evolved distinctly to serve a physiological function in trehalose metabolism.

Lipid production and mixotrophic growth features of cyanobacterial strains isolated from various aquatic sites.

The present study was conducted to determine the potential of five cyanobacteria strains isolated from aquatic zones to induce lipid production. The phylogenetic affiliation of the isolates was determined by 16S rRNA gene sequencing. Amongst the isolates, an efficient cyanobacterium, Synechococcus sp. HS01 showing maximal biomass and lipid productivity, was selected for further studies. In order to compare lipid productivity, the HS01 strain was grown in different media to screen potential significant culture ingredients and to evaluate mixotrophic cultivation. Mixotrophic cultivation of the strain using ostrich oil as a carbon source resulted in the best lipid productivity. GC analysis of fatty acid methyl esters of the selected cyanobacterial strain grown in media supplemented with ostrich oil showed a high content of C16 (palmitoleic acid and palmitic acid) and C18 (linoleic acid, oleic acid and linolenic acid) fatty acids of 42.7 and 42.8 %, respectively. Transmission electron micrographs showed that the HS01 cells exhibited an elongated rod-shaped appearance, either isolated, paired, linearly connected or in small clusters. According to initial experiments, ostrich oil, NaNO3 and NaCl were recognized as potential essential nutrients and selected for optimization of media with the goal of maximizing lipid productivity. A culture optimization technique using the response surface method demonstrated a maximum lipid productivity of 56.5 mg l(-1) day(-1). This value was 2.82-fold higher than that for the control, and was achieved in medium containing 1.12 g l(-1) NaNO3, 1 % (v/v) ostrich oil and 0.09 % (w/v) NaCl.

Characterization of a pH and detergent-tolerant, cold-adapted type I pullulanase from Exiguobacterium sp. SH3.

A pullulanase-encoding gene from psychrotrophic Exiguobacterium sp. SH3 was cloned and expressed in both E. coli and Bacillus subtilis. The functional recombinant enzyme (Pul-SH3) was purified as a His-tagged protein. Pul-SH3 was characterized to be a cold-adapted type I pullulanase with maximum activity at 45 °C. Using fluorescence spectroscopy, the melting temperature of Pul-SH3 was determined to be about 52 °C. The enzyme was able to hydrolyze pullulan, soluble starch, potato starch, and rice flour. It was exceptionally tolerant in the pH range of 4-11, exhibiting maximum activity at pH 8.5 and more than 60% of the activity in the pH range of 5-11. Being a detergent-tolerant pullulanase, Pul-SH3 retained 99, 89, and 54% of its activity at 10% concentration of Triton-X100, Tween 20, and SDS, respectively. The enzyme also exhibited an activity of 80.4 and 93.7% in the presence of two commercial detergents, Rika (7.5% v/v) and Fadisheh (2.5% w/v), respectively. The enzyme was even able to remain active by 54.5 and 85% after 10-day holding with the commercial detergents. Thermal stability of the enzyme could w on silica. Pul-SH3 with several industrially important characteristics seems desirable for cold hydrolysis of starch.

Calcium carbonate precipitation by strain Bacillus licheniformis AK01, newly isolated from loamy soil: a promising alternative for sealing cement-based materials.

The relevant experiments were designed to determine the ability of indigenous bacterial strains isolated from limestone caves, mineral springs, and loamy soils to induce calcium carbonate precipitation. Among all isolates examined in this study, an efficient carbonate-precipitating soil bacterium was selected from among the isolates and identified by 16S rRNA gene sequences as Bacillus licheniformis AK01. The ureolytic isolate was able to grow well on alkaline carbonate-precipitation medium and precipitate calcium carbonate more than 1 g L(-1). Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) analyses, and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX) examinations were performed in order to confirm the presence of calcium carbonate in the precipitate and to determine which polymorphs were present. The selected isolate was determined to be an appropriate candidate for application in a surface treatment of cement-based material to improve the properties of the mortar. Biodeposition of a layer of calcite on the surface of cement specimens resulted in filling in pore spaces. This could be an alternative method to improve the durability of the mortar. The kind of bacterial culture and medium composition had a profound impact on the resultant CaCO(3) crystal morphology.

Response surface optimization of biosurfactant produced by Pseudomonas aeruginosa MA01 isolated from spoiled apples.

A potent biosurfactant-producing bacterial strain isolated from spoiled apples was identified by 16S rRNA as Pseudomonas aeruginosa MA01. Compositional analysis revealed that the extracted biosurfactant was composed of high percentages of lipid (66%, w/w) and carbohydrate (32%, w/w). The surface tension of pure water decreased gradually with increasing biosurfactant concentration to 32.5 mN m(-1) with critical micelle concentration (CMC) value of 10.1 mg L(-1). The Fourier transform infrared spectrum of extracted biosurfactant confirmed the glycolipid nature of this natural product. Response surface methodology (RSM) was employed to optimize the biosynthesis medium for the production of MA01 biosurfactant. Nineteen carbon sources and 11 nitrogen sources were examined, with soybean oil and sodium nitrate being the most effective carbon and nitrogen sources on biosurfactant production, respectively. Among the organic nitrogen sources examined, yeast extract was necessary as a complementary nitrogen source for high production yield. Biosurfactant production at the optimum value of fermentation processing factor (15.68 g/L) was 29.5% higher than the biosurfactant concentration obtained before the RSM optimization (12.1 g/L). A central composite design algorithm was used to optimize the levels of key medium components, and it was concluded that two stages of optimization using RSM could increase biosurfactant production by 1.46 times, as compared to the values obtained before optimization.

Type 2 IDI performs better than type 1 for improving lycopene production in metabolically engineered E. coli strains.

In this study a comparison was made between type 1 and type 2 isopentenyl diphosphate isomerases (IDI) in improving lycopene production in Escherichia coli. The corresponding genes of Bacillus licheniformis and the host (i(Bl) and i(Ec), respectively) were expressed in lycopene producing E. coli strains by pTlyci(Bl) and pTlyci(Ec) plasmids, under the control of tac promoter. The results showed that the overexpression of i(Ec) improved the lycopene production from 33 ± 1 in E. coli Tlyc to 68 ± 3 mg/gDCW in E. coli Tlyci(Ec). In contrast, the expression of i(Bl) increased the lycopene production more efficiently up to 80 ± 9 mg/gDCW in E. coli Tlyci(Bl). The introduction of a heterologous mevalonate pathway to elevate the IPP abundance resulted in a lycopene production up to 132 ± 5 mg/gDCW with i(Ec) in E. coli Tlyci(Ec)-mev and 181 ± 9 mg/gDCW with i(Bl) in E. coli Tlyci(Bl)-mev, that is, 4 and 5.6 times respectively. When fructose, mannose, arabinose, and acetate were each used as an auxiliary substrate with glycerol, lycopene production was inhibited by different extents. Among auxiliary substrates tested, only citrate was an improving one for lycopene production in all strains with a maximum of 198 ± 3 mg/gDCW in E. coli Tlyci(Bl)-mev. It may be concluded that the type 2 IDI performs better than the type 1 in metabolic engineering attempts for isoprenoid production in E. coli. In addition, the metabolic engineering of citrate pathway seems a promising approach to have more isoprenoid accumulation in E. coli.

Metagenomic psychrohalophilic xylanase from camel rumen investigated for bioethanol production from wheat bran using Bacillus subtilis AP.

Bioethanol produced from lignocellulosic biomass is regarded as a clean and sustainable energy source. The recalcitrant structure of lignocellulose is a major drawback to affordable bioethanol production from plant biomass. In this study, a novel endo-1,4-xylanase, named Xyn-2, from the camel rumen metagenome, was characterized and evaluated for hydrolysis of agricultural wastes. The enzyme was identified as a psychrohalophilic xylanase with maximum activity at 20 °C, keeping 58% of the activity at 0 °C, and exhibiting twice as much activity in 0.5-4 M NaCl concentrations. Xyn-2 was able to hydrolyze wheat bran (100%), sunflower-seed shell (70%), wheat straw (56%), rice straw (56%), and rice bran (41%), in the relative order of efficiency. Besides, the ethanologenic B. subtilis AP was evaluated without and with Xyn-2 for bioethanol production from wheat bran. The strain was able to produce 5.5 g/L ethanol with a yield of 22.6% in consolidated bioprocessing (CBP). The contribution of Xyn-2 to ethanol production of B. subtilis AP was studied in an SSF system (simultaneous saccharification and fermentation) giving rise to a significant increase in ethanol production (p ≤ 0.001) to a final concentration of 7.3 g/L with a yield of 26.8%. The results revealed that the camel rumen metagenome might be an invaluable source of novel xylanolytic enzymes with potential application in lignocellulosic biomass valorization. At the same time, the results suggest that B. subtilis with a diverse carbon-source preference and sophisticated systems for production and secretion of enzymes might be a promising candidate for strain development for bioethanol production from plant biomass. It might be assumed that the fortification of B. subtilis enzymatic arsenal with select xylanolytic enzymes from camel rumen metagenome may have a great impact on bioethanol production.

Effect of MA01 rhamnolipid on cell viability and expression of quorum-sensing (QS) genes involved in biofilm formation by methicillin-resistant Staphylococcus aureus.

A group of biosurfactants, called rhamnolipids, have been shown to have antibacterial and antibiofilm activity against multidrug-resistant bacteria. Here, we examined the effect of rhamnolipid biosurfactants extracted from Pseudomonas aeruginosa MA01 on cell growth/viability, biofilm formation, and membrane permeability of methicillin-resistant Staphylococcus aureus (MRSA) ATCC6538 bacterial cells. The results obtained from flow cytometry analysis showed that by increasing the concentration of rhamnolipid from 30 to 120 mg/mL, the cell viability decreased by about 70%, and the cell membrane permeability increased by approximately 20%. In fact, increasing rhamnolipid concentration was directly related to cell membrane permeability and inversely related to cell survival. Microtiter plate biofilm assay and laser scanning confocal microscopy analysis revealed that rhamnolipid, at a concentration of 60 mg/mL, exerts a reducing effect on the biofilm formation of Staphylococcus aureus. Real-time PCR analysis for monitoring the relative changes in the expression of agrA, agrC, icaA, and icaD genes involved in biofilm formation and related to the quorum-sensing pathway after treatment with rhamnolipid indicated a reduced expression level of these genes, as well as sortase A gene. The results of the present study deepen our knowledge regarding the use of microbial natural products as promising candidates for therapeutic applications.

Anionic peroxidase production by Arnebia euchroma callus.

Arnebia euchroma callus, obtained from the root cell culture of an Iranian native specimen, has gained a doubling time of 63 H after regular subculturing on Linsmaier-Skoog (LS) medium containing sugar (50 g/L), 2,4-dichlorophenoxyacetic acid (10(-6) M), and kinetin (10(-5) M) under darkness at 25°C. Despite the observed somaclonal variations, peroxidase production by the A. euchroma calli has been stable over 4 years under the aforementioned conditions. Isoelectric focusing experiments revealed that the partially purified A. euchroma peroxidases (AePoxs) are mainly anionic with pI values of about 5.5 and 6.6. AePox reaches its optimal activity at 55°C and pH 7.5. Results of the various kinetic studies suggest that AePox belongs to the type III plant peroxidases with no activity for the oxidation of 3-indoleacetic acid, but seems to play a role in the lignin biosynthesis and H(2) O(2) regulation during the proliferation of the A. euchroma cells on LS medium. Comparing the biochemical properties of AePox with horseradish peroxidase and in view of the ease of solid cell culture, the A. euchroma callus could be considered as a source of plant peroxidase for some biotechnological applications.

Consolidated bioprocessing for bioethanol production by metabolically engineered Bacillus subtilis strains.

Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced cost-effectively by removing expense-intensive steps such as the enzymatic hydrolysis of substrate. Consolidated bioprocessing (CBP) is believed to be a practical solution combining saccharification and fermentation in a single step catalyzed by a microorganism. Bacillus subtills with innate ability to grow on a diversity of carbohydrates seems promising for affordable CBP bioethanol production using renewable plant biomass and wastes. In this study, the genes encoding alcohol dehydrogenase from Z. mobilis (adhZ) and S. cerevisiae (adhS) were each used with Z. mobilis pyruvate decarboxylase gene (pdcZ) to create ethanologenic operons in a lactate-deficient (Δldh) B. subtilis resulting in NZ and NZS strains, respectively. The S. cerevisiae adhS caused significantly more ethanol production by NZS and therefore was used to make two other operons including one with double copies of both pdcZ and adhS and the other with a single pdcZ but double adhS genes expressed in N(ZS)2 and NZS2 strains, respectively. In addition, two fusion genes were constructed with pdcZ and adhS in alternate orientations and used for ethanol production by the harboring strains namely NZ:S and NS:Z, respectively. While the increase of gene dosage was not associated with elevated carbon flow for ethanol production, the fusion gene adhS:pdcZ resulted in a more than two times increase of productivity by strain NS:Z as compared with NZS during 48 h fermentation. The CBP ethanol production by NZS and NS:Z using potatoes resulted in 16.3 g/L and 21.5 g/L ethanol during 96 h fermentation, respectively. For the first time in this study, B. subtilis was successfully used for CBP ethanol production with S. cerevisiae alcohol dehydrogenase. The results of the study provide insights on the potentials of B. subtilis for affordable bioethanol production from inexpensive plant biomass and wastes. However, the potentials need to be improved by metabolic and process engineering for higher yields of ethanol production and plant biomass utilization.

A Newly Characterized Potentially Probiotic Strain, Lactobacillus brevis MK05, and the Toxicity Effects of its Secretory Proteins Against MCF-7 Breast Cancer Cells.

Among seven strains of lactic acid bacteria (LAB) isolated from traditional dairy products, a Lactobacillus strain was identified through 16S rRNA gene sequencing and tentatively designated as Lactobacillus brevis MK05. This strain demonstrated the highest probiotic potential through biochemical analysis, including acid and bile salt resistance, as well as antibacterial activity. The collected cell-free supernatant (CFC) of L. brevis MK05 culture, compared with MRS broth with pH equal to the pH for CFC, revealed antimicrobial activity against Escherichia coli (ATCC 25922) and Staphylococcus aureus subsp. aureus (ATCC 25923), possibly due to the presence of antibacterial metabolites other than organic acids. This strain was, therefore, selected to assess the biological activity of its partially purified secretory proteins against MCF-7 cancer cells and normal fibroblast cells via the MTT assay. The partially purified cell-secreted proteins of this strain (hereafter referred to as Lb-PPSPs) showed a time and dose-dependent anti-cancer and apoptosis induction function. There was a remarkable decline in the survival rate of MCF-7 cells at doses equal to and higher than 0.5 mg/mL after 48 h. The changes in expression of the three genes involved in the apoptosis pathway (BAX, BCL-2, and BCL2L11) in MCF-7 cells treated with the Lb-PPSPs confirm its cytotoxic activity and apoptosis induction.

Evaluation of Bacterial Lipid Production: Quantitative and Qualitative Measurements: Tips and Guidelines.

Over the last decade, finding bacterial strains with ability to accumulate high concentrations of lipids has gained increasing interest, since these lipids may be used in different industries. Here we describe two methods for evaluation of lipid accumulation in cyanobacteria, following by our personal reflection on issues surrounding the use of these methods. First, we present the Bligh and Dyer protocol as a traditional extraction method using organic solvents for quantitative determination of lipids and next Nile red, a selective fluorescent stain, that has been used as a rapid approach for both qualitative and quantitative measurement of lipids.

An extreme halophilic xylanase from camel rumen metagenome with elevated catalytic activity in high salt concentrations.

An extreme halophilic xylanase, designated as XylCMS, was characterized by cloning and expression of the encoding gene from a camel rumen metagenome. XylCMS proved to be a GH11 xylanase with high identity to a hypothetical glycosyl hydrolase from Ruminococcus flavefaciens. XylCMS with a molecular weight of about 47 kDa showed maximum activity at pH 6 and 55 °C. The enzyme activity was significantly stimulated by NaCl in 1-5 M concentrations. Interestingly, the optimum temperature was not influenced by NaCl but the Kcat of the enzyme was enhanced by 2.7-folds at 37 °C and 1.2-folds at 55 °C. The Km value was decreased with NaCl by 4.3-folds at 37 °C and 3.7-folds at 55 °C resulting in a significant increase in catalytic efficiency (Kcat/Km) by 11.5-folds at 37 °C and 4.4-folds at 55 °C. Thermodynamic analysis indicated that the activation energy (Ea) and enthalpy (∆H) of the reaction were decreased with NaCl by 2.4 and threefold, respectively. From the observations and the results of fluorescence spectroscopy, it was concluded that NaCl at high concentrations improves both the flexibility and substrate affinity of XylCMS that are crucial for catalytic activity by influencing substrate binding, product release and the energy barriers of the reaction. XylCMS as an extreme halophilic xylanase with stimulated activity in artificial seawater and low water activity conditions has potentials for application in industrial biotechnology.

A bio-inspired strategy for the synthesis of zinc oxide nanoparticles (ZnO NPs) using the cell extract of cyanobacterium Nostoc sp. EA03: from biological function to toxicity evaluation.

Cyanobacteria, as one of the largest groups of phototrophic bacteria, have a high potential as an excellent source of fine chemicals and bioactive compounds, including lipid-like compounds, amino acid derivatives, proteins, and pigments. This study aimed to synthesize ZnO nanoparticles using the cell extract of the cyanobacterium Nostoc sp. EA03 (CEN-ZnO NPs) through a rapid and eco-friendly approach. The biosynthesized nanoparticles, CEN-ZnO NPs, were characterized by UV-Vis spectroscopy, X-ray diffraction (XRD), zeta potential measurement, differential scanning calorimetry (DSC)/thermogravimetric analysis (TGA), FTIR, SEM, TEM, and EDX spectroscopy. The UV-Vis spectrum showed an absorption peak at 370 nm. The star-shaped CEN-ZnO NPs, as observed in the TEM and SEM images, had an average diameter of 50-80 nm. MIC and MBC values for E. coli, P. aeruginosa and S. aureus, were determined to be, respectively, 2000, 2000, and 64 µg ml-1, and 2500, 2500 and 128 µg ml-1. Further analysis through confocal laser scanning microscopy (CLSM) provided the observable confirmation that the CEN-ZnO NPs stunted the bacterial growth, preventing the formation of exopolysaccharides. The AFM analysis of surface topography of bacterial biofilm samples treated with CEN-ZnO NPs showed a rugged topography in some parts of the biofilm surface, indicating the destruction of biofilms. In contrast, in the untreated control samples, the structured biofilms were flat and prominent. MTT assay indicated that CEN-ZnO NPs had less cytotoxicity on the MRC-5 lung fibroblast cells compared with the cancerous treated A549 cells. As the concentration of the CEN-ZnO NPs increased, the amount of ROS produced in the tested bacterial strains also increased. Analyzing the data obtained from flow cytometry showed that the higher concentrations of CEN-ZnO NPs lead to a reduction in the viability of P. aeruginosa PAO1, E. coli and S. aureus. The biosynthesized ZnO nanoparticles using Nostoc cell extracts exhibited different attributes, inspiring enough to be considered for further investigation.

Development of a novel method for the purification of C-phycocyanin pigment from a local cyanobacterial strain Limnothrix sp. NS01 and evaluation of its anticancer properties.

C-phycocyanin (C-PC) pigment, as a natural blue dye, has particular applications in various fields. It is a water-soluble protein which has anticancer, antioxidant and anti-inflammatory properties. Here, we introduce an efficient procedure for the purification of C-PC pigment, followed by conducting a comprehensive investigation of its cytotoxic effects on human breast cancer (MCF-7) cells and the underlying mechanisms. A novel four-step purification procedure including the adsorption of impurities with chitosan, activated charcoal, ammonium sulfate precipitation, and ion exchange chromatography was employed, achieving a high purity form of C-PC with purity index (PI) of 5.26. SDS-PAGE analysis showed the purified C-PC with two discrete bands, subunit α (17 kD) and ß (20 kD), as confirmed its identity by Native-PAGE. A highly purified C-PC was employed to evaluate its anticancer activity and underlying molecular mechanisms of action. The inhibitory effects of highly purified C-PC on the proliferation of human breast cancer cells (MCF-7) have detected by MTT assay. The IC50 values for 24, 48, and 72 hours of exposure to C-PC were determined to be 5.92, 5.66, and 4.52 µg/µl, respectively. Flow cytometric analysis of cells treated with C-PC, by Annexin V/PI double staining, demonstrated to induce MCF-7 cells apoptosis. Also, the results obtained from propidium iodide (PI) staining showed that MCF-7 cells treated with 5.92 µg/µl C-PC for 24 h would arrest at the G2 phase and 5.66 and 4.52 µg/µl C-PC for 48 and 72 h could induce cell cycle arrest at both G2 and S phases. The oxidative damage and mitochondrial dysfunction were evaluated to determine the possible pathways involved in C-PC-induced apoptosis in MCF-7 cells. Our findings clearly indicated that the treatment of MCF-7 cells with C-PC (IC50 for 24 h) increased the production of reactive oxygen species (ROS). Consequently, an increase in the lipid peroxidation (LPO) level and a reduction in the ATP level, mitochondrial membrane potential (MMP), glutathione (GSH) and its oxidized form (GSSG), occurred over time. The reduced expression levels of anti-apoptotic proteins, Bcl2 and Stat3, plus cell cycle regulator protein, Cyclin D1, using Real-Time PCR confirm that the C-PC-induced death of MCF-7 human breast cancer cells occurred through the mitochondrial pathway of apoptosis. Collectively, the analyses presented here suggest that C-PC has the potential so that to develop it as a chemotherapeutic anticancer drug.

Synthesizing, characterizing, and toxicity evaluating of Phycocyanin-ZnO nanorod composites: A back to nature approaches.

C-Phycocyanin pigment was purified from a native cyanobacterial strain using a novel consecutive multi-step procedure and utilized for the first time for the green synthesis of phycocyanin-zinc oxide nanorods (PHY-ZnO NRs) by a simple, low-cost and eco-friendly approach. The PHY-ZnO NRs were characterized using UV-vis spectroscopy, X-ray diffraction (XRD), zeta potential measurement, FTIR, SEM, TEM, differential scanning calorimetry (DSC), thermogravimetric (TGA), and EDX spectroscopy analysis. The UV-vis spectra showed an absorption band at 364 nm which is characteristic of ZnO nanoparticles (ZnONPs). The rod-shaped PHY-ZnO NRs observed in the TEM and SEM images had an average diameter size of 33 nm, which was in good agreement with the size calculated by XRD. The elemental analysis of PHY-ZnO NRs composition showed that three emission peaks of zinc metal and one emission peak of oxygen comprised 33.88% and 42.50%, respectively. The thermogram of PHY-ZnO NRs sample exhibited the weight loss of biosynthesized nanoparticles registered to be 3%, emphasizing the purity and heat stability of zinc oxide nanorods coated with phycocyanin pigment-protein. MTT assay indicated that PHY-ZnO NRs had a less cytotoxicity on fibroblast L929 compared to the ZnONRs-treated cells. A remarkable increase in ROS level was measured in cells treated with ZnO at final concentrations of 100, 200 and 500 µg/ml (78 ± 7, 99 ± 8 and 116 ± 11, respectively). When it comes to PHY-ZnO NRs, a protective effect for phycocyanin was detected which declined the level of ROS content as confirmed by fluorescent microscopy. The distinctive features of phycocyanin for surface functionalization of ZnO nanoparticles deserve to be deemed as a nano-drug candidate for further researches.