Journal of Microbial Biology
Year:2018 | Volume:7 | Issue:26 |Papers Count:10

Introduction: Evaluation of symbiosis between Arbuscular mycorrhizal fungi (AMF) and Saffron (Crocus Sativus L.) is important because this strategic plant encounters with many environmental stresses such as climatic and edaphic stresses during seasons and the AMF can let the crops increase their productivity along with the improvement of their resistance to stress factors and pathogens.Materials and methods: The spores of AMF around rhizosphere of saffron were studied in three fields of Gonabad, Khorasan province, Iran (2013-14). Moreover, the colonization of mycorrhizal fungi with saffron and sorghum trap were studied in three regions using morphologic and molecular methods by nested PCR and amplification of small subunit of rRNA gene fragments.Results: Three species of arbuscular mycorrhizal fungi, Scutellospora dipurpurescens, Funneliformis caledonius and Rhizophagus aggregatus were identified in the soil around rhizosphere of the saffron of three regions. The colonization of sorghum trap in the soil of saffron cultivation areas was among 21-41%, while the colonization in the natural Saffron field was 1.5% and just in one area. However, the nested PCR results revealed the colonization of Saffron in all 3 regions. These results showed the colonization of Saffron by Rhizophagus iranicus and F. caledonius.Discussion and conclusion: The genus and species diversity of AMF Saffron and Sorghum are different. Moreover, the hereby proposed molecular method is a more precise approach to identify AMF colonization with Saffron while classical methods may provide different and misleading results.

Introduction: Pseudomonas aeruginosa is a particular importance due to the numerous factors of pathogenicity and the prevalence of multi-resistance strains throughout the world. Therefore, the need to prevent and produce an effective vaccine seems necessary. The aim of this study was to use PLGA nanoparticles in the design of vaccine with alginate antigens, lipopolysaccharide, and exotoxin, A Pseudomonas aeruginosa. In this study, strain PAO1 of Pseudomonas aeruginosa is used. Then, antigens including, alginate, lipopolysaccharide, and exotoxin A were extracted. Then, lipopolysaccharide was detoxified by hot phenol method. Exotoxin A was purified by chromatography and detoxified with formalin. Then, antigens separately conjugated to PLGA. FT-IR and AFM methods were usedto confirm conjugation with nanoparticles. A rabbit model was used to study the pyrogenic effects of conjugates. The mortality effect of conjugates on mouse model was tested.Materials and methods: The success of conjugation based on the size and charge of the nanoparticle was confirmed. The presence of antigenic functional groups in the structure of the nanoparticle and the formation of a steric bond confirmed with FT-IR results and the corresponding courier form.Results: The 3D conjugate images of nanoparticles before and after conjugation showed an increase in the height of nanoparticle binding sites. The change from initial sharpness to puff after the conjugation confirmed the success of conjugation. Failure to observe the pyrogenic effects in the rabbit and no mortality observed in mice was proved.Discussion and conclusion: All of the results showed that conjugates were effective in immunization. Therefore, it can be a candidate vaccine with a great potential against Pseudomonas causing diseases.

Introduction: The uranium bioleaching process is performed using Acidithiobacillus ferrooxidans. This bacterium is capable of iron oxidation by an electron transport chain. One of the most important components of this chain is the cyc2 gene product that involved in the oxidation process of iron.Materials and methods: Evaluation of UV mutated (60, 120 and 180s) Acidithiobacillus sp. FJ2 cyc2 gene in the presence of uranium ore concentrations, has been implemented in this project. For this purpose, the original and mutated bacteria were cultivated in the presence of uranium ore concentrations (5, 10, 15, 25 and 50%). Uranium extraction, variation of pH and Eh values were measured at 24 h intervals. Then, when the uranium extraction yield reached to 100%, gene expressions of cyc2 original and mutated Acidithiobacillus sp. FJ2 were analyzed using Real-time PCR method.Results: The results of the experiments showed that, with increasing pulp density, the uranium extraction rate and oxidation activity of bacteria were reduced. In addition, the result of cyc2 gene expression showed that the target gene expression increases in the presence of uranium ore compared to sample with absence of uranium ore, and with further increase of pulp density, due to the toxicity of uranium, shows a decreasing trend.Discussion and conclusion: The results of this study indicated that the mutation in the bacterium has a positive effect on the uranium bioleaching process, which can play an important role in the process of uranium bioleaching at high concentrations. In addition, with increasing pulp density due to uranium toxicity, there is a decreasing trend in the process of uranium extraction, which indicates the important role of this factor in the uranium bioleaching process.

Introduction: In the recent years, a growing interest for the production of secretary recombinant proteins is seen. This is due to the advantages of recombinant protein production in the periplasm compared to the cytoplasm. However, signal peptides have a critical role in protein secretion as well as the applied technique for the extraction of the protein. Granulocyte colony stimulating factor (GCSF) is a type of colony stimulating factor that causes motivating of proliferation, differentiation and survival of neutrophiles and progenitor cells of these cells and are used to promote decreased neutrophils in some cancers after chemotherapy. The aim of this study was to evaluate the usage of different osmotic shock assays in order to achieve the highest amount of granulocyte colony stimulating factor (GCSF) in BL21 strain of E. coli.Materials and methods: The E. coli which contained pET22b- GCSF2- Intein2 expression vector was cultured in 4YT medium and was induced with IPTG 1Mm for protein production. It is necessary to mention that the pET22b has a pelB signal peptide that directs proteins to the periplasmic space. In the next step, three different methods of osmotic shocks were applied for the extraction of the obtained human recombinant protein. Finally, the isolated proteins were analyzed by SDS-PAGE and western blot techniques.Results: The results of this investigation indicated that GCSF was produced in both of the cytoplasmic and periplasmic spaces and the best method of osmotic shock for protein extraction is using Tris buffer and MgSO4.Discussion and conclusion: Regarding the results, it is concluded that the MgSO4 with Tris buffer create a good osmotic pressure and accordingly is a more effective way for G-CSF protein extraction. As a result, this method could be used for production and simple separation of recombinant drug proteins.

Introduction: Bioremediation is an effective, inexpensive and environmental friendly manner for removing oil pollutions. Studding the biodiversity of indigenous microorganisms and their function is very important for bioremediation strategy selection and performance. This study was aimed to investigate the rule of Bacillus species in bioremediation of diesel contaminated soil.Materials and Methods: Soil microcosms were prepared by adding 2 and 4% (W/W) of diesel to the soil. A control microcosm without diesel contamination was also set up. Microcosms were amended with nitrogen and phosphate sources and water (20%) and in a six month study period and parameters including diesel degradation, heterotrophic bacterial count and Bacillus spp. diversity by 16S rRNA PCR-DGGE were studied.Results: The results showed that in 2% microcosm up to 50% and in 4% microcosm up to 44.44% of diesel were degraded after six months. The increase of bacterial count was completely coincided with the highest diesel removal rate. The count of heterotrophic bacteria in 2 and 4% microcosms was elevated from 2x108 to 2x1011 and 2x1012 at the highest point, respectively. Accordingly, introduction of the diesel stimulated the native soil bacteria and the amount of pollution was determinative in bioremediation efficiency.Discussion and conclusion: Comparison of the pattern of DGGE bands variation between the microcosms showed that by entry of the contaminant into the soil, the diversity of Bacillus species was increased, indicating that Bacillus species has a particular role in diesel degradation. Simultaneous with decline of the pollution and microbial count of the soil, diversity of DGGE bands was decreased. Out of these findings we may conclude that addition of diesel as a carbon source to the soil increases the Bacillus spp. diversity at the beginning of bioremediation and afterwards by elimination of the pollutant, the diversity decreases gradually and shifts back to its original structure.

Introduction: Microorganisms in response to drug development, acquire resistance through a variety of mechanisms. The prevalence of resistance to fluoroquinolones (FQ), such as ciprofluxacin in Escherichia coli has increased markedly in recent years. Mutagenesis induced by SOS catalyzes the evolution of resistance to fluoroquinolones. A member of the SOS regulon is the dinI gene. Protein encoded by the gene DinI acts as positive and negative modulator of RecA performance. Previous studies showed recA gene expression in ciprofloxacin resistant Escherichia coli dinI+ in which mutants increased. The aim of this study was to investigate recA gene expression in dinI-mutant resistant to ciprofloxacin.Materials and Methods: For this purpose, dinI- mutant became ciprofloxacin resistant by encountering to increase amount of ciprofloxacin via stepwise method and be evaluated for MIC. Then, the expression of recA gene was determined in wild type, dinI- and a dinI+ mutants by real time PCR.Results: The results showed that a dinI- mutant acquired low level of resistance to ciprofloxacin and its MIC was 0.3 mg/ml. recAgene expression in the dinI- mutant was increased in comparison with wild type strain. However, the amount of increase was about one fourth of increase in dinI+ mutant.Discussion and conclusion: In conclusion, inactivation of dinI gene does not inhibit increase in recA gene expression and regulation of RecA activity is possibly complex and could be conducted in the absence of dinI by other regulatory proteins.

Introduction: Pesticides have been developed as powerful tools that apply for population feeding needs, all over the world. Pesticides’ overuse has become a serious issue about the environment. Among pesticides, diazinon is one of the organophosphorus inceticides that is used to control many different kinds of pests. There are a lot of researches about the toxicity effects of pesticides on the environment. However, one of the strategies to remove them from different environments is using degradation bacteria.Materials and methods: For screening vernacular bacterium that are able to degrade the pesticide the sampling has been done from different soils of agriculture and industrial places, and the mineral salt medium was used for extvation of bacteria. After enrichment of samples in the mineral salt media, the isolation was done in the soild medium and 10 different sterains were based on morphology characters extracted and were identified to species level. The concentration of diazinon in the presence of bacteria was determined as a high performance liquid chromatography (HPLC).Results: Among diazinon degradetion isolates S1 and S2 reduced the concentration of diazinon from 50 mg.l to 3.18 mg.l and 5.21 mg.l after 15 days, respectivly. The results indicated that S1 and S2 have higher efficiency to decreasing the amount of pesticide. Based on growing bacteria in the poor medium it’s shown that bacteria used diazinon as an energy source. Results indicated that amplification and sequencing of 16S rDNA of S1 and S2 isolated have the highest similarity with Paenibacillus and Pseudomonas, respectively.Discussion and conclusion: Results of this study revealed that degradation bacteria of diazinon exist in the places that used this insecticide and in the waste of industrial zone. It is expected that using these bacteria and biological resuscitation, it is possible to reduce some environmental hazardus issues of diazinon and also field application and determining the best formulation for bioramidation are essential in the future.

Introduction: Membrane-bound desaturases and related enzymes play a pivotal role in the biosynthesis of unsaturated fatty acids. Delta6 desaturase is a key enzyme in the biosynthesis of the unsaturated fatty acids. Mortierella alpina is an oleaginous fungus with active Delta 6 desaturase which hasbeengreatly considered recently.Materials and methods: In order to isolate and clone Δ6D gene from Mortierella alpina, after extraction of total RNA and synthesis cDNA, PCR amplification has been done using gene specific primers. The amplified fragment was cloned into the pBlueScriptSK+ containing seed specific promoter napin. Then the recombinant plasmid was transformed into E.coli DH5a by freezing and thawing method. The confirmed gene construct was cloned into the binary vector pBI121 and transformed into Agrobacterium LBA4404 in order to transform canola plants. Bioinformatics characterization of target gene was investigated by servers TMHMM, ProtParam and Psipred.Results: Correctness of cloning was confirmed by PCR with specific primers, enzymatic digestion and sequencing. The proliferation of a fragment with 830 bp using internal primer of napin promoter and Delta6 desaturase primer confirmed insertion of the gene along with napin promoter. Nucleotide sequencing results showed that cloned CDS includes 1374 nucleotides that will translate to a protein with 448 amino acids. Using bioinformatics analysis, presence of cytochrome b5 domain, three His-box, secondary and spatial structures, transmembrane and conserved domains were confirmed.Discussion and conclusion: Based on the results of BLAST analysis using nucleotide and protein sequences, and also presence of functional domains in the protein, it can be predicted that cloned CDS will show proper enzyme activity after transformation into plants. Confirming these results requires expression analysis of the gene in appropriate plant system and studying its function in the enzyme level.

Introduction: The algae are rich in minerals, vitamins, and in other nutrients and they are cultivated in order to produce valuable raw materials.Materials and methods: In this regard, the effect of 10% carbon dioxide concentration on growth factors, chemical composition, fatty acids profile, chlorophyll and carotene of two species Scenedesmus oblique and Haematocuccus pluvialis were studied.Results: Based on the results, the protein content was significantly higher in the S. oblique compared to the H. pluvialis (16.79% and 9.16%, respectively) (P<0.05). In contrast, the total lipid production was significantly higher in the H. pluvialis compared to the S. oblique (P<0.05). The saturated fatty acids percentage was significantly higher in the H. pluvialis in comparison to S. oblique (30.93% and 23.51%, respectively) (P<0.05). Also, the amount of chlorophyll a and total carotene was measured in the H. pluvialis significantly higher than the S. oblique (P<0.05). At the end of 20 days of the cultivation, the amount of biomass in the H. pluvialis was significantly higher than that produced at S. oblique (P<0.05).Discussion and conclusion: Although the presence of CO2 caused S. obliquus use the conditions for more production of biomass (0.18 g.L-1 DW), significant difference in biomass with H. pluvialis (0.17 g.L-1 DW) was not observed.

Introduction: Pectinase or pectinolytic enzymes are complex enzymes which include pectin methyl esterase (EC.3.1.1.11), pectin lyase (EC.4.2.2.2) and polygalacturonase (EC.3.3.1.15) which degrade pectin in the cell wall of plant cells. These enzymes have many industrial applications that some of them are active in extreme condition regarding to temperature, pH and salt concentration. In this study, the bacteria with an ability to produce pectinase enzyme in salty condition were identified and the corresponding gene was analyzed.Materials and methods: Strains from Urmia, Inche boron and Gomeshan Ponds were inoculated in the media containing pectin precursors. By analyzing the clear zones around the colonies based on I2/KI indicator, the positive strains were selected. Quantification of enzyme activity on all three types of pectinase was carried out by spectrophotometry. In order to molecularly screen the bacterium contained pectinase gene, the bacterium genome was amplified using appropriate primers.Results: Seventeen positive strains for pectinase (10 from Gomeshan Lake, 6 from Inche Boron Lake and 1 from where Urmia Lake) were identified among 130 studied samples. According to size of clear zone of enzyme activity in the qualitative test, activity level of all three pectinase enzymes in R2S25 strain of Inche Boron Lake was measured and the growth curve was obtained. Molecular study showed that all strains contain desired gene segment.Discussion and conclusion: Quantitative evaluation showed that production and activity of pectinase enzyme in R2S25 strain increased simultaneously with increasing the growth of selected strain in logarithmic phase. Molecular study also showed that the genuses of Martelella, Aeromocrobium, Planococcus, Marinobacter, Virgibacillus, Kocuria and Micrococcus contain the pectinase gene.