فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 40 (زمستان 1400)

Nowadays, probiotic bacteria such as Lactobacilli have been recognized as promising agents to prevent a large number of diseases, including cancer. This study aimed to evaluate the cytotoxic effects of Lactobacillus paracasei (IBRC_10784) extract on pancreatic cancer cell line ASPc-1 and to analyze the expression of bax, Bcl2, and DPC4 apoptotic genes.

This study was conducted on ASPC-1 pancreatic adenocarcinoma cell line to assess the effects of culture medium and Lactobacillus paracasei products on the expression of genes involved in apoptosis as well as DPC4 gene expression. Lactobacillus paracasei was cultured on specialized MSR broth medium and incubated for 48 hours in a CO2 incubator. After observing the growth of bacteria, the culture medium was centrifuged for 5 minutes at 9000 rpm and the supernatant was removed. Then, the supernatant was sterilized by a 0.22-micron syringe filter and was used in the subsequent steps. In the next step, AsPC-1 cells were treated with supernatant solution for 24 hours, followed by RNA extraction and cDNA synthesis. The Q-PCR reaction was performed using specific primers.

The findings of this research showed that the supernatant solution of Lactobacillus paracasei culture was able to change the expression of the studied genes, so that the expression of bax gene increased 1.86 times relative to the reference gene. Moreover, the expression of bcl-2 gene decreased by 4.56 folds compared to the reference gene. A notable point was the changing expression of DPC4 gene, which significantly increased to 2.67 folds in comparison to the reference gene (P<0.005).

According to the results of the present study, it seems that the increased expression of DPC4 tumor suppressor gene, which is one of the essential factors in TGF-β pathway, may be important in suppressing pancreatic cancer.

The radiation-resistant extremophile microorganisms can produce high amounts of carotenoids. However, the optimization of their metabolite production is critical for large-scale commercial purpose. This study aimed to investigate the production of carotenoid pigments by an extremophile strain phylogenetically close to the genus Cellulosimicrobium that has not been reported so far.

The influence of various carbon and nitrogen sources on the microbial biomass and pigment production of the studied strain were investigated using one-factor-at-a-time-approach. Antioxidant activity of the pigment was evaluated using free radical scavenging and ferric reducing antioxidant power. Besides, the antibacterial and cytotoxicity activity of the pigment extract were investigated using the agar well-diffusion method and cell metabolic activity assay, respectively.

The maximum amount of carotenoid pigment produced by the studied strain was achieved in the fermentation medium containing 1g/L yeast extract and 1g/L glucose (22.5 mg /L) that was 10.7 fold more than the unoptimized conditions (2.1 mg/L). The half-maximal effective concentration (EC50) values of the pigment were evaluated as much as 10.97 mg/mL and 6.02 μg/mL in the free radical scavenging and ferric reducing antioxidant power assay, respectively. Also, the pigments of this strain showed no toxic and inhibitory effect on the human fibroblast cell line.

As compared to previous studies, the carotenoid pigment extract of strain AZ displayed strong antioxidant activity. Therefore, the present study lays a foundation for future utilizations of Cellulosimicrobium strain AZ as a promising microorganism for commercial production of carotenoids in the food industry or sunscreens due to the antioxidant and non-cytotoxic activity of its carotenoid pigment.

Methanotrophs are microorganisms that play an important role in reducing up to 15% of environmental CH4 concentration. These bacteria can oxidize methane to methanol. Methane is an atmosphere-polluting greenhouse gas with increasing atmospheric concentrations and is considered a serious threat to the environment and the sustenance of life on the planet. Possible unfavorable future weather and the extensive use of different types of pesticides necessitate the use of methanotrophic bacteria that are resistant to these conditions. The present study focused on the isolation and specific study of Methylocystis methanotrophic bacteria.

The bacterial strains were isolated from different environments such as wetlands, rivers, and paddy fields. The isolated strains were concentrated with Methane gas enriched cultures and purified by serial dilution-extinction, alternating liquid, and solid subcultures. Then, molecular identification was performed and a suitable culture medium was designed for Methylocystis strains in the name of Methylocystis Culture (MOC). Strains were cultured in MOC to evaluate their viability in different conditions by changing important parameters such as the temperature, pH, salinity, and drought. In addition, they were affected by pesticides. The methane gas reduction in the upper space of the culture medium of each strain, in accordance with the control samples, is considered as the methane oxidation of that strain.

Methane gas oxidation rate was determined in vitro for the cultures of several strains. The growth curve of the strains was plotted. According to the results, Methylocystis methanotrophs had high survivability in different conditions and were resistant to common pesticides, and consumed more than 75% of methane gas over a period of 14 days.

Methylocystis methanotrophic bacteria can greatly reduce the release of methane into the atmosphere and alter the fate of the atmosphere.

Zymoseptoria tritici is a dimorphic pathogenic fungus in which switching growth pattern plays a crucial role in diseases development. Therefore, Tup1 protein, the global transcriptional repressor which is a well-known regulator of dimorphism might be a qualified candidate for preliminary study in the pathogen.

Using Saccharomyces cerevisiae Tup1p as a model, the functional domains of the protein were plotted using InterPro software. Then Zt.Tup1p tertiary structure were represented by accelrys DS visualize software. Finally, the in vitro and in vivo levels of expression of Zt.Tup were evaluated by real Time RT-PCR.

Zt.Tup1p included public structures available in the other Tup1-like eukaryotic proteins, with an N-terminal domain, a poorly conserved middle region and a C-terminal domain with seven WD repeats. Also, the results showed that the two glutamine-rich elements (Q1 and Q2) contrary to which was identified in the S. cerevisiae, do not exist in Zt.Tup1p structure. The results were indicated that the expression level of Zt.tup1 up-regulate during mycelial stage. In addition, Zt.Tup1p was partly up-regulated in 14 and 24th post-inoculation.

The presence of seven conserved WD repeats in C terminal domain of Zt.Tup1p and the other related fungi indicates that these functional domains play an important role in the life of these microorganisms. According to the data of the present study, the up-regulation of Zt.tup1 gene during mycelial stage suggests that it may play a major role in mycelium development than in conidia formation. Furthermore, it’s up-regulation during necrotrophy which are characterized by the development of pycnidia shows that the gene may play a key role in the growth and pathogenicity of Z. tritici. The present study can be considered as promising new leads for starting a new comprehensive survey for clarifying this gene detailed functions during the development and pathogenicity.

Endophytes are a large and diverse group of microorganisms living in the plants’ tissues and/or organs without causing any symptoms. Endophytes play an important role in plant protection against biotic and abiotic stress. The aim of the present study was to isolate and identify endophytic fungi in Corylus avellana L. and evaluate the possible effects of different tissues and geographical conditions on their communities.

Plant sampling of healthy tissues (leaf, stem, and root) of the hazelnut plants was carried out in Golestan, Mazandaran, Guilan, Ardabil, Zanjan, and Qazvin provinces in north and northwest of Iran. Following surface sterilization, endophytic fungi were isolated using standard culturing techniques and identified based on morphological characteristics. Representatives of each taxon were subjected to molecular identification based on ITS ‐rDNA, LSU, TEF1, and β-tubulin sequences. Data were analyzed using diversity indices and correspondence analysis (CA).

In total, 791 endophytic fungal isolates were isolated from healthy leaves (39%), stems (35.65%), and roots (25.16%). The isolates were classified into 24 fungal species belonging to seven orders in four fungal classes, including Dothideomycetes (Capnodiales and Pleosporales), Sordariomycetes (Diaporthales and Hypocreales) Eurotiomycetes (Eurotiales and Chaetothyriales), and Agaricomycetes (Polyporales). Ascomycota and Basidiomycota represented 96.2% and 3.8% of the isolates, respectively. The stem and root showed the highest and lowest total colonization frequency and diversity indices of endophytic fungi respectively. Also, the diversity indices of endophytes in Guilan were higher than those in other studied provinces.

The frequency and diversity of fungal endophytes in C. avellana are under the influence of factors such as geographical conditions, biodiversity, and tissue type. Different plant tissues, multiple sites, and related ecological indicators have helped define how maximum biodiversity may be found in a fungal endophyte population in a given plant species.

Glucose oxidase (GOX) is considered a commercial enzyme in the medicine, pharmaceutical, and food industries. Optimization of the fermentation medium and conditions is important to increase heterologous GOX production. The current study aimed to optimize GOX production in a ‎recombinant strain of Yarrowia lipolytica via statistical techniques.

Response surface methodology (RSM) as a statistical technique for the design of the experiment was used for the optimization of recombinant GOX production by Y. lipolytica. Then, the optimized conditions were used for the production of GOX in bioreactor cultivation.

GOX production was increased up to 570 U/L after optimization of the medium in flask culture which is 2 times more than the non-optimized medium. In the bioreactor culture using optimized medium containing glucose and sucrose as carbon sources, GOX production was reached 720 and 730 U/L after two and six days, respectively.

Results of the study provided valuable information about the statistical optimization of bioprocess development for the production of heterologous protein in Y. lipolytica.

Docosahexaenoic acid (a polyunsaturated fatty acid) plays an important role in the prevention of arteriosclerosis and cardiovascular diseases. Thraustochytrid protists, specifically Aurantiochytrium strains, are among the main microbial producers of omega-3 fatty acid. The aim of the present study was to enhance biomass, oil, and docosahexaenoic acid (DHA) content of Aurantiochytrium sp. by glucose or glycerol feeding in a 3L-fermentation system.

A native strain of Aurantiochytrium sp. qe-4 (KR091914.1) was used for DHA production. Glycerol and glucose were used as a carbon source in a defined medium. Cell growth, lipid, and DHA accumulation in Aurantiochytrium sp. were studied under fed-batch cultivation by using accurate feeding techniques in a 3 L fermentor.

The results of the present study indicated that at C/N ratio of 1.5 and medium composition of 30 g.L-1 glycerol, 10 g.L-1 peptone, and 10 g.L-1 yeast extract, the native strain of Aurantiochytrium produced 30.2 g.L-1 biomass, 8.8 g.L-1 lipid, and 1.7 g.L-1 DHA. Also at C/N ratio of 2 and medium composition of 40 g.L-1 glucose, 20 g.L-1 mono-sodium glutamate (MSG) and 6 g.L-1 yeast extract, 27.6 g.L-1 biomass, 12.5 g.L-1 lipid, and 1.45 g.L-1 DHA were produced. It was also shown that low dissolved oxygen less than 3% of saturation was efficient for DHA production.

During the fermentation process, dissolved oxygen values and carbon-to-nitrogen ratios are critical factors that influence growth and DHA synthesis in Aurantiochytrium. These results showed that the native strain of Aurantiochytrium had a better performance to produce DHA in a medium containing glycerol than glucose as the sole carbon source.

Nude mice are immunodeficient, commonly used model organisms for studying human diseases. Despite the high purchase price and maintenance cost of these animals, few studies have aimed to eradicate inevitable infectious complications which may arise during in-vivo studies. The current project is a practical guideline for detecting and elimination of possible infections developed in nude mice.

Followed by transplantation of human leukemic cell lines into C57BL/6 nude mice, body weights were monitored daily. After a sudden death of 7 mice during 4 consecutive days, all beddings were discarded and cages, food and water bottles were disinfected immediately. Automated blood culture analyses were carried out using radiometric BACTEC systems, and specific antibiotics were administered based on applied microbial growth inhibition assays.

Proteus vulgaris and Klebsiella pneumoniae were identified, respectively, according to the radiometric assays. According to the antimicrobial susceptibility tests, 132mg/kg ciprofloxacin was administered orally for 2 weeks, followed by 500mg/kg cefixime for one week. Meanwhile, low-dose cefixime (132 mg/kg) was applied as a prophylactic treatment. Automated blood cultures showed elimination of both bacterial infections followed by demand treatments. Low-dose antibiotic prophylaxis saved uninfected mice from developing infections.

Results showed that precise monitoring of nude mice health conditions help early detection of the possibly grown infections. Ciprofloxacin and cefixime are the antibiotics of choice for eradication of Proteus vulgaris and Klebsiella pneumoniae. On the other hand, low-dose antibiotic prophylactic treatment may rescue these expensive animals from possible infections and provide suitable environment for performing in-vivo experiments.

Hamedan province is one of the most important regions for cultivation of stone fruit trees, such as apricots (Prunus armeniaca) due to its suitable climatic conditions. Bacterial canker disease of stone fruit trees caused by Pseudomonas syringae pv. syringae, is one of the most serious diseases in this province, Iran and all over the world. The study was performed to identify the causative agent of the disease.

Samples of diseased apricots were collected in early spring and fall autumn of 2018. From the collected samples a total of 130 bacterial strains were isolated of which 65 representative’s strains were selected based on the color and type of colonies on the culture medium for further study. SDS-PAGE technique was used to group the representative’s strains. Pathogenicity of the representative’s strains were performed on apricot seedlings under greenhouse condition. The studied strains were identified based on their phenotypic features, using standard bacteriological method and followed by sequencing of the 16S rRNA encoding gene.

30 strains showed hypersensitivity reaction on geranium leaves.  According to the pattern of cellular soluble proteins on polyacrylamide gel (SDS-PAGE), the strains were divided into seven groups. BLAST of partial sequence of 16S rRNA encoding gene from this strain showed 98/7% similarity to P. syringae NCPPB 281. Another strain (FB46 strain) was identified as P. agglomerans due to 95.33% similarity with Pantoea agglomerans RSG19 by BLAST of 16S rRNA encoding gene sequence.

The results obtained are in agreement with of Vasebi et al. isolated P. syringae as a causative agent of apricot canker in East Azerbaijan province. P. agglomerans has also been reported as one of the factors associated with stone fruits, pome fruits and walnut trees in Alborz province. This is the first report for the presence of Pseudomonas syringae as a causative agent of apricot canker disease in Hamedan province. The bacterium Pantoea agglomerans is reported from the first time in the world as the causative agent which induce apricot canker symptoms.

The present study aimed to examine the effects of Lactobacillus acidophilus and Lactobacillus plantarum probiotics separately and synergistically on the immune system, growth factors, and disease resistance in the rainbow trout.

At in vitro level, bacteriocin extracts of the target bacteria were used in the antimicrobial test against the fish pathogen Aeromonas hydrophila. Next, their effects on the fish blood lymphocytes were evaluated. At in vivo level, after feeding the fish in each treatment with the lactobacilli at the concentration of 108 CFU g-1 for 60 days, the growth indices were calculated. Then, blood samples from the rainbow trout were used to analyze any increase in the number of lymphocytes. Furthermore, given the infectious challenge with the pathogen Chryseobacterium aquaticum, the survival rates of the fish were determined.

L. acidophilus had the best effect on the growth factors and significantly increased the number of fish blood lymphocytes (p <0.05). The broth microdilution assay also was shown to reduce the amount of A. hydrophila colonies by more than 90%. Treatment with this lactobacillus species produced the highest survival rate with this infectious challenge. Then, the synergistic and the L. plantarum treatments were analyzed, respectively, which each showed a significant increase in the indices under study.

According to obtained results, it might be concluded that the feeding by L. acidophilus could likely enhance the immune responses and growth indices in fish both separately and synergistically with L. plantarum.