فصلنامه دنیای میکروب ها
سال سوم شماره 1 (پیاپی 6، بهار 1389)

L-asparaginases enzyme is an effective drugs for treatment of lymphoblastic leukemia and Non-Hodgkin's lymphoma. Escherichia coli and Erwinia strains specially Erwinia chrysanthemi are more important producers of this enzyme. The aim of this study was survey on L-asparaginase enzyme obtained from Erwinia chrysanthemi DSM4610. After bacterial genomic DNA extraction, a PCR procedure was performed with specific primers, Echr Asn F and Echr Asn R1. At the next step, the amplified DNA segment were ligated in a pTZ57R/T vector and after transformation into E. coli DH5α, the recombinant plasmids were extracted. After sequencing, the results were analyzed by DNAMAN software. By designin its expression primers, the gene was cloned in a pAED4 vector and after transformation into E.coli BL21(DE3) cells, its expression was assessed by SDS-PAGE analysis. The sequence of the isolated gene from Erwinia chrysanthemi DSM4610 was different from other similar genes in Gene Bank and was assigned with the accession number: JF972567 in Gene Bank. The most expression was found in the condition of 0.5 mM of IPTG, LB broth media and 37 °C. SDS-PAGE analysis showed 37.5 KD protein. Results revealed that the L-asparaginase enzyme obtained of Erwinia chrysanthemi has proper features for further study as an antileukemia enzyme.

Nowadays, the immune cytokines, such as the chicken IL-2 (chIL-2) gene are incorporated into vaccination regimens used by the poultry industry. The cytokines can potentially enhance the efficiency of vaccines according to increase T lymphocyte proliferation, B lymphocyte development and natural killer cell (NK) activation. The aim of this study was extraction and cloning of Iranian chicken interleukine-2 (chIL-2). First of all, in order to extract chIL-2 gene, total cell RNAs were isolated by culturing the harvested splenocytes and using Trizol reagent. The chIL-2 specific mRNA was converted into cDNA using reverse Transcriptase (RT) and specific designed primers. Then, The PCR product was ligated into the PTZ57R/T plasmid (TA-cloning kit) and was transformed into the competent Top10 E. coli using heat shock method. A unique band of 668-bp was obtained after RT-PCR amplification. The results of restriction enzyme, colony PCR from transformed colonies and also gene sequencing confirmed the existence of desired gene in transformants bacteria. For the first time in Iran we could extract and clone the chIL-2 gene in bacteria and gene alignment. A direct sequencing test showed 99% similarity between this gene and the established data in NCBI

Bacillus thuringiensis (Bt) is a bacterium best known for its production of crystal-like bodies comprised of one or more crystal proteins in sporulation phase, which can be toxic to insects, nematodes and cancer cells. The purpose of this study is isolation, characterization and comparison of Cry proteins in the isolated strains obtained in different parts of Fars province. 25 soil samples were collected from six different habitats in Fars province using seven different methods. Characterization was performed based on biochemical tests, protein crystal morphology by phase-contrast microscope and variation of cry protein toxin using SDS-PAGE. The tests showed a highest count of Bt strains in soil samples obtained from Shiraz city. The Bt colonies were classified according to biochemical tests such as: sucrose and salicin fermentation, lecithinase production and esculin hydrolyzing. Results showed that although isolated strains have a variety of protein bands with different sizes, the proteins with 34KD was observed following proteinase K digestion in all the samples.

Biodiversity of yeasts in extreme habitats and applications in food industry attract many of researchers. The aim of this study was evaluation of invertase activity in isolated yeasts from saline soil. In this study, kinetic activity of invertase from 27 halophilic and halotolerant yeast strains isolated from salin soil of Qom and Arak were evaluated. After collection of soil samples, the yeasts were cultured on YPD Agar containing 5% salt. Out of 27 yeast strains, 2 halophilic and 6 halotolerant strains with invertase activity were assessed for selection of best strain. Assessment of invertase activity according to the Glucose Oxidase assay showed that the enzyme productivity of G5 strain is more powerful than other isolates. The optimum enzyme activity was obtained at 65C and pH 4.5. The highest enzymatic activity was observed in the medium containing 1.7mM sodium chloride. Na+, Mg2+ and Ca2+ ions (all chlorides and 5mM concentration) have stimulation effect on enzyme's activity. The Km of enzyme for sucrose was 20 mM. Thermal stability of enzyme in 65C was one hour and decrease of activity is not observed. According to the results of this study, G5 strain has able to produce invertase in different concentrations of different salts.

Shiga Toxin-Producing Escherichia coli (STEC) strains can infect humans via vegetal and animal food consumption and cause food poisoning with diarrhoeas, hemorrhagic colitis, haemolytic uraemic syndrome that can lead to death. The purpose of this study is isolation and identification of Escherichia coli O157:H7 virulence genes from Juice Purchase and vegetables in Shiraz. This cross- sectional study was performed on 89 samples of Juice Purchase and vegetables collected from Shiraz. Isolates were enriched in ECB with novobiocin medium in temperature 37C. After, in order to examine the fermentation of sorbitol and lactose the CT-SMAC and VRBA media and the activities of β-glucoronidase of separated bacteria were examined using the choromoagar medium. Then, the existence of E.coli O157:H7 was confirmed with the spesific antiserum. Finally, virulence genes stx1, stx2, eae and hly were tested by multiplex PCR and rfb O157 ang fliC H7 were tested by single PCR. Out of all examined samples 69 (77.53%) sorbitol negative bacteria were separated that after evaluation with specific antiserum 21 (23.60%) E.coli O157:H7 was detected. The molecular markers, stx1 genes in 3 samples and eae, stx1 in 1 sample were detected. Intensive studies of the sources, incidence, fate and transport of E.coli O157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human.

Bakground and Various strains of Staphylococcus aureus produce different bi-component toxins such as LUKE/D and PVL. This strains of S.aureus strains are associated with severe skin diseases, fatal pneumonia and osteomyelitis with high morbidity and mortality. The aim of this study was to determine the prevalence of genes encoding Leukocidins in Staphylococcus aureus strains resistant and sensitive to methicillin isolated from burn patients in, Ahvaz. This cross sectional study was performed on scar specimens of 203 burn patients hospitalized in Taleghani hospital. All samples were evaluated by traditional culture method and standard biochemical tests for detecting of S. aureus strains. After extracting DNA, mecA, PVL and LUKE/D genes were detected by using the polymerase chain reaction technique (PCR). S. aureus strains were isolated from 95 cases out of total studied samples (46.8%), betwwn them 83 strains (87.36%) were mecA positive. The prevalence of PVL and LUKE/D genes in MRSA strains were 7.23% and 66.26% respectively, while this prevalence were 33.3% for both genes in MSSA strains. Regarding to the high frequency of PVL and LUKE/D genes in MRSA strains, also to severe and lethal diseases caused by these bacteria, early diagnosis and proper treatment must be considered for the prevention of disease progress.

Q-fever is a zoonosis caused by an intracellular rickettsia, Coxiella burnetii, with worldwide distribution. This study was conducted to determine the seasonal prevalence rate of Coxiella burentii in raw milk samples obtained from different Cowpens in Isfahan. This cross-sectional study was performed from January 2009 to January 2010. Totally, 247 milk samples from 90 Cowpens were collected and were tested for C. burnetii using nested PCR assay. In this survey, 8 out of 247 (3.2%) cow milk samples were positive for C. burnetii. The prevalence of C. burnetii varied during different seasons. The highest incidence of C. burnetii observed in winter (8.5%). All 65 milk samples collected in summer were negative for C. burnetii. These results prove that cow milk could be considered as an important reservoir for C. burnetii infection in Iran.

Yellow rust is one of the most important diseases of wheat that the pathogen populations have many pathotypies in different environmental conditions and during different years. This study was conducted to identify the pathotypes of wheat yellow rust in different environmental conditions and during two successive years. In spring of 2008 and 2009, 29 isolates of wheat yellow rust leaves were collected from different parts of Iran base on the dispersion and the different environmental conditions. Urediniospores of collected leaves were multiplied on Bolani under greenhouse conditions and then pathotypes, virulence factors and frequency were detected base on inoculated differential sets responses to each isolates. Among 29 collected isolates, 19 pathotypes were identified. In some regions similar pathotypes observed including 6E2A+, 6E6A+, 6E130A+ and 6E150A+. In 2008, the most and the least virulence potency related to isolates Ahvaz2 and Gharakhil that could overcome 12 and 5 resistance genes of the host plant, respectively. In 2009, the most and the least virulence potency related to isolates from BayekolaII, ToroghII, Borojerd and ZarghanII that could overcome 10 and 4 resistance genes of the host plant, respectively. In two years, the majority of isolates with a high frequency percent showed virulence on plant/s with Yr2, Yr2+, Yr7, Yr7+ and YrA genes. No virulence was detected on plant/s with Yr 1, 3, 4, 5, 9+, 10, 15, CV, SP, SU and ND genes. Considering the fact that no virulence was detected for plant/s with Yr1, Yr3, Yr4 and Yr10 genes, these genes can be used in wheat breeding programs in all regions of Iran to control wheat yellow rust.