Ethanol as a carotenoid production stimulator in Dunaliella salina CCAP 19/18.

Dunaliella salina is a rich source of carotenoids. Carotenoid production is induced under specific conditions, i.e., high light intensity, high salt concentration, nutrient limitation, and suboptimal temperatures in this microalga. The control of environmental factors is vital for high productivity of carotenoids. In this paper, the effect of different ethanol concentrations in combination with nitrogen deficiency was investigated to induce carotenoid production in D. salina CCAP 19/18. Also, some biochemical and molecular parameters were investigated in response to ethanol in the cells. It was shown that ethanol at 0.5% concentration increased cell number but, at 5% concentration, reduced cell viability compared to the control. The highest carotenoid production was achieved at 3% ethanol concentration, which was 1.46 fold higher than the nitrogen deficiency condition. Investigation of the 3 carotenoid biosynthesis genes revealed that their expression levels increased at 3% ethanol concentration, and the phytoene synthase gene was the most upregulated one. Lipid peroxidation increased at both 3% and 5% ethanol concentrations. At 3% concentration, the activity of catalase and superoxide dismutase increased, but no significant changes were seen at 5% ethanol concentration. Peroxidase activity reduced at both 3% and 5% concentrations. Moreover, proline and reducing sugar content increased at 3% concentration while decreased at 5% ethanol concertation. The results showed that at 3% ethanol concentration, higher carotenoid productivity was associated with an increase in other intracellular responses (molecular and biochemical). Ethanol as a controllable element may be beneficial to increase carotenoid production even under inappropriate environmental conditions in D. salina.

A Comparative Taxonomic Profile of Microbial Polyethylene and Hydrocarbon-Degrading Communities in Diverse Environments.

BACKGROUND: Polyethylene (PE) is one of the most abundant plastic wastes which accumulates in marine and terrestrial environments. As microbial degradation has been a promising approach for the bioremediation of polluted environments, identification of the microbial community profile where these pollutants accumulate, has recently been in focus. OBJECTIVE: We have investigated the taxonomic and functional characteristics of polyethylene- degrading microorganisms in a plastic waste recycling site in Tehran, Iran. MATERIALS AND METHODS: We have analyzed and compared a 16S rRNA dataset from this study with 15 datasets from 4 diverse plastic and oil polluted habitats to identify and evaluate bacterial communities involved in bioremediation. RESULTS: Our findings reveal that Proteobacteria, Actinobacteria, Acidobacteria and Cloroflexi were the dominant phyla and Actinobacteria, Alphaproteobacteria, Gammaproteobacteria and Acidimicrobia were dominant classes in these samples. The most dominant Kegg Orthology associated with PE bioremediation in these samples are related to peroxidases, alcohol dehydrogenases, monooxygenases and dioxygenases. CONCLUSIONS: Long-term presence of contaminants in soil could lead to changes in bacterial phyla abundance, resulting in metabolic adaptations to optimize biological activity and waste management in a diverse group of bacteria.

Considerable increase in Poly(3-hydroxybutyrate) production via phbC gene overexpression in Ralstonia eutropha PTCC 1615.

Introduction: Poly(3-hydroxybutyrate) (PHB) is a well-known biodegradable polymer produced by some microorganisms and can be a suitable alternative for petrochemical plastics. PHB synthase encoded by phb C gene is the main enzyme in PHB biosynthesis pathway in Ralstonia eutropha. The aim of current study was the transformation of R. eutropha PTCC 1615 with its own phb C gene and evaluation of the overexpression effect on PHB accumulation. Methods: DNA fragment including phbC gene and its promoter and terminator regions, was isolated from R. eutropha PTCC 1615, inserted into pET28a(+) vector, and transferred to the competent bacteria using calcium chloride and heat shock method. The effect of the cloned gene expression on PHB production was investigated with absorption of crotonic acid produced through PHB dehydration. Statistical analyses were carried out by SPSS software. Results: PHB content of cells of the engineered strain was 1.4 times more than that of the native bacteria. This significant difference can be an important finding for improvement of biopolymer production. Conclusion: Overexpression of phb C, the critical gene in PHB biosynthesis pathway, in R. eutropha PTCC 1615 had considerable effect on PHB accumulation.

Natural overproduction of catalase by Kocuria sp. ASB 107: extraction and semi-purification.

BACKGROUND AND OBJECTIVES: Because of importance of catalase in various industries, efforts have been made to find more suitable bacterial sources for catalase production. Kocuria is one of well-known catalase-producing genus. This is the first report about a new catalase-overproducing bacterial strain, Kocuria sp. ASB 107. MATERIALS AND METHODS: Kocuria sp. ASB 107 had been isolated from Abe-Siah Spring in Ramsar in our previous report. The bacterial biomass freezed, thawed and then lysed by three different operations separately: ultrasound, lysing buffer and enzymatic digestion. The crude extract was subjected to ammonium sulfate precipitation (40 and 60% saturation). Quality and quantity of the semi-purification was checked by electrophoresis and measuring specific activity, respectively. RESULTS: Kocuria sp. ASB 107 can be lysed by a freeze-thaw stage followed by lysozyme digestion and not by lysing buffer and not by ultrasound. Surprisingly specific activity of catalase in crude extract from Kocuria sp. ASB 107 was measured to be 195, 370 U/mg protein which is too much higher than other bacterial strains. The bacterium showed a relatively long growth curve about 40 hours. Semi-purification using ammonium sulfate precipitation was led in an increased specific activity up to about 7×106 U/mg protein implying more than 3.6-fold purification. CONCLUSION: We have showed natural catalase-overproducing ability of Kocuria sp. ASB 107. Yield and purity of catalase from Kocuria sp. ASB 107 showed great potential in industrial application suggesting the strain as good source for mass production of catalase for treatment of H2O2-containing wastewater in comparison to other bacterial sources.

Enterobacterial repetitive intergenic consensus (ERIC) PCR based genetic diversity of Xanthomonas spp. and its relation to xanthan production.

BACKGROUND AND OBJECTIVE: The genus Xanthomonas is composed of phytopathogenic bacterial species. In addition to causing crops diseases, most of the Xanthomonas species especially Xanthomonas campestris produce xanthan gum via an aerobic fermentation process. Xanthan gum is, an important exopolysaccharide from Xanthomonas campestris, mainly used in the food, petroleum and other industries. the purpose of this study was assessment of relationship between genetic diversity and xanthan production in Xanthomonas spp. MATERIALS AND METHODS: In this study 15 strains of Xanthomonas spp. which had previously been isolated from soils of vegetable farms, were discriminated from each other using Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and 16S rDNA sequencing methods. Xanthan production of strains was measured in 250 ml flask. The results of ERIC PCR and xanthan production was compared. RESULTS: ERIC-PCR patterns not only could differentiate all Xanthomonas campestis from the control i.e. Xanthomonas translucent but also discriminate strains of Xanthomonas to three clusters with 40% similarity based on Jaccard's coefficient. This clustering of the strains was in agreement with other characteristics including xanthan production and biochemical features. DISCUSSION: The results showed that genomic fingerprinting conferred adequate genetic data for discriminating between strains of the species Xanthomonas campestris. The data indicated a partial relationship between ERIC-PCR patterns and xanthan production by the strains. CONCLUSION: Further development of experiments may result in making good prediction about xanthan production capability of the Xanthomonas strains on the basis of ERIC-PCR method.

Comparative Analysis of Denaturing Gradient Gel Electrophoresis and Temporal Temperature Gradient Gel Electrophoresis Profiles as a Tool for the Differentiation of Candida Species.

BACKGROUND: Candida species are usually opportunistic organisms that cause acute to chronic infections when conditions in the host are favorable. Accurate identification of Candida species is an essential pre-requisite for improved therapeutic strategy. Identification of Candida species by conventional methods is time-consuming with low sensitivity, yet molecular approaches have provided an alternative way for early diagnosis of invasive candidiasis. Denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient gel electrophoresis (TTGE) are polymerase chain reaction (PCR)-based approaches that are used for studying the community structure of microorganisms. By using these methods, simultaneous identification of multiple yeast species will be possible and reliable results will be obtained quickly. OBJECTIVES: In this study, DGGE and TTGE methods were set up and evaluated for the detection of different Candida species, and their results were compared. MATERIALS AND METHODS: Five different Candida species were cultured on potato dextrose agar medium for 24 hours. Next, total DNA was extracted by the phenol-chloroform method. Two sets of primers, ITS3-GC/ITS4 and NL1-GC/LS2 were applied to amplify the desired regions. The amplified fragments were then used to analyze DGGE and TTGE profiles. RESULTS: The results showed that NL1-GC/LS2 primer set could yield species-specific amplicons, which were well distinguished and allowed better species discrimination than that generated by the ITS3-GC/ITS4 primer set, in both DGGE and TTGE profiles. All five Candida species were discriminated by DGGE and TTGE using the NL1-GC/LS2 primer set. CONCLUSIONS: Comparison of DGGE and TTGE profiles obtained from NL1-GC/LS2 amplicons exhibited the same patterns. Although both DGGE and TTGE techniques are capable of detecting Candida species, TTGE is recommended because of easier performance and lower costs.

Analysis of vacA/cagA genotypes/status in Helicobacter pylori isolates from Iranian children and their association with clinical outcome.

BACKGROUND/AIM: More than 50% of Iranian children are infected with Helicobacterpylori; however, no data exist about the association of vacA/cagA genotype/status with disease outcomes in them. We analyzed association of vacA/cagA genotypes/status of children's isolates with gastric inflammation status as the first step in H. pylori pathogenesis. MATERIALS AND METHODS: Antral biopsies for culture and histopathology were taken from 328 children in 1997-2009. vacA (s, m) alleles and cagA statuses of the isolates were determined by PCR. Histopathology was performed according to the Sydney system; gastritis was scored as normal, mild, moderate, severe, and follicular. RESULTS: A total of 159 culture-positive cases, with no mixed infections, were enrolled in the study. Of them, 60% were cagA-positive; 21.4%, 37.1%, 16.3%, and 25.2% cases were slm1, slm2, s2m1, and s2m2, respectively. Histopathology showed normal (4.4%), mild- chronic (31.4%), moderate-chronic (38.4%), severe-chronic (10.7%), and follicular gastritis (15.1%) cases. Thirty-four (21.4%) of the children had ulcers. Correlation (P < 0.05) was observed between more severe (moderate, severe, follicular) status and both vacAs1 allele and cagA-positive status. No significant relation was observed between genotype/status of vacA/cagA and ulcers (P > 0.05). CONCLUSION: vacAs1 and cagA are associated with more severe gastric inflammation in Iranian children. Association ofvacAs1 and cagA with more severe pathology in Iran may be similar to that of other parts of the world.

Radio-resistance in psychrotrophic Kocuria sp. ASB 107 isolated from Ab-e-Siah radioactive spring.

A new isolate, Kocuria sp. ASB 107 from the Ab-e-Siah mineral radioactive spring (Ramsar, Mazandaran Province, Iran) was characterized on the basis of morphological and biochemical characteristics plus 16S rRNA gene sequencing. The isolate is most closely related to Kocuria rosea DSM 20447(T) (99.7% sequence similarity) and Kocuria polaris DSM 14382(T) (99.5%). This strain has some resistance to various genotoxic stresses, such as ionizing radiation, ultraviolet (256 nm- UV) and corona discharge. The 90% lethal doses (D(10)) for gamma-rays and 256 nm-UV are 2 kGy and 400 J m(-2), respectively, in definite cell concentration. Moreover, the resistance for a definite energy of corona discharge is 10 s, about 10 times greater than that of Escherichia coli. The growth temperature of the strain ASB 107 is 0-37 °C in TSB (tryptic soy broth). This study is the first report on the psychrotrophic radio-resistant bacteria belonging to the Kocuria genus isolated from Ab-e-Siah spring.

Development of an immunoaffinity method for purification of streptokinase.

BACKGROUND: Streptokinase is a potent activator of plasminogen to plasmin, the enzyme that can solubilize the fibrin network in blood clots. Streptokinase is currently used in clinical medicine as a thrombolytic agent. It is naturally secreted by ß-hemolytic streptococci. METHODS: To reach an efficient method of purification, an immunoaffinity chromatography method was developed that could purify the streptokinase in a single step with high yield. At the first stage, a CNBr-Activated sepharose 4B-Lysine column was made to purify the human blood plasminogen. The purified plasminogen was utilized to construct a column that could purify the streptokinase. The rabbit was immunized with the purified streptokinase and the anti-streptokinase (IgG) purified on another streptokinase substituted sepharose-4B column. The immunoaffinity column was developed by coupling the purified anti-Streptokinase (IgG) to sepharose 6MB-Protein A. The Escherichia coli (E.coli) BL21 (DE3) pLysS strain was transformed by the recombinant construct (cloned streptokinase gene in pGEX-4T-2 vector) and gene expression was induced by IPTG. The expressed protein was purified by immunoaffinity chromatography in a single step. RESULTS: The immunoaffinity column could purify the recombinant fusion GST-SK to homogeneity. The purity of streptokinase was confirmed by SDS-PAGE as a single band of about 71 kD and its biological activity determined in a specific streptokinase assay. The yield of the purification was about 94%. CONCLUSION: This method of streptokinase purification is superior to the previous conventional methods.