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Investigating the toxic effects of nonylphenol on Saccharomyces cerevisiae
Abstracts / New Biotechnology 33S (2016) S1–S213
genes encoding for a-amylase and -glucosidase which related to carbohydrate catabolism, inhibit expression of genes encoding for starch synthase and starch branching enzyme, up-regulate genes related to lipid synthesis, resulting in an increase of lipid content. The results supported metabolite results indicated that under phosphorus-limiting condition, carbon is partitioned toward lipid production from carbohydrate. The transcriptome provided an unprecedented opportunity to discover all related-genes associated with lipid metabolic pathway in Scenedesmus sp., which will be useful for engineering economic algae species aimed at biodiesel production. http://dx.doi.org/10.1016/j.nbt.2016.06.1399
P33-5 Artificial CRISPR cassette for studying type I-C CRISPR-Cas system in Porphyromonas gingivalis Michal Burmistrz ∗ , Jose Ignacio Rodriguez Martinez, Krzysztof Pyrc
S197
investigated this response in P. putida in more detail. In this study, we sought to identify the components of the SOS response that links exposure to the fluoroquinolone antibiotic norfloxacin with the transcriptional regulation of lexA and recA expression in P. putida and E. coli. To this end, a standardized test to quantify SOS expression in individual bacterial cells was developed by assembling a promoter-gfp reporter system in a vector plasmid. Also, P. putida lexA and recA null mutants and their derivatives complemented with E. coli orthologous genes were constructed. Then, the PrecA and PlexA promoters from E. coli and P. putida were tested in these strains for response to norfloxacin. Expression of the recA and lexA genes under antibiotic treatment using the thereby constructed reporter fusions was analyzed in single cells by flow cytometry. Based on fluorescence intensity, the induction of RecA by DNA damage in P. putida was markedly lower than E. coli. The different response to DNA damage between the two bacterial species may explain their very dissimilar abilities to bring about homologous DNA recombination. In addition, it is plausible that DNA damage could be quenched by the background metabolism of P. putida, prone to counteract the mutagenic effects of reactive oxygen species. http://dx.doi.org/10.1016/j.nbt.2016.06.1401
Jagiellonian University, Poland The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) is an unique system that provides prokaryotic organisms with hereditary and adaptive immunity against foreign nucleic acids. This system is widely spread among Bacteria and Archea. Porphyromonas gingivalis is a human pathogen causing periodontitis, which is also linked with rheumatoid arthritis, and cardiovascular disease. It’s genome contains four CRISPR arrays and two cas gene operons of type III-B and I-C. Recently, we showed that the type I-C CRISPR-Cas system of P. gingivalis is functional and confirmed that Protospacer Adjecent Motif (PAM) element is required for its activity. To characterize regulatory elements of this CRISPR-Cas it was necessary to introduce multiple different, yet specific mutations into this genomic region. To facilitate the process we designed a plasmidbased delivery system, which contains artificial, easily modifiable CRISPR array, as well as flanking regions enabling replacement of original array with artificial one using homologous recombination. Obtained mutants were selected against erythromycin and verified with genomic DNA sequencing. Functional assay revealed that substituted CRISPR array is incorporated by CRISPR-Cas machinery. It is witnessed by production of functional crRNA for spacers of both natural and artificial origin and in vivo degradation of plasmids containing protospacers with correct PAM sequence. http://dx.doi.org/10.1016/j.nbt.2016.06.1400
P33-6 DNA damage elicits a different SOS response in Pseudomonas putida and Escherichia coli Özlem Akkaya 1,∗ , Pablo Ivan Nikel 2 , Danilo Pérez-Pantoja 2 , Víctor de Lorenzo 2 1
Gebze Technical University; Centro Nacional de Biotecnología (CNB-CSIC), Turkey 2 Centro Nacional de Biotecnología (CNB-CSIC), Spain The details of the SOS system have been worked out from studies of Escherichia coli, the SOS response of the Pseudomonas putida is presently not well understood. Given the potential of this species as a host for redox reactions of biotechnological interest, we have
P33-7 Investigating the toxic effects of nonylphenol on Saccharomyces cerevisiae Bulent Mertoglu ∗ , Kazim Yalcin Arga Marmara University, Turkey Various chemicals released continuously into the environment have a growing danger to human and wildlife health. Nonylphenol (NP), an endocrine disrupting chemical, is largely used in various household and industrial applications, formulation of pharmacological and agricultural drugs and production of textile and paper pulp. Recently, NP was detected in adipose tissue, breast milk, human blood and serum, seafood, and baby foods. Therefore, analysis and understanding the possible toxic effects of such chemical are of vital importance to identify and prevent its adverse effects on ecological system. In the present study, it was aimed to investigate toxic effects of NP on Saccharomyces cerevisiae, a widely used model organism for toxic assessments. Within this scope, yeast cells were cultivated on yeast extract-peptone-glucose (YPD) at 30 ◦ C and 180 rpm to optical density (OD600 ) of 0.8 ± 0.05 (mid-exponential phase). Cultures were exposed to various concentrations of NP for 3 h. To determine the inhibition rates, cell viability was determined with 0.01% methylene blue-2% sodium citrate solution using hemocytometer. It was found that NP has different inhibition rates on yeast cells depending on the concentration used. It was observed that the maximum sub-lethal dose of NP is 5 mg/L with 68.4% inhibition rate, while the minimum inhibition concentration is 1 mg/L NP with 11.5% inhibition rate. In conclusion, these results suggest that yeast cells may respond to different concentrations of NP via distinct mechanisms at the molecular level. Upon obtained data, future study is planned to investigate the effects of NP at transcriptome level using RNA-sequencing method. http://dx.doi.org/10.1016/j.nbt.2016.06.1402
genes encoding for a-amylase and -glucosidase which related to carbohydrate catabolism, inhibit expression of genes encoding for starch synthase and starch branching enzyme, up-regulate genes related to lipid synthesis, resulting in an increase of lipid content. The results supported metabolite results indicated that under phosphorus-limiting condition, carbon is partitioned toward lipid production from carbohydrate. The transcriptome provided an unprecedented opportunity to discover all related-genes associated with lipid metabolic pathway in Scenedesmus sp., which will be useful for engineering economic algae species aimed at biodiesel production. http://dx.doi.org/10.1016/j.nbt.2016.06.1399
P33-5 Artificial CRISPR cassette for studying type I-C CRISPR-Cas system in Porphyromonas gingivalis Michal Burmistrz ∗ , Jose Ignacio Rodriguez Martinez, Krzysztof Pyrc
S197
investigated this response in P. putida in more detail. In this study, we sought to identify the components of the SOS response that links exposure to the fluoroquinolone antibiotic norfloxacin with the transcriptional regulation of lexA and recA expression in P. putida and E. coli. To this end, a standardized test to quantify SOS expression in individual bacterial cells was developed by assembling a promoter-gfp reporter system in a vector plasmid. Also, P. putida lexA and recA null mutants and their derivatives complemented with E. coli orthologous genes were constructed. Then, the PrecA and PlexA promoters from E. coli and P. putida were tested in these strains for response to norfloxacin. Expression of the recA and lexA genes under antibiotic treatment using the thereby constructed reporter fusions was analyzed in single cells by flow cytometry. Based on fluorescence intensity, the induction of RecA by DNA damage in P. putida was markedly lower than E. coli. The different response to DNA damage between the two bacterial species may explain their very dissimilar abilities to bring about homologous DNA recombination. In addition, it is plausible that DNA damage could be quenched by the background metabolism of P. putida, prone to counteract the mutagenic effects of reactive oxygen species. http://dx.doi.org/10.1016/j.nbt.2016.06.1401
Jagiellonian University, Poland The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) is an unique system that provides prokaryotic organisms with hereditary and adaptive immunity against foreign nucleic acids. This system is widely spread among Bacteria and Archea. Porphyromonas gingivalis is a human pathogen causing periodontitis, which is also linked with rheumatoid arthritis, and cardiovascular disease. It’s genome contains four CRISPR arrays and two cas gene operons of type III-B and I-C. Recently, we showed that the type I-C CRISPR-Cas system of P. gingivalis is functional and confirmed that Protospacer Adjecent Motif (PAM) element is required for its activity. To characterize regulatory elements of this CRISPR-Cas it was necessary to introduce multiple different, yet specific mutations into this genomic region. To facilitate the process we designed a plasmidbased delivery system, which contains artificial, easily modifiable CRISPR array, as well as flanking regions enabling replacement of original array with artificial one using homologous recombination. Obtained mutants were selected against erythromycin and verified with genomic DNA sequencing. Functional assay revealed that substituted CRISPR array is incorporated by CRISPR-Cas machinery. It is witnessed by production of functional crRNA for spacers of both natural and artificial origin and in vivo degradation of plasmids containing protospacers with correct PAM sequence. http://dx.doi.org/10.1016/j.nbt.2016.06.1400
P33-6 DNA damage elicits a different SOS response in Pseudomonas putida and Escherichia coli Özlem Akkaya 1,∗ , Pablo Ivan Nikel 2 , Danilo Pérez-Pantoja 2 , Víctor de Lorenzo 2 1
Gebze Technical University; Centro Nacional de Biotecnología (CNB-CSIC), Turkey 2 Centro Nacional de Biotecnología (CNB-CSIC), Spain The details of the SOS system have been worked out from studies of Escherichia coli, the SOS response of the Pseudomonas putida is presently not well understood. Given the potential of this species as a host for redox reactions of biotechnological interest, we have
P33-7 Investigating the toxic effects of nonylphenol on Saccharomyces cerevisiae Bulent Mertoglu ∗ , Kazim Yalcin Arga Marmara University, Turkey Various chemicals released continuously into the environment have a growing danger to human and wildlife health. Nonylphenol (NP), an endocrine disrupting chemical, is largely used in various household and industrial applications, formulation of pharmacological and agricultural drugs and production of textile and paper pulp. Recently, NP was detected in adipose tissue, breast milk, human blood and serum, seafood, and baby foods. Therefore, analysis and understanding the possible toxic effects of such chemical are of vital importance to identify and prevent its adverse effects on ecological system. In the present study, it was aimed to investigate toxic effects of NP on Saccharomyces cerevisiae, a widely used model organism for toxic assessments. Within this scope, yeast cells were cultivated on yeast extract-peptone-glucose (YPD) at 30 ◦ C and 180 rpm to optical density (OD600 ) of 0.8 ± 0.05 (mid-exponential phase). Cultures were exposed to various concentrations of NP for 3 h. To determine the inhibition rates, cell viability was determined with 0.01% methylene blue-2% sodium citrate solution using hemocytometer. It was found that NP has different inhibition rates on yeast cells depending on the concentration used. It was observed that the maximum sub-lethal dose of NP is 5 mg/L with 68.4% inhibition rate, while the minimum inhibition concentration is 1 mg/L NP with 11.5% inhibition rate. In conclusion, these results suggest that yeast cells may respond to different concentrations of NP via distinct mechanisms at the molecular level. Upon obtained data, future study is planned to investigate the effects of NP at transcriptome level using RNA-sequencing method. http://dx.doi.org/10.1016/j.nbt.2016.06.1402