Journal: PLoS ONE

Article Title: Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

doi: 10.1371/journal.pone.0027472

Figure Lengend Snippet: (A) Growth (··•··), catalase activity (–Δ–) and pimaricin production (−▪−) of S. natalensis ATCC 27448 in YEME medium. Growth phases are indicated by solid lines at the top of the graph. Vertical bars indicate standard deviation of the mean values. Data are the average of triplicates from three independent experiments. (B)Native PAGE of cell extracts (30 µg protein per lane) from S. natalensis stained for catalase activity. S. natalensis was grown in YEME medium and samples were collected during the defined four growth phases (see ): RG1, RG2, S/P and S/NP. Arrows show the two detectable catalase activity bands. (C) Native PAGE stained for catalase activity of cell extracts from S. natalensis ATCC 27448 cultures collected during RG1 phase after 1 mM H 2 O 2 insult (+).

Article Snippet: In this work we addressed the role of ROS homeostasis, in particular intracellular H 2 O 2 levels, and of the adaptive response triggered by H 2 O 2 , on the production of pimaricin by S. natalensis ATCC 27448.

Techniques: Activity Assay, Standard Deviation, Clear Native PAGE, Staining

Journal: PLoS ONE

Article Title: Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

doi: 10.1371/journal.pone.0027472

Figure Lengend Snippet: Transcription profiles of the monofuncional catalase ( katA1 ) and alkyl hydroperoxide reductase ( ahpC ) encoding genes and their transcriptional regulators, catR and oxyR , in S. natalensis ATCC 27448 upon a H 2 O 2 insult. The transcription of ahpC, oxyR , katA1 and catR was evaluated by RT-qPCR from S. natalensis grown in YEME medium and samples collected 30 min after 1 mM H 2 O 2 treatment (+) or an equal volume of water as the untreated control (-).H 2 O 2 treatments were applied independently during the early exponential phase (RG1) and late exponential phase (RG2). The Mean Normalized Fold Expression (±standard errors) of the target genes was calculated relative to the transcription of the reference genes (16 S rDNA and lysA ) and the reaction internal normalization was performed using the sample from cells collected immediately before H 2 O 2 addition to the culture broth (not shown). Results (average of triplicates and standard deviation) are representative of three independent experiments.

Article Snippet: In this work we addressed the role of ROS homeostasis, in particular intracellular H 2 O 2 levels, and of the adaptive response triggered by H 2 O 2 , on the production of pimaricin by S. natalensis ATCC 27448.

Techniques: Quantitative RT-PCR, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

doi: 10.1371/journal.pone.0027472

Figure Lengend Snippet: S. natalensis ATCC 27448 was grown in iron-supplemented YEME medium, and pimaricin specific yield (per mg of total protein) measured at 72 h. 1 mM (final concentration) H 2 O 2 was added to the culture broth either at the RG1, RG2 or S/P phase. Data are the means from three independent experiments. To assess the presence of significant differences between the tested growth phases, a one-way ANOVA was performed followed by post-hoc test (Tukey test; GraphPad Prism) in which each condition was compared to the control situation (pimaricin production without H 2 O 2 addition; 100%). *, statistically significant (P<0.01); ns, not statistically significant.

Article Snippet: In this work we addressed the role of ROS homeostasis, in particular intracellular H 2 O 2 levels, and of the adaptive response triggered by H 2 O 2 , on the production of pimaricin by S. natalensis ATCC 27448.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

doi: 10.1371/journal.pone.0027472

Figure Lengend Snippet: (A) Native PAGE of cell extracts (30 µg protein per lane) from S. natalensis ATCC 27448 (upper panel) and S. natalensis CAM.02 (lower panel) stained for SOD activity. S. natalensis strains were grown in YEME medium and cells collected on the four previously defined growth stages. NiSO 4 20 µM (final concentration) was added to the YEME medium for the induction of sodN expression. (B) Intracellular H 2 O 2 levels in S natalensis ATCC 27448 (WT) and SodF defective mutant (CAM.02) at RG2 growth phase. Values are means from two independent experiments. To assess the presence of significant differences between the tested condition, a one-way ANOVA was performed followed by post-hoc test (Tukey test; GraphPad Prism) in which each condition was compared to the control situation (wild-type strain intracellular H 2 O 2 levels in Ni non-supplemented cultures; 100%). *, statistically significant (P<0.01).

Article Snippet: In this work we addressed the role of ROS homeostasis, in particular intracellular H 2 O 2 levels, and of the adaptive response triggered by H 2 O 2 , on the production of pimaricin by S. natalensis ATCC 27448.

Techniques: Clear Native PAGE, Staining, Activity Assay, Concentration Assay, Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

doi: 10.1371/journal.pone.0027472

Figure Lengend Snippet: Strains and plasmids used in this study.

Article Snippet: In this work we addressed the role of ROS homeostasis, in particular intracellular H 2 O 2 levels, and of the adaptive response triggered by H 2 O 2 , on the production of pimaricin by S. natalensis ATCC 27448.

Techniques: Clone Assay, Conjugation Assay, Mutagenesis, Plasmid Preparation

Journal: PLoS ONE

Article Title: Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

doi: 10.1371/journal.pone.0027472

Figure Lengend Snippet: (A) Growth (··•··), catalase activity (–Δ–) and pimaricin production (−▪−) of S. natalensis ATCC 27448 in YEME medium. Growth phases are indicated by solid lines at the top of the graph. Vertical bars indicate standard deviation of the mean values. Data are the average of triplicates from three independent experiments. (B)Native PAGE of cell extracts (30 µg protein per lane) from S. natalensis stained for catalase activity. S. natalensis was grown in YEME medium and samples were collected during the defined four growth phases (see ): RG1, RG2, S/P and S/NP. Arrows show the two detectable catalase activity bands. (C) Native PAGE stained for catalase activity of cell extracts from S. natalensis ATCC 27448 cultures collected during RG1 phase after 1 mM H 2 O 2 insult (+).

Article Snippet: Once confirmed the genetic identity of the PCR products by sequencing, they were labelled with digoxigenin and used as probes for screening a S. natalensis ATCC 27448 cosmid library .

Techniques: Activity Assay, Standard Deviation, Clear Native PAGE, Staining

Journal: PLoS ONE

Article Title: Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

doi: 10.1371/journal.pone.0027472

Figure Lengend Snippet: Transcription profiles of the monofuncional catalase ( katA1 ) and alkyl hydroperoxide reductase ( ahpC ) encoding genes and their transcriptional regulators, catR and oxyR , in S. natalensis ATCC 27448 upon a H 2 O 2 insult. The transcription of ahpC, oxyR , katA1 and catR was evaluated by RT-qPCR from S. natalensis grown in YEME medium and samples collected 30 min after 1 mM H 2 O 2 treatment (+) or an equal volume of water as the untreated control (-).H 2 O 2 treatments were applied independently during the early exponential phase (RG1) and late exponential phase (RG2). The Mean Normalized Fold Expression (±standard errors) of the target genes was calculated relative to the transcription of the reference genes (16 S rDNA and lysA ) and the reaction internal normalization was performed using the sample from cells collected immediately before H 2 O 2 addition to the culture broth (not shown). Results (average of triplicates and standard deviation) are representative of three independent experiments.

Article Snippet: Once confirmed the genetic identity of the PCR products by sequencing, they were labelled with digoxigenin and used as probes for screening a S. natalensis ATCC 27448 cosmid library .

Techniques: Quantitative RT-PCR, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

doi: 10.1371/journal.pone.0027472

Figure Lengend Snippet: S. natalensis ATCC 27448 was grown in iron-supplemented YEME medium, and pimaricin specific yield (per mg of total protein) measured at 72 h. 1 mM (final concentration) H 2 O 2 was added to the culture broth either at the RG1, RG2 or S/P phase. Data are the means from three independent experiments. To assess the presence of significant differences between the tested growth phases, a one-way ANOVA was performed followed by post-hoc test (Tukey test; GraphPad Prism) in which each condition was compared to the control situation (pimaricin production without H 2 O 2 addition; 100%). *, statistically significant (P<0.01); ns, not statistically significant.

Article Snippet: Once confirmed the genetic identity of the PCR products by sequencing, they were labelled with digoxigenin and used as probes for screening a S. natalensis ATCC 27448 cosmid library .

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Crosstalk between ROS Homeostasis and Secondary Metabolism in S. natalensis ATCC 27448: Modulation of Pimaricin Production by Intracellular ROS

doi: 10.1371/journal.pone.0027472

Figure Lengend Snippet: (A) Native PAGE of cell extracts (30 µg protein per lane) from S. natalensis ATCC 27448 (upper panel) and S. natalensis CAM.02 (lower panel) stained for SOD activity. S. natalensis strains were grown in YEME medium and cells collected on the four previously defined growth stages. NiSO 4 20 µM (final concentration) was added to the YEME medium for the induction of sodN expression. (B) Intracellular H 2 O 2 levels in S natalensis ATCC 27448 (WT) and SodF defective mutant (CAM.02) at RG2 growth phase. Values are means from two independent experiments. To assess the presence of significant differences between the tested condition, a one-way ANOVA was performed followed by post-hoc test (Tukey test; GraphPad Prism) in which each condition was compared to the control situation (wild-type strain intracellular H 2 O 2 levels in Ni non-supplemented cultures; 100%). *, statistically significant (P<0.01).

Article Snippet: Once confirmed the genetic identity of the PCR products by sequencing, they were labelled with digoxigenin and used as probes for screening a S. natalensis ATCC 27448 cosmid library .

Techniques: Clear Native PAGE, Staining, Activity Assay, Concentration Assay, Expressing, Mutagenesis