Fig 1.
Survival of microaerobic cultures to GSNO exposure.
a. Growth curves of P. extremaustralis in microaerobiosis with different GSNO concentrations. Values represent the mean ± SD of 6 independent experiments. * denotes significant differences using 1-way ANOVA for each time point (P < 0.05) b. Cultures were exposed to GSNO during 1h and survival in LB plates was determined. Circles represent individual values of independent experiments. Error bars represent the standard deviation of the mean. Different letters denote significant differences using 1-way ANOVA with Tukey’s multiple comparisons test (P < 0.05).
Fig 2.
Effect of GSNO on gene expression profile.
a. Classification of differentially expressed genes (P<0.05) after GSNO exposure in microaerobiosis (m-NS vs. m-C) into functional categories. Hypothetical transcripts (with no inferred function) are not displayed. Green and red bars represent up- and down-regulated genes, respectively. b. Over-expressed genes related to iron metabolism. c. Nitrosylable proteins predicted with Target Pathogen in the differential expressed genes compared to total nytrosilable proteins in P. extremaustralis genome. DE: Differentially expressed genes. Fisher’s exact test, P = 0.0323.
Fig 3.
Metabolic pathway associated with inositol catabolism in Pseudomonas extremaustralis 14-3b: Expression under nitrosative stress and genetic organization.
a. Gene expression and metabolic route related to inositol catabolism under m-NS Genes and arrows shown in green indicate up-regulated functions under m-NS, whereas those in blue represent functions with no expression differences between both conditions. b. Analysis of genes involved in inositol in P. extremaustralis in comparison with P. protegens Pf-5 and P. syringae pv. syringae B728a. (*) The same genetic organization was observed in USBA 515, DSM17835T, 2E-UNGS, CSW01,1906 and NQ5 P. extremaustralis strains. Compounds: KMI, 2-keto-myo-inositol; THcHDO, 3, 3D-(3,4/5) trihydroxycyclohexane-1,2-dione; 5DG, 5-deoxy glucuronic acid; DKG, 2-deoxy-5-keto-d-gluconic acid; DKGP, DKG 6-phosphate; DHAP, dihydroxyacetone phosphate; MSA, malonic semialdehyde; acetyl-CoA, acetyl coenzyme A; DHAP, dihydroxyacetone phosphate; G3P, glyceraldeyde-3-phosphate; TCA, tricarboxylic acid cycle. Genes: iolG1, myo-inositol dehydrogenase; iolG2, scyllo-inositol dehydrogenase; iolE, 2KMI dehydratase; iolD, THcHDO hydrolase; iolB, 5DG isomerase; iolC/J, DKG kinase + aldolase; iolA, MSA dehydrogenase; iatA, sugar ABC transporter ATP-binding protein; iatP, ABC transporter permease; ibpA, sugar ABC transporter substrate-binding protein.
Fig 4.
Physiological features of P. extremaustralis using myo-inositol o glucose as carbon source.
a. Photograph of microaerobic myo-inositol or glucose supplemented cultures showing the biofilm in the surface. b. Microaerobic growth measured by optical density (OD600nm) or Cellular dry weight (DW). c. Biofilm formation index (ACV/APC) in myo-inositol or glucose. d. Effect of carbon sources on pyoverdine production. RFU: Relative Fluorescence Units. Fluorescence units were normalized to the cell dry weight/ml. Error bars represent the standard deviation of the mean. In all cases asterisks (*) indicate significant differences (P<0.05) using an Unpaired t- test.
Fig 5.
Nitrosative and combined nitro-oxidative stress resistance in P. extremaustralis.
a. Survival after GSNO exposure. b. Nitrite production in cultures supplemented with myo-inositol or glucose and different nitrate concentrations. c. Inhibition halos of cultures performed with myo-inositol or glucose at different nitrate concentrations exposed to H2O2 (Combined nitro-oxidative stress). Error bars represent the standard deviation of the mean. In all cases, different letters represent significative differences (P<0.05) using a 2-Way ANOVA with Tukey’s multiple comparisons test.