KatG and KatB localization and their <i>oxyR</i> growth promoting activity.

<p><b>A–D. </b><i>V. cholerae</i> containing either P<i>_BAD</i>-<i>katG-flag</i> (<b>A, C</b>) or P<i>_BAD</i>-<i>katB-flag</i> (<b>B, D</b>) were grown on LB agar plates containing 0.1% arabinose. Western-blot analysis to detect FLAG-tagged KatG and KatB as well as a cytoplasmic protein control HapR in the cell pellets (from 25 µl of OD₆₀₀ = 3.0 cultures) and culture supernatants (concentrated from 1 ml of OD₆₀₀ = 3.0 cultures). Anti-FLAG antibody and anti-HapR antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053383#pone.0053383-Liu1" target="_blank">[20]</a> were used. <b>E.</b> Purified KatG and KatB restored the <i>oxyR</i> growth defect. Overnight cultures of <i>oxyR</i> mutants were inoculated 1∶1000 into fresh LB containing the concentrations of purified KatG-His₆ or KatB-His₆ indicated and shaken at 37°C. OD₆₀₀ was measured after 8-hr incubation and compared to wildtype growth.</p

The effect of co-culturing catalase-positive and catalase-negative cells on <i>oxyR</i> growth in different environments.

<p><b>A.</b> Overnight cultures of wildtype (circles), <i>katG-katB</i> (triangles), and <i>oxyR</i> mutants (squares) were inoculated alone or together at 1∶1000 into fresh LB medium and shaken at 37°C for 8 hrs. CFU of live cells were then determined by serial dilution and plating on LB agar plates containing appropriate antibiotics and 0.5 µM purified KatG. A spontaneous rifamycin-resistant <i>oxyR</i> mutant was used to facilitate selection. Each symbol represents CFU from one culture. <b>B.</b> Approximately 10⁶ single or mixed cells indicated were intragastrically inoculated into 6-day-old CD-1 mice. After 18-hr incubation, bacterial numbers colonized in small intestines were determined as described in <b>A.</b> The data shown are from three independent experiments and each symbol represents CFU recovered from one mouse intestine. <b>C and D.</b> 3-month old zebrafish were placed in 1% NaCl water containing approximately 10⁴/ml of bacterial cells indicated for 24 hrs. The zebrafish intestines were removed after surface sterilization by 70% ethanol and the colonization of fish intestines was determined as described in A and reported in <b>C.</b> The number of bacteria in the salt water was determined as described in A and reported in <b>D.</b> The data shown are from three independent experiments and each symbol represents CFU recovered from one fish intestine (C) or recovered from one container of salt water containing zebrafish (D).</p

Aerobic growth and H₂O₂ resistance of wildtype, <i>oxyR</i>, and catalase mutants.

<p><b>A.</b> Overnight cultures of <i>V. cholerae</i> strains were inoculated at 1∶1000 into fresh LB and shaken at 37°C for 6 hrs. Viable cells were counted by serial dilution and plating on LB agar plates containing 1/10 (v/v) cell-free supernatants prepared from wildtype cultures. <b>B.</b> Disc diffusion plate assays. Approximately 10⁸ bacterial cells were mixed with top LB agar and discs saturated with 6 M H₂O₂ were placed in the middle. The plates were incubated at 37°C for 8 hrs and the diameter of the inhibition zone was measured for each strain. Data are mean and s.d. of three independent experiments.</p

The expression and production of catalase.

<p><b>A.</b> Overnight cultures of wildtype or <i>oxyR</i> mutants containing promoter-<i>luxCDABE</i> transcriptional fusion plasmids were inoculated at 1∶20 into fresh LB containing appropriate antibiotics and shaken at 37°C until mid-log phase. When indicated, additional H₂O₂ (50 µM) was added and all cultures were incubated for 1 hr. Luminescence was then measured and reported as light units/OD₆₀₀. <b>B.</b> Overnight cultures of wildtype, <i>oxyR,</i> and <i>katG/katB</i> mutants were inoculated at 1∶20 into fresh LB containing appropriate antibiotics and shaken at 37°C until mid-log phase. When indicated, additional H₂O₂ (100 µM) was added and all cultures were incubated for 1 hr. The cell lysates were then subjected to a catalase activity assay. Data are mean and s.d. of three independent experiments.</p

Growth of <i>oxyR</i> mutants in the absence and in the presence of stationary-phase cultural supernatants.

<p><b>A.</b> Wildtype, <i>oxyR</i> mutants, and <i>oxyR</i> (P<i>_BAD-oxyR</i>) grown on LB agar plates containing 0.01% arabinose without and with 1/10 (v/v) cell-free supernatants (see Methods for preparation) after overnight incubation at 37°C. <b>B. and C.</b> Wildtype, <i>oxyR</i> mutants, and <i>oxyR</i> (P<i>_BAD-oxyR</i>) grown in LB liquid (<b>B</b>) and M9 minimal medium (<b>C</b>). Overnight cultures were inoculated 1∶1000 into fresh LB or M9-glucose medium containing 0.01% arabinose without and with 1/10 (v/v) cell-free supernatants and shaken at 37°C. OD₆₀₀ was measured at the time points indicated. Data are mean and s.d. of three independent experiments.</p

Additional file 1 of GhIMP10D, an inositol monophosphates family gene, enhances ascorbic acid and antioxidant enzyme activities to confer alkaline tolerance in Gossypium hirsutum L.

Additional file 1: Supplementary Table S1. Gene locus ID and their proposed names of all observed species and the gene characteristics in G. hirsutum. Supplementary Table S2. Duplicated gene pairs in 10 combinations (Ga-Ga, Ga-Gb, Ga-Gr, Gb-Gb, Gb-Gr, Gh-Gh, Gh-Ga, Gh-Gb, Gh-Gr and Gr-Gr). Supplementary Table S3. Non-synonymous (Ka) and synonymous (Ks) divergence values for Ga-Ga, Ga-Gb, Ga-Gr, Gb-Gb, Gb-Gr, Gh-Gh, Gh-Ga, Gh-Gb, Gh-Gr and Gr-Gr. Supplementary Table S4. Primer pairs used for this experiment