Table 1.
Characteristics of the herbicides ametryn and clomazone.
Figure 1.
Maximum-parsimony phylogenetic tree constructed from the 16S rRNA gene.
A total of 600 bp nucleotide of Pseudomonas spp. from RDP database were used. Burkhloderia cariophilli and B. plantarii served as outgroup. Bootstrap values were 1000 repetitions. Bars indicate the number of evolutionary steps with diverging sequences. Strains CC07 and 4C07 are shown inside the boxes.
Figure 2.
Growth curve of bacterial strains in the presence of 0 mM (control), 25 mM ametryn, 9 mM clomazone and 20 mM of each the herbicide.
(A) Strain CC07 and (B) Strain 4C07. Values represent the means from three replicates ±SEM.
Figure 3.
Lipid peroxidation (MDA content) of bacteria exposed to the herbicides.
Values of MDA content (nmol g−1 fr. wt) represent the means from three replicates ±SEM. Means with different letters are significantly different (P<0.05) by one-way analysis of variance (ANOVA) and Tukey's test.
Figure 4.
GSH content (nmol g-1 fr. wt) of bacteria exposed to the herbicides.
Values represent the means from three replicates ±SEM. Means with different letters are significantly different (P<0.05) by one-way analysis of variance (ANOVA) and Tukey's test.
Figure 5.
SDS-PAGE protein profiles of bacteria exposed to the herbicides.
Lane 1, protein molecular mass markers (220 to 20 kDa). Lanes 2, 3, 4 and 5, strain CC07 grown in the presence of 0 mM (control), 25 mM ametryn, 9 mM clomazone and 20 mM of each the herbicide, respectively. Lanes 6, 7, 8 and 9, strain 4C07 grown in the presence of 0 mM (control), 25 mM ametryn, 9 mM clomazone and 20 mM of each the herbicide, respectively. Arrows indicate selected variations in intensity or appearance/disappearance depending on the treatment tested. I and II indicate protein bands of 225 kDa and 58 KDa that are depleted specifically in the presence of clomazone.
Figure 6.
Activity staining for SOD following non-denaturing PAGE of extracts from cultured bacterial cells.
(A) First lane is a bovine SOD standard, lanes 1, 2, 3 and 4 are strain CC07 grown in the presence of 0 mM (control), 25 mM ametryn, 9 mM clomazone and 20 mM of each herbicide, respectively. (B) Lanes 5, 6, 7 and 8 are strain 4C07 grown in the presence of 0 mM (control), 25 mM ametryn, 9 mM clomazone and 20 mM of each herbicide, respectively. Arrows indicate sequentially numbered SOD bands (I–II) that are independent of the bacterial strain. (C) Activity staining for SOD of strain CC07 (1, 2 and 3) and strain 4C07 (4, 5 and 6), used for classification of SOD isoenzymes. Lanes 1 and 4, control SOD activity. Lanes 2 and 5, SOD activity with 2 mM potassium cyanide treatment; lanes 3 and 6; 5 mM H2O2 treatment. Arrows indicate SOD bands that are sequentially numbered (I–II) according to Fig. 6A and B.
Figure 7.
Activity staining for CAT following non-denaturing PAGE of extracts from cultured bacterial cells.
Lane M is a bovine CAT standard, lanes 1, 2, 3 and 4, strain CC07 grown in the presence of 0 mM (control), 25 mM ametryn, 9 mM clomazone and 20 mM of each herbicide, respectively. Lanes 5, 6, 7 and 8, strain 4C07 grown in the presence of 0 mM (control), 25 mM ametryn, 9 mM clomazone and 20 mM of each herbicide, respectively. Arrows indicate sequentially numbered CAT bands for strains CC07 (I–III) and 4C07 (I–IV).
Figure 8.
(A) and (B) Specific activity of GR, expressed as µmol min−1 mg−1 protein. Values are the means of three replicates ±SEM. Means with different letters are significantly different (P<0.05) by one-way analysis of variance (ANOVA) and Tukey's test. (C) Activity staining for GR following non-denaturing PAGE of extracts of cultured bacterial cells. Lane M is a bovine GR standard, lanes 1, 2, 3 and 4, strain CC07 grown in the presence of 0 mM (control), 25 mM ametryn, 9 mM clomazone and 20 mM of each herbicide, respectively. Lanes 5, 6, 7 and 8, strain 4C07 grown in the presence of 0 mM (control), 25 mM ametryn, 9 mM clomazone and 20 mM of each herbicide, respectively. Arrows indicate sequentially numbered GR bands (I–VI) that are independent of the bacterial strain.
Figure 9.
GST specific activity, expressed as units mg−1 protein.
Values represent the means from three replicates ±SEM. Means with different letters are significantly different (P<0.05) by one-way analysis of variance (ANOVA) and Tukey's test.
Figure 10.
General view of the antioxidant system involving enzymatic and non-enzymatic components for the strains CC07 and 4C07 based on the data obtained in the present work.
SOD (superoxide dismutase); CAT (catalase); GST (glutathione S-transferase); GR (glutathione reductase); GSSG (oxidized glutathione); GSH (reduced glutathione); Am (ametryn); Cl (clomazone); Am+Cl (combined herbicides). Blue arrows indicate decreases in enzymatic activity and glutathione content; red arrows indicate increases in enzymatic activity and glutathione content; the absence of symbols indicates no alterations. All changes are relative to the untreated control.