R nonmethylotrophic bacteria containing XoxF homologues.Figure 2. MDH activity in strain

R nonmethylotrophic bacteria containing XoxF homologues.Figure 2. MDH activity in strain AM1 grown on the methanol and succinate media with Ca2+ and/or La3+. Cells were grown aerobically in succinate (white bar) and methanol (gray bar) media with 30 mM La3+ and/or Ca2+ at 30uC. Results are shown as means with standard deviations (n = 3). doi:10.1371/JI 101 journal.pone.0050480.gFigure 3. Expression of mxaF-xylE and xoxF1-xylE promoter fusions in M. extorquens strain AM1. The mxaF and xoxF1 promoter activities were determined by measuring catechol 2,3-dioxygenase activity (XylE) in crude cell extracts of strains grown in methanol (black bar) or succinate (white bar) media containing 30 mM La3+ and/or Ca2+. Results are shown as means with standard deviations (n = 3). doi:10.1371/journal.pone.0050480.gXoxF1 Is La3+-Dependent MDHFigure 4. SDS AGE analysis (A) and molecular weight (B) of purified MDH from strain AM1 grown on methanol/Ca2++La3+ medium. A, Lane 1, marker proteins; lane 2, purified MDH. B, Marker proteins were: 1, ovoalbumin (43 kDa); 2, conalbumin (75 kDa); 3, aldolase (158 kDa); and 4, ferritin (440 kDa). doi:10.1371/journal.pone.0050480.gIt has been reported that XoxF1 purified from strain DmxaF harboring the pCM80-xoxF-His vector grown on Ca2+-containing medium exhibits low MDH activity (Vmax value for methanol is 0.015 U/mg) [21]. La3+-dependent XoxF1, however, exhibited significant specific activity for methanol (10.0 U/mg), with levels over fifteen times higher than those in purified Ca2+-Avasimibe induced MxaFI from cells grown on Ca2+-containing medium (0.66 U/mg) (data not shown). Strain DmxaF grown in the methanol/Ca2+ medium had little MDH activity despite high expression levels of the xoxF1 gene (Fig. 3). Moreover, Ca2+-induced XoxF1 was a monomer [21], while La3+-containing XoxF1 was a homo-dimer of a-subunits only (Fig. 4). Thus it can be hypothesized that La3+ can facilitate the dimerization of XoxF1 protein, which in the absence of La3+ is an inactive monomeric apo-enzyme. Similarly, since there was no fraction showing any MDH activity except for that containing XoxF1, MxaFI may be inactive in cells grown in the presence of La3+, although their hetero-tetramerization has not been examined (data not shown). It has been reported that Ca2+dependent enzymes and other proteins, e.g., horseradish peroxidase, might be inhibited by La3+ [27?0]. Thus we hypothesize that La3+ may inhibit MxaFI posttranslational activation and/or its activity. Taken together, our work suggests that the posttranslational activation of XoxF1 and that of MxaFI require La3+ and Ca2+, respectively, and that strain AM1 has the ability to generate MDH, either XoxF1 or MxaF, depending on which metal is present, for methanol 23977191 metabolism. La3+ is one of the REEs, which are relatively abundant in the earth’s crust (35 mg/g for La3+, 66 mg/g for Ce3+, and 40 mg/g for Nd3+); in fact, the abundance of Ce3+ is almost equal to those of much more commonly studied elements in the environment, such as Cu and Zn [26]. La3+ exists in all plants examined, with levels ofaround 0.178?.1 mg/g dry mass in leaves, which is in the same range as Mn (0.5?5.6 mg/g dry mass) and Fe (1.33?.5 mg/g dry mass) [25]. Meanwhile, Delmotte et al. have reported that XoxF is highly expressed in bacterial phyllosphere communities in situ, with a prevalence similar to that of MxaF as demonstrated by shotgun proteomics [1]. Therefore, the Methylobacterium species, as major plant epiphytes, would be re.R nonmethylotrophic bacteria containing XoxF homologues.Figure 2. MDH activity in strain AM1 grown on the methanol and succinate media with Ca2+ and/or La3+. Cells were grown aerobically in succinate (white bar) and methanol (gray bar) media with 30 mM La3+ and/or Ca2+ at 30uC. Results are shown as means with standard deviations (n = 3). doi:10.1371/journal.pone.0050480.gFigure 3. Expression of mxaF-xylE and xoxF1-xylE promoter fusions in M. extorquens strain AM1. The mxaF and xoxF1 promoter activities were determined by measuring catechol 2,3-dioxygenase activity (XylE) in crude cell extracts of strains grown in methanol (black bar) or succinate (white bar) media containing 30 mM La3+ and/or Ca2+. Results are shown as means with standard deviations (n = 3). doi:10.1371/journal.pone.0050480.gXoxF1 Is La3+-Dependent MDHFigure 4. SDS AGE analysis (A) and molecular weight (B) of purified MDH from strain AM1 grown on methanol/Ca2++La3+ medium. A, Lane 1, marker proteins; lane 2, purified MDH. B, Marker proteins were: 1, ovoalbumin (43 kDa); 2, conalbumin (75 kDa); 3, aldolase (158 kDa); and 4, ferritin (440 kDa). doi:10.1371/journal.pone.0050480.gIt has been reported that XoxF1 purified from strain DmxaF harboring the pCM80-xoxF-His vector grown on Ca2+-containing medium exhibits low MDH activity (Vmax value for methanol is 0.015 U/mg) [21]. La3+-dependent XoxF1, however, exhibited significant specific activity for methanol (10.0 U/mg), with levels over fifteen times higher than those in purified Ca2+-induced MxaFI from cells grown on Ca2+-containing medium (0.66 U/mg) (data not shown). Strain DmxaF grown in the methanol/Ca2+ medium had little MDH activity despite high expression levels of the xoxF1 gene (Fig. 3). Moreover, Ca2+-induced XoxF1 was a monomer [21], while La3+-containing XoxF1 was a homo-dimer of a-subunits only (Fig. 4). Thus it can be hypothesized that La3+ can facilitate the dimerization of XoxF1 protein, which in the absence of La3+ is an inactive monomeric apo-enzyme. Similarly, since there was no fraction showing any MDH activity except for that containing XoxF1, MxaFI may be inactive in cells grown in the presence of La3+, although their hetero-tetramerization has not been examined (data not shown). It has been reported that Ca2+dependent enzymes and other proteins, e.g., horseradish peroxidase, might be inhibited by La3+ [27?0]. Thus we hypothesize that La3+ may inhibit MxaFI posttranslational activation and/or its activity. Taken together, our work suggests that the posttranslational activation of XoxF1 and that of MxaFI require La3+ and Ca2+, respectively, and that strain AM1 has the ability to generate MDH, either XoxF1 or MxaF, depending on which metal is present, for methanol 23977191 metabolism. La3+ is one of the REEs, which are relatively abundant in the earth’s crust (35 mg/g for La3+, 66 mg/g for Ce3+, and 40 mg/g for Nd3+); in fact, the abundance of Ce3+ is almost equal to those of much more commonly studied elements in the environment, such as Cu and Zn [26]. La3+ exists in all plants examined, with levels ofaround 0.178?.1 mg/g dry mass in leaves, which is in the same range as Mn (0.5?5.6 mg/g dry mass) and Fe (1.33?.5 mg/g dry mass) [25]. Meanwhile, Delmotte et al. have reported that XoxF is highly expressed in bacterial phyllosphere communities in situ, with a prevalence similar to that of MxaF as demonstrated by shotgun proteomics [1]. Therefore, the Methylobacterium species, as major plant epiphytes, would be re.