Article Figures & Data
Figures
- FIG. 1.
Molecular characterization of BphP3 B BTAi1. (A) Comparative genomic analysis of the region surrounding the pucBA operon and BphP3 B BTAi1 (BrBphP3) in the Bradyrhizobium BTAi1 and ORS278 strains and in R. palustris CGA009. The values given between the genes correspond to the percentages of identity of the corresponding proteins. The arrows indicate the localization of the primers used to search for pucBA and BphP genes in different photosynthetic bradyrhizobia. Abbreviations: TF, transcriptional factor; HP, hypothetical protein. (B) Predicted domain structure of BphP3B BTAi1 (BrBphP3.BTAi1) and TFBTAi1 (TF.BTAi1). HK, histidine kinase domain; HisKa, phosphoacceptor domain; HATPase, ATP binding domain; RR, response regulator domain; HTH, helix-turn-helix domain. (C) Phylogenetic analysis of the BphP family based on an alignment of the GAF domain. The sequences were aligned by using the CLUSTALX software program, and the tree was generated by the neighbor-joining method and displayed using the NJPLOT software program. Bootstrap values, expressed as percentages of 1,000 replications, are given at the branching points. The end points of the GAF domain of each sequence were determined by Pfam analysis (1). Species abbreviations: At, Agrobacterium tumefaciens; Ath, Arabidospsis thaliana; Br, Bradyrhizobium sp.; Dr, Deinococcus radiodurans; Pa, Pseudomonas aeruginosa; Pf, Pseudomonas fluorescens; Pp, Pseudomonas putida; Ps, Pseudomonas syringae; Rc, Rhodospirillum centenum; Rl, Rhizobium leguminosarum; Rp, R. palustris; Rr, Rhodospirillum rubrum; Rs, Rhodobacter sphaeroides; Xa, Xanthomonas axonopodis; Xc, Xanthomonas campestris.
- FIG. 2.
Spectral characterization of the His-tagged BphP3B BTAi1 recombinant protein. Absorption spectra of purified BphP3B BTAi1 are as follows: spectrum a (brown line), recorded after 30 min of dark adaptation following a 770-nm preillumination; spectrum b (red line), after 705-nm illumination; spectrum c (blue line), after 2 min of dark after 705-nm illumination; spectrum d (black line), after 30 min of dark after 705-nm illumination.
- FIG. 3.
BphP3B BTAi1 acts as a light-regulated histidine kinase. (A) Effect of light conditions on kinase activity of BphP3B BTAi1. The chromoprotein was converted preferentially to its Pfr (a) or Pr (b) forms. The maximal amount of the Pfr form was obtained by 15 min of preillumination at 705 nm, followed by 15 min of dark adaptation, and that of the Pr form by illumination at 770 nm. Proteins were incubated with [γ-32P]ATP for 15 min. The reaction products were separated by SDS-PAGE, and the gel was subjected to autoradiography (top) or stained by Coomassie blue (bottom). (B) Effect of redox conditions on kinase activity of BphP3B BTAi1. For the redox effect, the sample was subjected to the following conditions: a 15 min preillumination with 705-nm light, followed by a 15-min dark adaptation; DTT (1 mM) (a) or ferricyanide (1 mM) (b) was used. (C) Phosphotransfer between BphP3B BTAi1 (BrBphP3) and the Rpa1489 recombinant protein. BphP3B BTAi1 was preferentially placed in its Pfr form by a 705-nm illumination followed by 15 min of dark adaptation. Lane a, BphP3B BTAi1 alone; lane b, BphP3B BTAi1 plus Rpa1489. (D) Sequence analysis of the pucBA promoter region of BTAi1 showing the presence of Rpa1489 and PpsR binding sites.
- FIG. 4.
BphP3B BTAi1 controls the peripheral LH synthesis in Bradyrhizobium BTAi1. (A) Absorption spectra of Bradyrhizobium BTAi1 cells (WT) grown under semiaerobic conditions, subjected to illumination at various wavelengths (648 nm, green line; 732 nm, red line; 769 nm, blue line). (B) Wavelength dependence of the synthesis of RC/LH1 (red line) or of the peripheral LH complexes (green line) or of the expression of a pucBA-lacZ fusion (blue line). (C) Absorption spectra of Bradyrhizobium BTAi1 cells (WT) grown under 770-nm illumination (green line) or under 700- plus 770-nm light (blue line). (D) Absorption spectra of Bradyrhizobium strain BTAi1 (ΔBphP3B) cells (deletion mutant) grown under 770-nm illumination (green line) or under 700- plus 770-nm lights (blue line).
- FIG. 5.
Absorption and fluorescence emission spectra of intact cells of Bradyrhizobium BTAi1 grown under two different light conditions. (A) Low-temperature (77 K) absorption spectra of intact cells of Bradyrhizobium BTAi1 grown under 770-nm light (blue) or 700- plus 770-nm lights (red). The absorption spectra have been normalized to the same concentration of RCs. (B) Absorption spectra (continuous lines) and emission spectra (dashed lines), recorded at room temperature, of cells grown under 770-nm light or 700- plus 770-nm light are in blue and red, respectively. Recording of the fluorescence emission spectra was performed under 380-nm excitation. The absorption and fluorescence spectra have been normalized to the same concentration of RCs.
