Bacteriophytochrome controls carotenoid-independent response to photodynamic stress in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7

Abstract

Ever since the discovery of the role of bacteriophytochrome (BphP) in inducing carotenoid synthesis in Deinococcus radiodurans in response to light the role of BphPs in other non-photosynthetic bacteria is not clear yet. Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours a pair of BphPs out of which AbBphP1 is a homolog of AtBphP1 of Agrobacterium tumefaciens. By overexpression, purification, biochemical and spectral characterization we have shown that AbBphP1 is a photochromic bacteriophytochrome. Phenotypic study of the ΔAbBphP1 mutant showed that it is required for the survival of A. brasilense on minimal medium under red light. The mutant also showed reduced chemotaxis towards dicarboxylates and increased sensitivity to the photooxidative stress. Unlike D. radiodurans, AbBphP1 was not involved in controlling carotenoid synthesis. Proteome analysis of the ΔAbBphP1 indicated that AbBphP1 is involved in inducing a cellular response that enables A. brasilense in regenerating proteins that might be damaged due to photodynamic stress.

Figure 1

(a) Phylogeny of bacteriophytochromes from different bacteria based on amino acid sequences retrieved from NCBI (accession numbers are provided in supplementary file).Neighbor-joining tree was built in MEGA version 5.04 with a 1000 bootstrap replications and Poisson model. The two bacteriophytochromes of A. brasilense Sp7 are marked in light blue. (b) Organization of genes around bacteriophytochrome (BphP) encoding gene in A. brasilense Sp7 and other bacteria. Direction of arrow indicates the orientation of genes. The nucleotide (nt) shows the distance between overlapping (minus sign) and closely associated gene.

Figure 2. Photochromic properties and zinc fluorescence of AbBphP1 apoprotein (A) and holoprotein (H).

Visual appearance of purified apo- and holo-proteins (a), SDS-PAGE of apo- and holo-proteins (c) and fluorescence of apo- and holo-proteins in UV light after zinc staining (b).

Figure 3. Spectral characterization of recombinant AbBph1 protein.

(a) UV-Visible spectra of holoprotein in the dark and after irradiation with red (695 nm) light. (b) Slow reversion to dark state of the recombinant holoprotein after irradiation with red light. (c) Fast reversion to dark state of the recombinant holoprotein after irradiation with red light followed by far-red light irradiation.

Figure 4

Characterization of oligomeric states of AbBphP1 by size exclusion chromatography (SEC) of holoprotein (a) and apoprotein (b).FMN was used as a loading indicator, 120 ml -140 ml elution volume indicates FMN fraction. SDS-PAGE with Coomassie (left) and Zn (right) staining of glutaraldehyde (G) cross-linked SEC purified AbBphP1 holoprotein (c).

Figure 5. Absorption spectra of methanolic extracts of carotenoids showing effect of darkness and white light on carotenoid content of A. brasilense Sp7 and ΔAbBphP1 mutant.

Figure 6

(a) Growth curve of A. brasilense Sp7 and ΔAbBphP1 mutant in the dark and in red light in LB medium and minimal medium with 38 mM malate.(b) Swarm plates showing chemotaxis of A. brasilense Sp7, ΔAbBphP1 and ΔAbBphP1 (pSnK5) towards 10 mM malate in red light. (c) Epifluorescence microscopy images of A. brasilense Sp7 grown in dark (i) and red light (ii); ΔAbBphP1 mutant gown in dark (iii) and red light (iv) after staining with DAPI and propidium iodide as described in supplemental material.

Figure 7. Sensitivity of A. brasilense Sp7, ΔAbBphP1 and ΔAbBphP1 (pSnK5) to 5 mM toluidine blue plus light shown by zone of inhibition diameter on 1.5 % LB agar.

Each bar represents the mean diameter of the zone of inhibition recorded in three independent assays performed in triplicate. Error bars indicate SD.