Agrobacterium tumefaciens soxR is involved in superoxide stress protection and also directly regulates superoxide-inducible expression of itself and a target gene

Abstract

Inactivation of Agrobacterium tumefaciens soxR increases sensitivity to superoxide generators. soxR expression is highly induced by superoxide stress and is autoregulated. SoxR also directly regulates the superoxide-inducible expression of atu5152. Taken together, the physiological role of soxR and the mechanism by which it regulates expression of target genes make the A. tumefaciens SoxR system different from other bacterial systems.

FIG. 1.

Determination of the resistance levels toward superoxide generators in A. tumefaciens strains. A, levels of resistance of exponential-phase A. tumefaciens strains to superoxide generators were determined (22) by mixing exponential-phase cells with molten top agar and overlaying this mixture on LB plates. Sterile 6-mm-diameter paper discs soaked with 5 μl of 1.0 M paraquat or 1.0 M MD were placed on the surface of the cell lawn, and the zones of growth inhibition were measured after 24 h of incubation at 30°C. B, level of resistance of the stationary-phase cells to MD killing was determined using a plate sensitivity assay (22). Serial dilutions of stationary-phase cells were made in 50 mM sodium phosphate buffer (pH 7.0), and 10 μl of each dilution was spotted onto an LB agar plate containing 400 or 500 μM MD. The plates were incubated at 30°C for 24 h before the results were read. Percentage survival is defined as the number of cells grown on plates containing MD divided by the number of cells grown on plates without MD multiplied by 100. The significances of differences among treatments were statistically determined using one-way analysis of variance and post hoc pairwise comparison with the least significant difference test. An asterisk indicates a P value of <0.01 compared with results for the NTL4 strain. NTL4, A. tumefaciens parental strain; PW01, soxR mutant; PW01/pSoxR, PW01 harboring pSoxR.

FIG. 2.

Menadione induced soxR expression and identification of soxR promoter and regulatory regions. A, Northern blot of the total RNA (10 μg) samples prepared from exponential-phase cultures of A. tumefaciens; uninduced (UN) or induced with 250 μM H₂O₂ (H₂O₂), 200 μM t-butyl hydroperoxide (tBOOH), or 200 μM menadione (MD) for 15 min. These samples were then separated, blotted, and hybridized with a ³²P-labeled soxR-specific probe. The level of 16S rRNA as a loading control is shown underneath the autoradiograph of the Northern blot. B, primer extension of RNA extracted from uninduced (UN) or MD-induced (MD) cultures was previously described (2). The experiment was performed using the ³²P-labeled oligonucleotide primer BT551 (5′ ATGGATGGTGGAAACGGC3′). C, T, A, and G are sequence ladders. The arrowhead and +1 indicate the soxR transcription start site. C, regulatory regions of soxR. The −35 and −10 regions are shown in bold capital letters. RBS and Met represent the ribosome binding site and the translation start site of soxR, respectively. A putative SoxR box that shared homology with the E. coli SoxR binding site is shown in small letters. +1 indicates the transcription start site.

FIG. 3.

In vivo promoter analysis and binding of SoxR to the promoter fragment. A, in vivo soxR promoter analysis. Exponential-phase cultures of A. tumefaciens strains harboring pP_soxR (NTL4, PW01, NTL4/pSoxR, or PW01/pSoxR) uninduced (UN) or treated with oxidants (as described in the legend to Fig. 2A) were harvested for crude lysate preparation and analysis of β-galactosidase activity (16). For anaerobic cultures, cells were grown anaerobically for 8 h (7), and MD was then added to the induced culture, which was returned to anaerobic conditions for 30 min before crude lysate preparation and enzyme assay. β-Galactosidase activity is expressed in μmol p-nitrophenol generated at 25°C in 1 min per mg protein (18). An asterisk represents a P value of <0.01 compared with results for the uninduced condition. B, SoxR binding to the soxR promoter. Gel mobility shift reactions were carried out by adding 3 fmol of labeled probe (³²P-labeled 207-bp soxR promoter fragment) to a 25-μl reaction mixture [20 mM Tris (pH 7.0), 50 mM KCl, 1 mM EDTA, 5% glycerol, 50 μg ml⁻¹ bovine serum albumin, 5 μg ml⁻¹ calf thymus DNA, 0.5 mg ml⁻¹ poly(dI/dC)]. Purified oxidized SoxR (0 to 100 ng protein) was added, and the reaction mixture was incubated at 25°C for 15 min. Protein-DNA complexes were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel in 0.5× Tris-borate-EDTA buffer at 4°C. UP indicates reaction mixtures containing 1 μg unlabeled soxR promoter in addition to SoxR (80 ng). P_soxR−22 is the 45-bp-deleted soxR promoter fragment use in the binding reactions. F indicates free probe; B indicates bound probe.

FIG. 4.

soxR directly regulates atu5152. A, primer extension analysis of atu5152. Primer extension was performed on total RNA samples isolated from uninduced (UN) or menadione-induced (MD) cultures. The BT1271 (5′CGGCTCCACCAGGCGTGTT3′) primer was used. B, regulatory regions of atu5152. The promoter −10 and −35 regions are shown in bold. The transcription start site (+1) and a putative SoxR box between the conserved promoter regions are shown. RBS and Met represent the ribosome binding site and the translation initiation site of atu5152, respectively. C, in vivo atu5152 promoter analysis. Exponential-phase cultures of A. tumefaciens NTL4 and PW01 harboring pP₅₁₅₂ were treated with oxidants either aerobically or anaerobically as for Fig. 3A. Crude lysate preparations and β-galactosidase activity assays were performed as previously described (16). An asterisk represents a P value of <0.01 compared with results for the uninduced condition. D, binding of SoxR to the atu5152 promoter. A gel mobility shift assay was performed using an increased amount of purified oxidized SoxR (ng protein) as described for Fig. 3C except that the 212-bp ³²P-labeled atu5152 promoter fragment was used.