Thioredoxin can influence gene expression by affecting gyrase activity

Abstract

The expression of many genes of facultatively photosynthetic bacteria of the genus Rhodobacter is controlled by the oxygen tension. Among these are the genes of the puf and puc operons, which encode proteins of the photosynthetic apparatus. Previous results revealed that thioredoxins are involved in the regulated expression of these operons, but it remained unsolved as to the mechanisms by which thioredoxins affect puf and puc expression. Here we show that reduced TrxA of Rhodobacter capsulatus and Rhodobacter sphaeroides and oxidized TrxC of R.capsulatus interact with DNA gyrase and alter its DNA supercoiling activity. While TrxA enhances supercoiling, TrxC exerts a negative effect on this activity. Furthermore, inhibition of gyrase activity strongly reduces puf and puc expression. Our results reveal a new signaling pathway by which oxygen can affect the expression of bacterial genes.

Figure 1

Interaction of Trx and gyrase B subunit (GyrB) revealed by the yeast-two-hybrid system. (A) Screening results for the interaction of Trx with the GyrB. Clone 280 was screened out from the library of R.sphaeroides with trxA (Rs trxA) as a bait gene. Clone 29 was screened out from the library of R.capsulatus with trxC (Rc trxC) as a bait gene. The position and the coverage are marked by black bars within the gyrB gene. RcgyrB: gyrB of R.capsulatus; RsgyrB: gyrB of R.sphaeroides. (B) Quantification of the β-galactosidase activity. The activity was measured in yeast strain AH109 with the cotransformed plasmids containing the binding domain of GAL4 plus the cloned trx gene and the activation domain of GAL4 plus the part of gyrB gene. pos.: Positive control with pGADT7-7 and pGBKT7-53. RctrxA, RctrxC: trxA and trxC genes from R.capsulatus. RstrxA: trxA gene from R.sphaeroides. gyr392: cognate of clone 280 in R.capsulatus. Neg.: negative control with pGADT7 and pGBKT7-Lam.

Figure 2

GST pulldown showing the interaction of oxidized TrxC (A and B) and reduced TrxA (C and D) with GyrB of R.capsulatus in vitro. Anaerobically (An) and aerobically (A) grown cell extracts, His-tagged TrxC protein and GST–GyrB fusion protein were prepared as described (see Materials and Methods). TrxA in cell extracts (total protein 20 μg, determined with Bradford) and His-tagged TrxC (50 μM) were reduced with 20 mM β-mercaptoethanol (β-ME), or oxidized with 1 mM H₂O₂ in 10 μl at room temperature for 30 min, then subjected to thiol-trapping by AMS (Molecular Probes) or GST pulldown. (A) Examination of disulfide bond formation of His-tagged TrxC after β-ME or H₂O₂ treatment by AMS modification. (B) GST pulldown to detect the interaction of GyrB with TrxC. The upper panel shows the immunological detection of TrxC; the bottom panel shows silver-stained GST and GST–GyrB proteins. (C) Examination of disulfide bond formation of TrxA in anaerobic low-light (100 mmol m⁻² s⁻¹) and aerobic dark grown cells in vivo (upper panel) and after β-ME or H₂O₂ treatment (lower panel) by AMS modification. (D) GST pulldown to detect the interaction of GyrB with TrxA. The upper panel shows the immunological detection of TrxA, the bottom panel, silver stained GST and GST–GyrB proteins.

Figure 3

Characterization of the trxA mutant of R.capsulatus. (A) Effect of TrxA on the expression of photosynthesis genes, i.e. the puf and puc operons. Cultures were grown aerobically until the optical density (OD₆₆₀) reached 0.4 to 0.5, and then shifted to low oxygen tension. Total RNA was prepared and northern blot analysis was performed as described in Materials and Methods. (B) Quantification of the effect of TrxA on puf expression from the northern result (A). Closed square: trxA mutant; open square: wild type. (C) Absorption spectra of whole cells of wild type (solid line) and trxA mutant (dotted line). Cultures were grown semiaerobically in malate medium supplemented with appropriate antibiotics overnight. Sample preparation and measurement were carried out as described in Materials and Methods.

Figure 4

TrxA affects gyrase supercoiling activity in R.sphaeroides. Exponential (OD₆₆₀ = 0.6) or stationary (OD₆₆₀ = 1.4) phase cells were harvested. Half of the cells were resuspended in sonication buffer I [25 mM Tris–HCl, pH 7.5, 150 mM KCl, 2 mM phenylmethylsulfonyl-fluoride (PMSF), 10% glycerol] for the supercoiling assay, and the other half in sonication buffer II (50 mM Tris–HCl, pH 8.0, 250 mM NaCl) for detection of TrxA protein levels. Cell extracts were prepared as described in Materials and Methods. (A) Supercoiling activity of gyrase in cell extracts of wild-type WS8 and trxA mutant TK1. Two micrograms of total protein (cell extract) as determined by Bradford assay were incubated at room temperature in buffer (50 mM Tris–HCl, pH 7.5, 2 mM DTT, 20 mM KCl, 1.5 mM ATP, 5 mM spermidine, 10 mM MgCl₂, 50 μg/ml BSA) with 0.2 μg of relaxed plasmid pBluescript DNA, for 2 h or overnight at 37°C. SDS (0.2%) was added to stop the reaction, and the samples were analyzed by 0.8% agarose gel electrophoresis. Odd numbers: 2 h incubation; even numbers: overnight incubation. A, aerobic condition; SA, semiaerobic condition; Exp, exponential phase; Sta, stationary phase. M, DNA marker; C, control without cell extract. (B) TrxA levels of WS8 and TK1 in different growth condition analyzed with western blot.

Figure 5

TrxA and TrxC show opposing effects on the supercoiling activity of DNA gyrase. The exact method is described in the legend to Figure 4. Cell extracts used in this study are from semiaerobically grown culture in exponential phase. (A) Supercoiling activity of the cell extracts of SB1003 (WT) and trx mutants (trxA, trxC) of R.capsulatus. (B) Protein levels of GyrB, TrxA and TrxC in wild type and trx mutants of R.capsulatus analyzed by western blot. (C) Supercoiling assay with E.coli cell extracts. Wild type: Aegis256. trxC: Aegis257. trxA: Aegis262. C, relaxed pBluescript plasmid by DNA topisomerase I as a control, without cell extracts. (D) Supercoiling assays with purified gyrase from E.coli and TrxA from E.coli (E. c) and R.sphaeroides (R. s). C, relaxed plasmid without gyrase addition as in (C). To all other reactions 0.5 U of gyrase were added. Lanes 1–4: 0.0, 0.16, 0.5, 1.0 μM TrxA from E.coli; lanes 5–8: 0.0, 0.16, 0.5, 1.0 μM TrxA from R.sphaeroides.

Figure 6

Effect of gyrase inhibitor novobiocin (Novo) on formation of the photosynthesis apparatus. Novo was added at the time point 0 as the oxygen tension in the cultures was reduced. (A) Novo inhibited accumulation of bacteriochlorophyll. Sample preparation and the measurement were described in Materials and Methods. Diamond, control without Novo; square, Novo addition (30 μM, Novo30); triangle: Novo addition (75 μM, Novo75). (B) Northern blot analysis of puf mRNA levels in wild-type and mutant strain TK1 of R.sphaeroides with or without addition of novobiocin. The 2.7 kb pufBALMX and the 0.5 kb pufBA mRNA segments are marked.

Figure 7

Effect of gyrase inhibitor novobiocin (Novo) on bacteriochlorophyll accumulation (A), expression of puc and puf genes (B) and gyrB expression (C) of R.capsulatus. Novo was added at the time point 0 as the oxygen tension in the cultures was reduced. (A) Bacteriochlorophyll accumulation was inhibited during the Novo treatment after the oxygen tension was reduced. Bacteriochlorophyll content (A770) was normalized to the optical density of the culture. (B) Novo reduced the expression of photosynthesis genes as analyzed by northern blot. rRNA was probed as a control. For the control without Novo treatment see Figure 3 and Li et al. (11). (C) Quantification of gyrB expression during Novo treatment (30 μM) analyzed by RT–PCR. The inset shows the RT–PCR product (312 bp) of the gyrB gene.

Figure 8

Schematic model for signal transduction from thioredoxin to gene expression in Rhodobacter. The redox switch of thioredoxins caused by a change in oxygen tension alters the supercoiling activity of gyrase, which further affects gene expression. A, TrxA; C, TrxC; GyrB₂A₂, gyrase tetramer with the two subunits, A and B.