Tricksy business: transcriptome analysis reveals the involvement of thioredoxin A in redox homeostasis, oxidative stress, sulfur metabolism, and cellular differentiation in Bacillus subtilis

Abstract

Thioredoxins are important thiol-reactive proteins. Most knowledge about this class of proteins is derived from proteome studies, and little is known about the global transcriptional response of cells to various thioredoxin levels. In Bacillus subtilis, thioredoxin A is encoded by trxA and is essential for viability. In this study, we report the effects of minimal induction of a strain carrying an IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible trxA gene (ItrxA) on transcription levels, as determined by DNA macroarrays. The effective depletion of thioredoxin A leads to the induction of genes involved in the oxidative stress response (but not those dependent on PerR), phage-related functions, and sulfur utilization. Also, several stationary-phase processes, such as sporulation and competence, are affected. The majority of these phenotypes are rescued by a higher induction level of ItrxA, leading to an approximately wild-type level of thioredoxin A protein. A comparison with other studies shows that the effects of thioredoxin depletion are distinct from, but show some similarity to, oxidative stress and disulfide stress. Some of the transcriptional effects may be linked to thioredoxin-interacting proteins. Finally, thioredoxin-linked processes appear to be conserved between prokaryotes and eukaryotes.

FIG. 1.

Western blot detection of TrxA protein in wild-type B. subtilis 168 and the ItrxA strain grown in the presence of 25 or 100 μM IPTG.

FIG. 2.

Flow cytometric analysis of xylose-induced expression of a redox-sensitive GFP in the ItrxA mutant upon induction with 25, 100, or 500 μM of IPTG. (A) Fluorescence distribution of a typical experiment; (B) mean fluorescence of a culture based on flow cytometric data.

FIG. 3.

(A) The transcriptional response of the nucA-nin genome region in the ItrxA mutant cells; (B) the transcriptional response of the cah-ycg genome region in the ItrxA mutant cells. Thin black arrows indicate no significant difference (n-fold) from the wild type. Thick gray arrows indicate a transcriptional response upon thioredoxin A depletion (P < 0.025). Differences (n-fold) in expression levels are given next to the gene names.

FIG. 4.

The effect of thioredoxin A depletion on the transcription of genes involved in sulfur uptake and utilization. Gene names are indicated in italics. Alternative gene names for some of the genes are given in the text (see “Sulfur uptake and utilization”). Regulation (n-fold) of genes in ItrxA 25 cells compared to that of the wild type is given in parentheses next to each gene name. When gene names are in parentheses too, P values did not meet our criteria. SAH, S-adenosyl homocysteine; SRH, S-ribosyl homocysteine. Question marks indicate the involvement of an unknown factor or a postulated function.

FIG. 5.

Growth of the ItrxA mutant in chemically defined medium with cysteine (squares) or without cysteine (triangles). Medium was supplemented with 25 μM (open symbols) or 100 μM (closed symbols) of IPTG. Times (t) are given in hours after inoculation.