S. pneumoniae Thiol peroxidase (TpxD) is one of the key enzymes used to cope with oxidative stress conditions. Previously we demonstrated that TpxD expression is modulated in response to H2O2 and is involved in the regulation of the psa operon. In the current study we focus on TpxD function as a sensor and global regulator in the bacterial response to H2O2. By using microarray assays we show that TpxD expression is necessary for the activation of bacterial global gene response to H2O2. The mechanism underlying TpxD regulatory function was further elucidated by replacing the catalytic Cys58 with alanine. This point mutation prevented tpxD up-regulation by H2O2, as well as the effect of H2O2 on additional genes that were affected by H2O2 in the WT but not in DtpxD, signifying that this cysteine is crucial for TpxD signaling activity. We then tried to identify a candidate transcription factor which regulates tpxD expression under H2O2 stress. Bioinformatics identified a putative CodY 15 bp binding site in the proximal upstream region of tpxD coding sequence. Genetic engineering techniques and EMSA assays confirmed the presence of an active CodY box. Indeed, no up-regulation of TpxD was noticed in DcodY challenged with H2O2. These data identify CodY as the transcription factor modulating tpxD expression under H2O2 stress. We propose a model of gene regulation under oxidative stress conditions which places TpxD as an intermediary between H2O2 and the global bacterial response to oxidative stress.
