Evidence that Oxidative Stress Induces spxA2 Transcription in Bacillus anthracis Sterne through a Mechanism Requiring SpxA1 and Positive Autoregulation

Abstract

Bacillus anthracis possesses two paralogs of the transcriptional regulator, Spx. SpxA1 and SpxA2 interact with RNA polymerase (RNAP) to activate the transcription of genes implicated in the prevention and alleviation of oxidative protein damage. The spxA2 gene is highly upregulated in infected macrophages, but how this is achieved is unknown. Previous studies have shown that the spxA2 gene was under negative control by the Rrf2 family repressor protein, SaiR, whose activity is sensitive to oxidative stress. These studies also suggested that spxA2 was under positive autoregulation. In the present study, we show by in vivo and in vitro analyses that spxA2 is under direct autoregulation but is also dependent on the SpxA1 paralogous protein. The deletion of either spxA1 or spxA2 reduced the diamide-inducible expression of an spxA2-lacZ construct. In vitro transcription reactions using purified B. anthracis RNAP showed that SpxA1 and SpxA2 protein stimulates transcription from a DNA fragment containing the spxA2 promoter. Ectopically positioned spxA2-lacZ fusion requires both SpxA1 and SpxA2 for expression, but the requirement for SpxA1 is partially overcome when saiR is deleted. Electrophoretic mobility shift assays showed that SpxA1 and SpxA2 enhance the affinity of RNAP for spxA2 promoter DNA and that this activity is sensitive to reductant. We hypothesize that the previously observed upregulation of spxA2 in the oxidative environment of the macrophage is at least partly due to SpxA1-mediated SaiR repressor inactivation and the positive autoregulation of spxA2 transcription. T ranscription factors of pathogenic bacteria can be important virulence determinants, since they control genes that specify toxins, proteins functioning in nutrient acquisition within the hostile host environment, and products that operate by subverting the host innate immunity (1-4). For example, in Bacillus anthracis, the causative agent of the zoonotic infectious disease, anthrax, the plasmid-borne atxA gene encodes a transcription factor required for virulence, since it is necessary for the activation of genes for toxin and protective capsule production (5, 6). Other Grampositive bacteria possess genes encoding one or more forms of the ArsC (arsenate reductase) family protein, Spx, which is an RNA polymerase (RNAP)-binding protein directing the expression of large regulons that include genes necessary for pathogenesis (7-11). Studies of streptococci, Listeria monocytogenes, Staphylococcus aureus, and Enterococcus spp. have demonstrated the requirement for Spx proteins for virulence and viability (7,8,(12)(13)(14)(15). IMPORTANCE Regulators of transcription initiationSpx was detected in studies of B. subtilis, in which it is encoded by a gene that is under complex transcriptional control involving four RNAP forms and two repressors (16)(17)(18)(19). The complex control of spx transcription operates in response to various stress conditions, such as oxidative and cell envelope stress. Spx protein levels increase under conditions caus...

Regulatory circuits controlling Spx levels in Streptococcus mutans

“…In addition to what appears to be a secondary/ back-up role in the activation of oxidative stress responses, global transcriptional analysis suggested that SpxA2 may participate in the regulation of processes associated with cell envelope homeostasis (Kajfasz et al, 2010;. After the initial discovery of two Spx-like regulators in S. mutans, several studies revealed the presence of two Spx paralogs in other streptococci as well as in Listeria monocytogenes and Bacillus anthracis (Turlan et al, 2009;Chen et al, 2012;Barendt et al, 2013;Zheng et al, 2014;Barendt et al, 2016;Port et al, 2017). In addition to the conserved role in activation of oxidative stress responses, Spx regulation has been more recently linked to activation of other types of stress, including antibiotic, osmotic and cell envelope stresses (Renzoni et al, 2011;Jousselin et al, 2013;Baek et al, 2015;Villanueva et al, 2016;Rojas-Tapias and Helmann, 2018a;Nilsson et al, 2019).…”

Integrated Transcriptomic and Proteomic Analyses Reveal the Role of NprR in Bacillus anthracis Extracellular Protease Expression Regulation and Oxidative Stress Responses

“…RNA-seq analysis indicated that the expression level of the spxA1 (AW20_RS08630) and spxA2 (AW20_RS25945) genes significantly downregulated in the nprR deletion mutant strain. Both SpxA1 and SpxA2 are closely related to the oxidative stress responses of B. anthracis , and spxA1 is essential for peroxide resistance and participates in disulfide stress tolerance ( Barendt et al, 2013 , 2016 ). SpxA1 is also involved in oxidative stress response of many gram-positive bacteria ( Chen et al, 2012 ; Kajfasz et al, 2017 ; Nilsson et al, 2019 ; Cesinger et al, 2020 ).…”

Roles and regulation of Spx family transcription factors in Bacillus subtilis and related species

“…The activity of SpxA2 is regulated at the transcriptional level, in addition to likely regulation by regulated proteolysis. Induction of the operon encoding spxA2 can be activated by either SpxA1 or SpxA2 in response to oxidative stress, such as might be encountered in an activated macrophage (Barendt, Birch, Mbengi, & Zuber, 2016). This induction is needed to overcome the activity of SaiR, which acts as a repressor of spxA2 (M. M. Nakano et al, 2014).…”