The Activity of  V, an Extracytoplasmic Function   Factor of Bacillus subtilis, Is Controlled by Regulated Proteolysis of the Anti-  Factor RsiV

Abstract

During growth in the environment, bacteria encounter stresses which can delay or inhibit their growth. To defend against these stresses, bacteria induce both resistance and repair mechanisms. Many bacteria regulate these resistance mechanisms using a group of alternative factors called extracytoplasmic function (ECF) factors. ECF factors represent the largest and most diverse family of factors. Here, we demonstrate that the activation of a member of the ECF30 subfamily of ECF factors, V in Bacillus subtilis, is controlled by the proteolytic destruction of the anti-factor RsiV. We will demonstrate that the degradation of RsiV and, thus, the activation of V requires multiple proteolytic steps. Upon exposure to the inducer lysozyme, the extracellular domain of RsiV is removed by an unknown protease, which cleaves at site 1. This cleavage is independent of PrsW, the B. subtilis site 1 protease, which cleaves the anti-factor RsiW. Following cleavage by the unknown protease, the N-terminal portion of RsiV requires further processing, which requires the site 2 intramembrane protease RasP. Our data indicate that the Nterminal portion of RsiV from amino acid 1 to 60, which lacks the extracellular domain, is constitutively degraded unless RasP is absent, indicating that RasP cleavage is constitutive. This suggests that the regulatory step in RsiV degradation and, thus, V activation are controlled at the level of the site 1 cleavage. Finally, we provide evidence that increased resistance to lysozyme decreases V activation. Collectively, these data provide evidence that the mechanism for V activation in B. subtilis is controlled by regulated intramembrane proteolysis (RIP) and requires the site 2 protease RasP.

Activation without Proteolysis of Anti-<i>σ</i> Factor RsiV of the Extracytoplasmic Function <i>σ</i> Factor <i>σ</i><sup>V</sup> in a Glucolipid-Deficient Mutant of <i>Bacillus subtilis</i>

“…A rasP::tet mutant showed a basal level σ V activity in the absence of HEWL ( Figure 1(c)). Whereas the addition of 100 ng/mL HEWL activated σ V several-fold in rasP + cells, the σ V activity level in the rasP::tet mutant did not change in the presence of 100 ng/mL HEWL (Figure 1(c)), indicating that the activation of σ V by HEWL requires RasP [5]. On the other hand, the approximately two-fold activation of σ V in the ∆ugtP mutant did not change when, in addition, rasP was disrupted (Figure 1(c)), indicating that RasP has nothing to do with the σ V activation by the lack of glucolipids.…”

“…The zinc chelator 1,10-phenanthroline (PT) inhibits the site-2 protease RasP [5], as it does many other site-2 metalloproteases [28]. When ugtP + cells were challenged by hen egg white lysozyme (HEWL) at low concentration (100 ng/mL), …”

“…Escherichia coli has a much simpler membrane lipid composition than B. subtilis. The membrane contains three kinds of phospholipids (phosphatidylethanolamine, phosphatidylglycerol and cardiolipin), except for the outer leaflet of the outer membrane, which is composed of lipopolysaccharide [17] only two-to three-fold, whereas treatment with a high concentration (100 µg/ mL) of lysozyme causes ~65-fold activation [5]. In this study we explored the question of how σ V is activated in the B. subtilis ∆ugtP mutant.…”

“…Among these ECF σ factors, σ V primarily responds to lysozyme [4]. It is activated by stepwise proteolytic destruction of the anti-σ factor RsiV via regulated intramembrane proteolysis (RIP) [5]. Lysozyme directly binds to RsiV and induces the cleavage of its C-terminal extracytoplasmic portion by signal peptidase (site-1 cleavage).…”

The atypical two-subunit σ factor from Bacillus subtilis is regulated by an integral membrane protein and acid stress

“…It has been suggested that PrsW, the site-1 protease, is the sensor of the environmental stressor (Ellermeier & Losick, 2006), but further research into the mechanism suggests the involvement of a negative regulator of proteolysis in sensing the stress signal (Heinrich et al, 2009). More recently, the regulation of s V by its anti-s factor RsiV has been investigated (Hastie et al, 2013(Hastie et al, , 2014). Like the s W stress response, the s V stress response chemically remodels cell envelope structure providing resistance to its inducer, lysozyme (Guariglia-Oropeza & Helmann, 2011;Ho et al, 2011).…”