Expression of, and in vivo stressosome formation by, single members of the RsbR protein family in Bacillus subtilis

Abstract

The Bacillus subtilis stressosome is a 1.8 MDa complex that is the focal point for activating the bacterium's general response to physical stress. In vitro studies demonstrated that the stressosome's core element can be formed from one or more of a family of paralogous proteins (RsbRA, -RB, -RC and -RD) onto which the system's activator protein (RsbT) and its principal inhibitor (RsbS) are bound. The RsbR components of the stressosome are envisioned to be the initial receptors of stress signalling with the stressosome structure itself serving as a device to integrate multiple stress signals for a coordinated response. In the current work, we examine several of the in vivo characteristics of the RsbR family members, including their expression and ability to form stressosomes to regulate s B . Translational fusions of lacZ to each rsbR paralogue revealed that rsbRA, -RB and -RC are expressed at similar levels, which remain relatively constant during growth, ethanol stress and entry into stationary phase. rsbRD, in contrast, is expressed at a level that is only slightly above background during growth, but is induced to 30 % of the rsbRA expression level following ethanol stress. Velocity sedimentation analyses of B. subtilis extracts from strains expressing single rsbR paralogues demonstrated that each incorporates RsbS into fast-sedimenting complexes. However, consistent with rsbRD's lower expression, the RsbRDdependent RsbS complexes were present at only 20 % of the level of the complexes seen in a wild-type strain. The lower stressosome level in the RsbRD strain is still able to hold RsbT's activity in check, implying that the RsbR/S component of stressosomes is normally in excess for the control of RsbT. Consistent with such a notion, reporter gene and Western blot assays demonstrate that although RsbT is synthesized at the same rate as RsbRA and RsbS, RsbT's ultimate level in growing B. subtilis is only 10 % that of RsbRA. Apparently, RsbT's inherent structure and/or its passage between the stressosome and its activation target compromises its persistence.

Differentiation of Function among the RsbR Paralogs in the General Stress Response of Bacillus subtilis with Regard to Light Perception

“…3D). The latter observation might be explained by a lower expression level of rsbRD, as has recently been suggested (35), although our Western blot does not suggest large differences in protein concentration. However, despite the minor roles of RsbRC and RsbRD, the additional presence of either one allows RsbRA to overcome the negative effect of RsbRB (Fig.…”

“…6) (23), again indicating normal functionality of the stressosomes. Third, a recent study found the expression levels of rsbRA and rsbRB to be similar (35). The availability of sufficient protein is also suggested by the Western blot shown in Fig.…”

Rate of environmental change determines stress response specificity

“…S8). For example, it was reported that RsbT could become unstable once released from the stressosome, which would prevent the system from maintaining a strong activation level after a step increase in stress (47). Activation of σ B requires an element that can transduce the total level of stress rapidly enough to "outrun" this negative feedback loop.…”

Substitutions in the Presumed Sensing Domain of the Bacillus subtilis Stressosome Affect Its Basal Output but Not Response to Environmental Signals

“…Genetic and biochemical studies have shown that the B. subtilis stressosome comprises at least three protein species: the RsbT serine/threonine kinase, the RsbS antagonist, and one or more members of the RsbR coantagonist family (1,9,13,24,32). In vivo each stressosome appears to contain a mixture of the four partially redundant RsbR coantagonists, RsbRA, RsbRB, RsbRC, and RsbRD, and in vitro these coantagonists can dynamically exchange into the complex (13,24).…”