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ςB, the general stress response ς factor ofBacillus subtilis, is activated when intracellular ATP levels fall or the bacterium experiences environmental stress. Stress activates ςB by means of a collection of regulatory kinases and phosphatases (the Rsb proteins), which catalyze the release of ςB from an anti-ς factor inhibitor. By using the yeast dihybrid selection system to identify B. subtilisproteins that could interact with Rsb proteins and act as mediators of stress signaling, we isolated the GTP binding protein, Obg, as an interactor with several of these regulators (RsbT, RsbW, and RsbX).B. subtilis depleted of Obg no longer activated ςB in response to environmental stress, but it retained the ability to activate ςB by the ATP responsive pathway. Stress pathway components activated ςB in the absence of Obg if the pathway’s most upstream effector (RsbT) was synthesized in excess to the inhibitor (RsbS) from which it is normally released after stress. Thus, the Rsb proteins can function in the absence of Obg but fail to be triggered by stress. The data demonstrate that Obg, or a process under its control, is necessary to induce the stress-dependent activation of ςB and suggest that Obg may directly communicate with one or more ςB regulators.

general stress response sigma factor of Bacillus subtilis, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates B through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of B from an anti-B factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the B stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude B. subtilis extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of B . B. subtilis strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of B . Neither the energy-responsive activation of B nor stress-dependent chaperone gene induction (a B -independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of B are shown to be active in the RplK ؊ strain but fail to be triggered by stress. The data demonstrate that the B. subtilis ribosomes provide an essential input for the stress activation of B and suggest that the ribosomes may themselves be the sensors for stress in this system.

Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity. The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change withinlon leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.

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