In Vivo Mutational Analysis of YtvA from Bacillus subtilis

Abstract

LOV2 domains (1), members of the superfamily of PAS domains (2, 3), are abundant in all domains of life and were first identified in plant phototropins (4). These photoreceptors regulate stomatal opening, phototropism, etc. and contain two N-terminal LOV domains that confer light regulation on the C-terminal Ser/Thr kinase domain (4). They also occur in bacteria, in which YtvA from Bacillus subtilis has been best characterized (for a review, see e.g. Ref. 5). Its N-terminal LOV domain binds FMN and shows the typical LOV photochemistry (6, 7): covalent adduct formation between a cysteine and the FMN chromophore. A linker helix, denoted J␣ (7), connects the LOV domain to a STAS domain. The latter domain is present in many regulators of the general stress response of B. subtilis (8,9). Stress via the addition of salt or ethanol (for a review, see Ref.10) and blue light (11, 12) activates the general stress response via the environmental pathway, which integrates various signals via a large multiprotein complex, called the stressosome (13,14). YtvA, which mediates light activation of B (11, 12, 15), co-purifies with other STAS domain proteins in the stressosomes (16). When cells are stressed, STAS domains of several stressosome proteins (e.g. RsbS and RsbR) are phosphorylated by another intrinsic stressosome component, the serine/threonine kinase RsbT (9,14,17,18). Next, RsbT is released from the complex to trigger RsbU, a protein phosphatase, thus (indirectly) activating B (19). Phosphorylation of YtvA, however, has never been detected. Rather, it has been demonstrated in vitro that YtvA shows light-dependent GTP binding, presumably at its NTP-binding site in the STAS domain (20).Little is known about the mechanism of signal transmission in and by YtvA, except that in the C62A mutant, photochemistry in vitro (12) and light activation of B in vivo (12, 15) are abolished. More detailed information is available for LOV domains of phototropins. A conserved glutamine, which is in hydrogen-bonding contact with the isoalloxazine ring of FMN, rotates its side chain by 180 o upon covalent adduct formation (21). Replacement of this residue by leucine in the LOV2 domain of Phy3 from Adiantum results in a considerable reduction of the light-induced structural change (22). The corresponding mutation in phototropin 1 from Arabidopsis impairs autophosphorylation activity (23). The signal generated in the LOV2 domain is transmitted to the downstream kinase domain of phototropin 1 of Avena sativa through disruption of the interaction between its central ␤-sheet and the C-terminal linker region, the J␣-helix (24).Here, we study the mechanism of activation of YtvA in vivo, i.e. light-induced activation of the B response, with site-directed mutagenesis. We focus on three regions of the protein, the flavinbinding pocket, the ␤-sheet of the LOV domain, and the GTPbinding site, and on potential phosphorylation sites of the STAS domain. We demonstrate that light-activated GTP binding is crucial for functional YtvA. A computational approac...

Blue-Light Inhibition of Listeria monocytogenes Growth Is Mediated by Reactive Oxygen Species and Is Influenced by σ B and the Blue-Light Sensor Lmo0799

“…One possible interpretation of these data was that oxygen levels in the medium were influenced by the population cell density, and that this variable might influence the sensitivity to blue light, since other studies have reported that light can lead to the generation of reactive oxygen species (ROS) (25,35). To investigate this hypothesis, we measured the inhibitory effect of blue light on cells grown in BHI medium containing the ROS scavenger dimethylthiourea (DMTU).…”

“…A deletion mutant, ⌬lmo0799, and a missense mutant with an alanine replacing the conserved cysteine at position 56 of the Lmo0799 protein (C56A) were constructed. The lmo0799 C56A mutant was constructed to genetically test the idea that this residue is essential for the light-sensing function of the protein, as proposed by others (20,21,35). Two phenotypes known to be associated with the loss of Lmo0799 function were tested: derepressed motility in the presence of light and loss of ring formation in response to light-dark cycles (17,18).…”

Solution Structure of the Guanine Nucleotide-binding STAS Domain of SLC26-related SulP Protein Rv1739c from Mycobacterium tuberculosis

“…The percent quench varied from 19 (for GTP) and 49% for GDP (Table 2). These data suggest a nucleotide interaction surface in Rv1739C STAS that directly or indirectly perturbs Trp 50 and/or the three Tyr residues through conformational effects on adjacent or interposed residues.…”

“…4A, Rv1739c STAS was photolabeled by a ␥-biotinylated derivative of 8-N 3 -GTP, and this photolabeling was prevented in the presence of excess unlabeled GTP. The presence of a unique Trp residue, Trp 50 (aa 486 of Rv1739c holoprotein), in Rv1739c STAS and Tyr residues at STAS positions 7, 19, and 21 ( Fig. 3E and supplemental Fig.…”

“…Whereas SpoIIAA hydrolyzed GTP (22), YtvA had undetectable GTP hydrolase activity (47,50). Rv1739c STAS-associated GTPase activity was detectable as slow inorganic .…”