Differentiation of function among the RsbR paralogs in the general stress response of Bacillus subtilis with regard to light perception.

The general stress response of Bacillus subtilis can be activated by a wide range of signals, including low intensities of visible light. It is regulated by a dedicated σ factor via a complex signal transduction pathway that makes use of stressosomes: hetero-oligomeric complexes that include one or more of the RsbR proteins (RsbRA, RsbRB, RsbRC, and RsbRD). The response to blue light is mediated by the photoreceptor YtvA. We show here which of the four RsbR proteins are necessary for the activation of the σ(B) response by blue light. Experiments performed with single-, double-, and triple-deletion strains in the rsbR genes show that RsbRB and RsbRA function antagonistically, with the former being a negative regulator and the latter a positive regulator of the YtvA-dependent light activation of the stress response. A strain with RsbRB as the only RsbR protein is unable to respond to light-activation of σ(B). Furthermore, RsbRC and RsbRD can replace RsbRA's function only in the absence of RsbRB. This differentiation of function is confined to light stress, since strains with RsbRA or RsbRB as the only RsbR protein behave similarly in our experimental conditions in response to physicochemical stresses. Interestingly, RsbRB's absence is sufficient to result in light activation of the general stress response at wild-type expression levels of ytvA, while it was previously reported that YtvA could only activate σ(B) when overproduced, or when cells are supplemented with an additional environmental stress.

Red light activates the sigmaB-mediated general stress response of Bacillus subtilis via the energy branch of the upstream signaling cascade.

The sigma(B)-dependent general stress response in the common soil bacterium Bacillus subtilis can be elicited by a range of stress factors, such as starvation or an ethanol, salt, or heat shock, via a complex upstream signaling cascade. Additionally, sigma(B) can be activated by blue light via the phototropin homologue YtvA, a component of the environmental branch of the signaling cascade. Here we use a reporter-gene fusion to show that sigma(B) can also be activated by red light via the energy branch of its upstream signaling cascade. Deletion mutagenesis and homologous overproduction experiments indicate that the RsbP protein (composed of an N-terminal Per-ARNT-Sim [PAS] domain and a C-terminal PP2C-type phosphatase domain) is involved in the red light response. This second light input pathway functions complementarily to YtvA; it shows broader spectral sensitivity but requires higher light intensities. These results are confirmed by transcriptome analyses, which show that both light effects result in upregulation of the sigma(B) regulon, with minimal activation of other responses.

In vivo mutational analysis of YtvA from Bacillus subtilis: mechanism of light activation of the general stress response.

The general stress response of Bacillus subtilis can be activated by stimuli such as the addition of salt or ethanol and with blue light. In the latter response, YtvA activates sigma(B) through a cascade of Rsb proteins, organized in stressosomes. YtvA is composed of an N-terminal LOV (light, oxygen, and voltage) domain and a C-terminal STAS (sulfate transporter and anti-sigma factor) domain and shows light-modulated GTP binding in vitro. Here, we examine the mechanism of YtvA-mediated activation of sigma(B) in vivo with site-directed mutagenesis. Constitutive off and constitutive on mutations have been identified. Disruption of GTP binding in the STAS domain eliminates light activation of sigma(B). In contrast, modification of sites relevant for phosphorylation of STAS domains does not affect the stress response significantly. The data obtained are integrated into a model for the structure of full-length YtvA, which presumably functions as a dimer.

Novel s-triazine-degrading bacteria isolated from agricultural soils of central Chile for herbicide bioremediation

s-Triazine-degrading bacterial strains were isolated from long-term simazine-treated agricultural soils of central Chile. The number of culturable heterotrophic bacteria of these agricultural soils (7 x 10(6) CFU/g of dry soil) was not affected by simazine application on field. The simazine-degrading bacterial strains P51, P52 and C53 were isolated by enrichment in minimal medium using simazine as the sole nitrogen source. Resting cells of strains P51 and P52 degraded >80 percent of simazine within 48 hrs, whereas strain C53 was able to remove >60 percent of the herbicide. The atzA and atzD genes of the s-triazine upper and lower catabolic pathways were detected in strains P51 and C53, while only atzD gene was observed in strain P52. To compare the bacterial 16S rRNA gene sequence structure, ARDRA were performed using the restriction enzymes Msp1 and Hha1. ARDRA indicated that strain P52 was a different ribotype than C53 and P51 strains. For further characterization the novel isolates were identified by 16S rRNA gene sequencing. Strains C53 and P51 belong to the genus Stenotrophomonas and the strain P52 belongs to the genus Arthrobacter . s -Triazine-degrading bacterial strains isolated from contaminated soils could be used as biocatalysts for bioremediation of these herbicides.

On the role of aromatic side chains in the photoactivation of BLUF domains.

BLUF (blue-light sensing using FAD) domain proteins are a novel group of blue-light sensing receptors found in many microorganisms. The role of the aromatic side chains Y21 and W104, which are in close vicinity to the FAD cofactor in the AppA BLUF domain from Rhodobacter sphaeroides, is investigated through the introduction of several amino acid substitutions at these positions. NMR spectroscopy indicated that in the W104F mutant, the local structure of the FAD binding pocket was not significantly perturbed as compared to that of the wild type. Time-resolved fluorescence and absorption spectroscopy was applied to explore the role of Y21 and W104 in AppA BLUF photochemistry. In the Y21 mutants, FADH*-W* radical pairs are transiently formed on a ps time scale and recombine to the ground state on a ns time scale. The W104F mutant shows a spectral evolution similar to that of wild type AppA but with an increased yield of signaling state formation. In the Y21F/W104F double mutant, all light-driven electron-transfer processes are abolished, and the FAD singlet excited-state evolves by intersystem crossing to the triplet state. Our results indicate that two competing light-driven electron-transfer pathways are available in BLUF domains: one productive pathway that involves electron transfer from the tyrosine, which leads to signaling state formation, and one nonproductive electron-transfer pathway from the tryptophan, which leads to deactivation and the effective lowering of the quantum yield of the signaling state formation. Our results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from the conserved tyrosine to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination.

Light-induced flipping of a conserved glutamine sidechain and its orientation in the AppA BLUF domain.

The AppA BLUF domain is a blue light photoreceptor containing flavin. Conserved glutamine 63 is necessary for the photocycle of the protein, and its side chain has been proposed to flip in response to blue light illumination. Recently published crystal structures of AppA WT and the AppA mutant C20S describe contradictory conclusions regarding the orientation of the conserved glutamine 63 side chain in the dark. Here, we present evidence from NMR spectroscopy confirming light-induced flipping of the glutamine side chain to form a strong hydrogen bond between the glutamine 63 side chain carbonyl group and the tyrosine 21 side chain hydroxyl proton in the light-induced state. Our conclusions are consistent with published data from UV/vis absorbance and FTIR spectroscopy, as well as the crystal structure of AppA WT.

Blue light activates the sigmaB-dependent stress response of Bacillus subtilis via YtvA.

Here we present evidence for a physiologically relevant light response mediated by the LOV domain-containing protein YtvA in the soil bacterium Bacillus subtilis. The loss and overproduction of YtvA abolish and enhance, respectively, the increase in sigma(B)-controlled ctc promoter activity at moderate light intensities. These effects were absent in the dark and in red light but present under blue-light illumination. Thus, activation of the general stress response in B. subtilis is modulated by blue light.