Role of the PAS sensor domains in the Bacillus subtilis sporulation kinase KinA

Histidine kinases are sophisticated molecular sensors that are used by bacteria to detect and respond to a multitude of environmental signals. KinA is the major histidine kinase required for initiation of sporulation upon nutrient deprivation in Bacillus subtilis. KinA has a large N-terminal region (residues 1 to 382) that is uniquely composed of three tandem Per-ARNT-Sim (PAS) domains that have been proposed to constitute a sensor module. To further enhance our understanding of this "sensor" region, we defined the boundaries that give rise to the minimal autonomously folded PAS domains and analyzed their homo- and heteroassociation properties using analytical ultracentrifugation, nuclear magnetic resonance (NMR) spectroscopy, and multiangle laser light scattering. We show that PAS(A) self-associates very weakly, while PAS(C) is primarily a monomer. In contrast, PAS(B) forms a stable dimer (K-d [dissociation constant] o

Fluctuations in spo0A Transcription Control Rare Developmental Transitions in Bacillus subtilis

Phosphorylated Spo0A is a master regulator of stationary phase development in the model bacterium Bacillus subtilis, controlling the formation of spores, biofilms, and cells competent for transformation. We have monitored the rate of transcription of the spo0A gene during growth in sporulation medium using promoter fusions to firefly luciferase. This rate increases sharply during transient diauxie-like pauses in growth rate and then declines as growth resumes. In contrast, the rate of transcription of an rRNA gene decreases and increases in parallel with the growth rate, as expected for stable RNA synthesis. The growth pause-dependent bursts of spo0A transcription, which reflect the activity of the spo0A vegetative promoter, are largely independent of all known regulators of spo0A transcription. Evidence is offered in support of a “passive regulation” model in which RNA polymerase stops transcribing rRNA genes during growth pauses, thus becoming available for the transcription of spo0A. We show that the bursts are followed by the production of phosphorylated Spo0A, and we propose that they represent initial responses to stress that bring the average cell closer to the thresholds for transition to bimodally expressed developmental responses. Measurement of the numbers of cells expressing a competence marker before and after the bursts supports this hypothesis. In the absence of ppGpp, the increase in spo0A transcription that accompanies the entrance to stationary phase is delayed and sporulation is markedly diminished. In spite of this, our data contradicts the hypothesis that sporulation is initiated when a ppGpp-induced depression of the GTP pool relieves repression by CodY. We suggest that, while the programmed induction of sporulation that occurs in stationary phase is apparently provoked by increased flux through the phosphorelay, bet-hedging stochastic transitions to at least competence are induced by bursts in transcription

The Tertiary Structure at 1.59 \AA Resolution and the Proposed Amino Acid Sequence of a Family-11 Xylanase from the Thermophilic Fungus Paecilomyces varioti Bainier

We report the crystal structure at 1.59 \AA and the proposed amino acid sequence of an endo-1,4-$\beta$-xylanase (PVX) from the thermophilic fungus Paecilomyces varioti Bainier (PvB), stable up to $75^o C$. This fungus is attracting clinical attention as a pathogen causing post-surgical infections. Its xylanase, known as a skin-contact allergen, is the first protein from this fungus whose three-dimensional structure has been elucidated. The crystals of PVX conform to the space group $P2_12_12_1$ with $a = 38.76 \AA$, $b = 54.06 \AA$ and $c = 90.06 \AA$. The structure was solved by molecular replacement techniques using polyalanine coordinates of the Thermomyces lanuginosus xylanase (PDB code 1YNA) and a careful model building based on the amino acid sequence known for two trypsin-digested peptide fragments (17 residues), the sequence and structural alignment of family-11 xylanases and electron density maps. The final refined model has 194 amino acid residues and 128 water molecules, with a crystallographic R-factor of 19.07 % and a free R-factor of 21.94%. The structure belongs to an all-$\beta$ fold, with two curved $\beta$-sheets, forming the cylindrical active-site cleft, and a lone $\alpha$-helix, as present in other family-11 xylanases. We have carried out a quantitative comparison of the structure and sequence of the present thermophilic xylanase (PVX) with other available native structures of mesophiles and thermophiles, the first such detailed analysis to be carried out on family-11 xylanases. The analysis provides a basis for the rationalisation of the idea that the “hinge” region is made more compact in thermophiles by the addition of a disulphide bridge between Cys110 and Cys154 and a N-H···O hydrogen bond between Trp159 near the extremity of the lone α-helix and Trp138 on $\beta$-strand B8. This work brings out explicitly the presence of the C-H···O and the C-H···$\pi$ type interactions in these enzymes. A complete description of structural stability of these enzymes needs to take account of these weaker interactions

Alleviation of salinity stress by EDTA chelated-biochar and arbuscular mycorrhizal fungi on maize via modulation of antioxidants activity and biochemical attributes

Abstract Salinity stress adversely affects agricultural productivity by disrupting water uptake, causing nutrient imbalances, and leading to ion toxicity. Excessive salts in the soil hinder crops root growth and damage cellular functions, reducing photosynthetic capacity and inducing oxidative stress. Stomatal closure further limits carbon dioxide uptake that negatively impact plant growth. To ensure sustainable agriculture in salt-affected regions, it is essential to implement strategies like using biofertilizers (e.g. arbuscular mycorrhizae fungi = AMF) and activated carbon biochar. Both amendments can potentially mitigate the salinity stress by regulating antioxidants, gas exchange attributes and chlorophyll contents. The current study aims to explore the effect of EDTA-chelated biochar (ECB) with and without AMF on maize growth under salinity stress. Five levels of ECB (0, 0.2, 0.4, 0.6 and 0.8%) were applied, with and without AMF. Results showed that 0.8ECB + AMF caused significant enhancement in shoot length (~ 22%), shoot fresh weight (~ 15%), shoot dry weight (~ 51%), root length (~ 46%), root fresh weight (~ 26%), root dry weight (~ 27%) over the control (NoAMF + 0ECB). A significant enhancement in chlorophyll a, chlorophyll b and total chlorophyll content, photosynthetic rate, transpiration rate and stomatal conductance was also observed in the condition 0.8ECB + AMF relative to control (NoAMF + 0ECB), further supporting the efficacy of such a combined treatment. Our results suggest that adding 0.8% ECB in soil with AMF inoculation on maize seeds can enhance maize production in saline soils, possibly via improvement in antioxidant activity, chlorophyll contents, gas exchange and morphological attributes