Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.     

The STIM1 CTID domain determines access of SARAF to SOAR to regulate Orai1 channel function

Ca²⁺ influx by store-operated Ca²⁺ channels (SOCs) mediates all Ca²⁺-dependent cell functions, but excess Ca²⁺ influx is highly toxic. The molecular components of SOC are the pore-forming Orai1 channel and the endoplasmic reticulum Ca²⁺ sensor STIM1. Slow Ca²⁺-dependent inactivation (SCDI) of Orai1 guards against cell damage, but its molecular mechanism is unknown. Here, we used homology modeling to identify a conserved STIM1(448–530) C-terminal inhibitory domain (CTID), whose deletion resulted in spontaneous clustering of STIM1 and full activation of Orai1 in the absence of store depletion. CTID regulated SCDI by determining access to and interaction of the STIM1 inhibitor SARAF with STIM1 Orai1 activation region (SOAR), the STIM1 domain that activates Orai1. CTID had two lobes, STIM1(448–490) and STIM1(490–530), with distinct roles in mediating access of SARAF to SOAR. The STIM1(448–490) lobe restricted, whereas the STIM1(490–530) lobe directed, SARAF to SOAR. The two lobes cooperated to determine the features of SCDI. These findings highlight the central role of STIM1 in SCDI and provide a molecular mechanism for SCDI of Orai1.     

Garden-waste-vermicompost leachate alleviates salinity stress in tomato seedlings by mobilizing salt tolerance mechanisms

The incidence of salinity-induced plant stress as a result of natural and anthropogenic factors in arid and semi-arid agricultural lands is great. In South Africa alone, 9 % of irrigated agricultural land is salt-affected. Commercial fertilizers used for improving soil nutrient levels are costly and affect the quality, lifespan and sustainability of soil and water resources. Organic farming practices are based on cost-effective and environmentally-aware management systems. Vermicompost leachate (VCL) is a vermicompost-derived liquid product that has become recognised as a suitable soil amendment product. Commercial tomato (Lycopersicon esculentum Mill var. Heinz-1370) seedlings were subjected to sodium chloride (NaCl) concentrations of 0, 25, 50 and 100 mM and were treated with 1:10 (v/v) WizzardWorms VCL prepared in Hoagland’s nutrient solution under greenhouse conditions. Morphological characters of VCL-treated tomato seedlings showed improved root growth and stimulated overall aboveground growth with significantly higher numbers of leaves, greater stem thickness and increased leaf area, even at a high NaCl-tested concentration (100 mM). The accumulation of compatible solutes such as proline and total soluble sugars indicate an induced salt tolerance or adaptive mechanism in VCL-treated tomato seedlings. The current investigation demonstrates the potential of an organic liquid to maximise tomato productivity by improving seedling growth performance under salt stress conditions.     

Urban land use limits regional bumble bee gene flow

Potential declines in native pollinator communities and increased reliance on pollinator‐dependent crops have raised concerns about native pollinator conservation and dispersal across human‐altered landscapes. Bumble bees are one of the most effective native pollinators and are often the first to be extirpated in human‐altered habitats, yet little is known about how bumble bees move across fine spatial scales and what landscapes promote or limit their gene flow. In this study, we examine regional genetic differentiation and fine‐scale relatedness patterns of the yellow‐faced bumble bee, Bombus vosnesenskii, to investigate how current and historic habitat composition impact gene flow. We conducted our study across a landscape mosaic of natural, agricultural and urban/suburban habitats, and we show that B. vosnesenskii exhibits low but significant levels of differentiation across the study system (F_ST = 0.019, D_est = 0.049). Most importantly, we reveal significant relationships between pairwise F_ST and resistance models created from contemporary land use maps. Specifically, B. vosnesenskii gene flow is most limited by commercial, industrial and transportation‐related impervious cover. Finally, our fine‐scale analysis reveals significant but declining relatedness between individuals at the 1–9 km spatial scale, most likely due to local queen dispersal. Overall, our results indicate that B. vosnesenskii exhibits considerable local dispersal and that regional gene flow is significantly limited by impervious cover associated with urbanization.     

Catalytic activity of Peptidase N is required for adaptation of Escherichia coli to nutritional downshift and high temperature stress

Peptidase N (PepN), the sole M1 family member in Escherichia coli, displays broad substrate specificity and modulates stress responses: it lowers resistance to sodium salicylate (NaSal)-induced stress but is required during nutritional downshift and high temperature (NDHT) stress. The expression of PepN does not significantly change during different growth phases in LB or NaSal-induced stress; however, PepN amounts are lower during NDHT stress. To gain mechanistic insights on the roles of catalytic activity of PepN in modulating these two stress responses, alanine mutants of PepN replacing E264 (GAMEN motif) and E298 (HEXXH motif) were generated. There are no major structural changes between purified wild type (WT) and mutant proteins, which are catalytically inactive. Importantly, growth profiles of ΔpepN upon expression of WT or mutant proteins demonstrated the importance of catalytic activity during NDHT but not NaSal-induced stress. Further fluorescamine reactivity studies demonstrated that the catalytic activity of PepN is required to generate higher intracellular amounts of free N-terminal amino acids; consequently, the lower growth of ΔpepN during NDHT stress increases with high amounts of casamino acids. Together, this study sheds insights on the expression and functional roles of the catalytic activity of PepN during adaptation to NDHT stress.     

Implications of biofuel policy‐driven land cover change for rainfall erosivity and soil erosion in the United States

Large‐scale conversion of traditional agricultural cropping systems to biofuel cropping systems is predicted to have significant impact on the hydrologic cycle. Changes in the hydrologic cycle lead to changes in rainfall and its erosive power, and consequently soil erosion that will have onsite impacts on soil quality and crop productivity, and offsite impacts on water quality and quantity. We examine regional change in rainfall erosivity and soil erosion resulting from biofuel policy‐induced land use/land cover (LULC) change. Regional climate is simulated under current and biofuel LULC scenarios for the period 1979–2004 using the Weather Research Forecast (WRF) model coupled to the NOAH land surface model. The magnitude of change in rainfall erosivity under the biofuel scenario is 1.5–3 times higher than the change in total annual rainfall. Over most of the conterminous United States (~56%), the magnitude of the change in erosivity is between ?2.5% and +2.5%. A decrease in erosivity of magnitude 2.5–10% is predicted over 23% of the area, whereas an increase of the same magnitude is predicted over 14% of the area. Corresponding to the changes in rainfall erosivity and crop cover, a decrease in soil loss is predicted over 60% of the area under the biofuel scenario. In Kansas and Oklahoma, the states in which a large fraction of land area is planted with switchgrass under the biofuel scenario, soil loss is estimated to decrease 12% relative to the baseline. This reduction in soil loss is due more to changes in the crop cover factor than changes in rainfall or rainfall erosivity. This indicates that the changes in LULC, due to future cellulosic biofuel feedstock production, can have significant implications for regional soil and water resources in the United States and we recommend detailed investigation of the trade‐offs between land use and management options.     

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance,Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Background

Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress.

Methodology

The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.

Results

The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.

Conclusions

Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.     

Landscape genetics of a tropical rescue pollinator

Pollination services are increasingly threatened by the loss and modification of natural habitats, posing a risk to the maintenance of both native plant biodiversity and agricultural production. In order to safeguard pollination services, it is essential to examine the impacts of habitat degradation on the population dynamics of key pollinators and identify potential “rescue pollinators” capable of persisting in these human-altered landscapes. Using a landscape genetic approach, we assessed the impact of landscape structure on genetic differentiation in the widely-distributed tropical stingless bee Trigona spinipes (Apidae: Meliponini) across agricultural landscape mosaics composed of coffee plantations and Atlantic forest fragments in southeastern Brazil. We genotyped 115 bees at 16 specific and highly polymorphic microsatellite loci, developed using next-generation sequencing. Our results reveal that T. spinipes is capable of dispersing across remarkably long distances, as we did not find genetic differentiation across a 200 km range, nor fine-scale spatial genetic structure. Furthermore, gene flow was not affected by forest cover, land cover, or elevation, indicating that reproductive individuals are able to disperse well through agricultural landscapes and across altitudinal gradients. We also found evidence of a recent population expansion, suggesting that this opportunistic stingless bee is capable of colonizing degraded habitats. Our results thus suggest that T. spinipes can persist in heavily-altered landscapes and can be regarded as a rescue pollinator, potentially compensating for the decline of other native pollinators in degraded tropical landscapes.