Core Stress Response Research Articles

IraM remodels the RssB segmented helical linker to stabilize σs against degradation by ClpXP

Upon Mg2+ starvation, a condition often associated with virulence, enterobacteria inhibit the ClpXP-dependent proteolysis of the master transcriptional regulator, σs, via IraM, a poorly understood antiadaptor that prevents RssB-dependent loading of σs onto ClpXP. This inhibition results in σs accumulation and expression of stress resistance genes. Here, we report on the structural analysis of RssB bound to IraM, which reveals that IraM induces two folding transitions within RssB, amplified via a segmented helical linker. These conformational changes result in an open, yet inhibited RssB structure in which IraM associates with both the C-terminal and N-terminal domains of RssB and prevents binding of σs to the 4-5-5 face of the N-terminal receiver domain. This work highlights the remarkable structural plasticity of RssB and reveals how a stress-specific RssB antagonist modulates a core stress response pathway that could be leveraged to control biofilm formation, virulence, and the development of antibiotic resistance.

EIF3d controls the persistent integrated stress response

Cells respond to intrinsic and extrinsic stresses by reducing global protein synthesis and activating gene programs necessary for survival. Here, we show that the integrated stress response (ISR) is driven by the non-canonical cap-binding protein eIF3d that acts as a critical effector to control core stress response orchestrators, the translation factor eIF2α and the transcription factor ATF4. We find that during persistent stress, eIF3d activates the translation of the kinase GCN2, inducing eIF2α phosphorylation and inhibiting general protein synthesis. In parallel, eIF3d upregulates the m6A demethylase ALKBH5 to drive 5' UTR-specific demethylation of stress response genes, including ATF4. Ultimately, this cascade converges on ATF4 expression by increasing mRNA engagement of translation machinery and enhancing ribosome bypass of upstream open reading frames (uORFs). Our results reveal that eIF3d acts in a life-or-death decision point during chronic stress and uncover a synergistic signaling mechanism in which translational cascades complement transcriptional amplification to control essential cellular processes.

Honey bee sHSP are responsive to diverse proteostatic stresses and potentially promising biomarkers of honey bee stress

The pollination services provided by the honey bee are critical in both natural and agricultural ecosystems. Honey bee colonies in the United States have suffered from an increased rate of die-off in recent years, stemming from a complex set of interacting stresses that remain poorly described. Defining specific common cellular processes and cellular stress responses impacted by multiple stressors represent a key step in understanding these synergies. Proteotoxic stresses negatively impact protein synthesis, folding, and degradation. Diverse proteotoxic stresses induce expression of genes encoding small heat shock proteins (sHSP) of the expanded lethal (2) essential for life (l(2)efl) gene family. In addition to upregulation by the Integrated Stress Response (ISR), the Heat Shock Response (HSR), and the Oxidative Stress Response (OSR), our data provide first evidence that sHSP genes are upregulated by the Unfolded Protein Response (UPR). As these genes appear to be part of a core stress response that could serve as a useful biomarker for cellular stress in honey bees, we designed and tested an RT-LAMP assay to detect increased l(2)efl gene expression in response to heat-stress. While this assay provides a powerful proof of principle, further work will be necessary to link changes in sHSP gene expression to colony-level outcomes, to adapt our preliminary assay into a Point of Care Testing (POCT) assay appropriate for use as a diagnostic tool for use in the field, and to couple assay results to management recommendations.

Whole-genome sequencing, genome mining, metabolic reconstruction and evolution of pentachlorophenol and other xenobiotic degradation pathways in Bacillus tropicus strain AOA-CPS1.

A pentachlorophenol degrading bacterium was isolated from effluent of a wastewater treatment plant in Durban, South Africa, and identified as Bacillus tropicus strain AOA-CPS1 (BtAOA). The isolate degraded 29% of pentachlorophenol (PCP) within 9 days at an initial PCP concentration of 100 mg L-1 and 62% of PCP when the initial concentration was set at 350 mg L-1. The whole-genome of BtAOA was sequenced using Pacific Biosciences RS II sequencer with the Single Molecule, Real-Time (SMRT) Link (version 7.0.1.66975) and analysed using the HGAP4-de-novo assembly application. The contigs were annotated at NCBI, RASTtk and PROKKA prokaryotic genome annotation pipelines. The BtAOA genome is comprised of a 5,246,860-bp chromosome and a 58,449-bp plasmid with a GC content of 35.4%. The metabolic reconstruction for BtAOA showed that the organism has been naturally exposed to various chlorophenolic compounds including PCP and other xenobiotics. The chromosome encodes genes for core processes, stress response and PCP catabolic genes. Analogues of PCP catabolic gene (cpsBDCAE, and p450) sequences were identified from the NCBI annotation data, PCR-amplified from the whole genome of BtAOA, cloned into pET15b expression vector, overexpressed in E. coli BL21 (DE3) expression host, purified and characterized. Sequence mining and comparative analysis of the metabolic reconstruction of the BtAOA genome with closely related strains suggests that the operon encoding the first two enzymes in the PCP degradation pathway were acquired from a pre-existing pterin-carbinolamine dehydratase subsystem. The other two enzymes were recruited via horizontal gene transfer (HGT) from the pool of hypothetical proteins with no previous specific function, while the last enzyme was recruited from pre-existing enzymes from the TCA or serine-glyoxalase cycle via HGT events. This study provides a comprehensive understanding of the role of BtAOA in PCP degradation and its potential exploitation for bioremediation of other xenobiotic compounds.

Anticipatory Stress Responses and Immune Evasion in Fungal Pathogens.

In certain niches, microbes encounter environmental challenges that are temporally linked. In such cases, microbial fitness is enhanced by the evolution of anticipatory responses where the initial challenge simultaneously activates pre-emptive protection against the second impending challenge. The accumulation of anticipatory responses in domesticated yeasts, which have been termed 'adaptive prediction', has led to the emergence of 'core stress responses' that provide stress cross-protection. Protective anticipatory responses also seem to be common in fungal pathogens of humans. These responses reflect the selective pressures that these fungi have faced relatively recently in their evolutionary history. Consequently, some pathogens have evolved 'core environmental responses' which exploit host signals to trigger immune evasion strategies that protect them against imminent immune attack.

Effects of Soil Microbes on Forest Recovery to Climax Community through the Regulation of Nitrogen Cycling

Microbes, as important regulators of ecosystem processes, play essential roles in ecosystem recovery after disturbances. However, it is not clear how soil microbial communities and functions change and affect forest recovery after clear-cutting. Here, we used metagenome sequencing to systematically analyse the differences in soil microbial community composition, functions, and nitrogen (N) cycling pathways between primary Korean pine forests (PF) and secondary broad-leaved forests (SF) formed after clear-cutting. Our results showed that the dominant phyla of the two forest types were consistent, but the relative abundance of some phyla was significantly different. Meanwhile, at the genus level, the fold-changes of rare genera were larger than the dominant and common genera. The genes related to microbial core metabolic functions, virulence factors, stress response, and defence were significantly enriched in SF. Additionally, based on the relative abundance of functional genes, a schema was proposed to analyse the differences in the whole N cycling processes between the two forest types. In PF, the stronger ammoniation and dissimilatory nitrate reduction (DNRA) and the weaker nitrification provided a genetic explanation for PF dominated by ammonium (NH4+) rather than nitrate (NO3−). In SF, the weaker DNRA, the stronger nitrification and denitrification, the higher soil available phosphorus (AP), and the lower nitrogen to phosphorus ratio (N/P) comprehensively suggested that SF was faced with a greater degree of N limitation. These results offer insights into the potential relationship between soil microbes and forest recovery, and aid in implementing proper forestry management.

Genome-wide Identification, Expression Profiling and Promoter Analysis of Trehalose-6-Phosphate Phosphatase Gene Family in Rice

Trehalose-6-phosphate phosphatase (TPP) plays a key role in trehalose metabolism in plants. Here, we performed comprehensive in silico analyses and identified 12 OsTPPs (Oryza sativa TPPs) utilizing various bioinformatics tools. Phylogenetic tree, accomplished with OsTPPs and TPPs from 11 monocot and dicot species, was divided mainly into two clades, each clade containing six OsTPPs. Exon–intron distribution was related to phylogenetic clades. All OsTPPs are distributed within nine chromosomes (chr.), except Chr. 1, Chr. 5 and Chr. 11. OsTPPs were found to be stable in nature according to the 3-D structure prediction. Cis-regulatory elements (CREs) were also analyzed using 2 kb upstream of start codon for each gene to predict their biological functions. We categorized all CREs in five distinct groups based on core elements, stress response, cellular development, hormonal regulation, and unknown function, distributed in a range of 3–14 CREs in each group. Interestingly, our expression analysis showed that OsTPPs were more upregulated in response to drought and cold stresses compared to salt stress. Abundance of stress-related CREs found signifies TPPs’ possible role in stress response, which may facilitate to find related transcription factors and unveil complex molecular mechanisms during stress response.

Application of pulsed electric fields for the biocompatible extraction of proteins from the microalga Haematococcus pluvialis

This study aims to employ a pulsed electric field (PEF) treatment for the biocompatible (non-destructive) extraction of proteins from living cells of the green microalga Haematococcus pluvialis. Using a field strength of 1 kV cm−1, we achieved the extraction of 10.2 µg protein per mL of culture, which corresponded to 46% of the total amount of proteins that could be extracted by complete destructive extraction (i.e. the grinding of biomass with glass beads). We found that the extraction yield was not improved by stronger field strengths and was not dependent on the pulse frequency. A biocompatibility index (BI) was defined as the relative abundance of cells that remained alive after the PEF treatment. This index relied on measurements of several physiological parameters after a PEF treatment. It was found that at 1 kV cm−1 that cultures recovered after 72 h. Therefore, these PEF conditions constituted a good compromise between protein extraction efficiency and culture survival. To characterize the PEF treatment further at a molecular level, mass spectrometry-based proteomics analyses of PEF-prepared extracts was used. This led to the identification of 52 electro-extracted proteins. Of these, only 16 proteins were identified when proteins were extracted with PEF at 0.5 cm−1. They belong to core metabolism, stress response and cell movement. Unassigned proteins were also extracted. Their physiological implications and possible utilization in food as alimentary complements are discussed.

Transcriptional Basis for Differential Thermosensitivity of Seedlings of Various Tomato Genotypes.

Transcriptional reprograming after the exposure of plants to elevated temperatures is a hallmark of stress response which is required for the manifestation of thermotolerance. Central transcription factors regulate the stress survival and recovery mechanisms and many of the core responses controlled by these factors are well described. In turn, pathways and specific genes contributing to variations in the thermotolerance capacity even among closely related plant genotypes are not well defined. A seedling-based assay was developed to directly compare the growth and transcriptome response to heat stress in four tomato genotypes with contrasting thermotolerance. The conserved and the genotype-specific alterations of mRNA abundance in response to heat stress were monitored after exposure to three different temperatures. The transcripts of the majority of genes behave similarly in all genotypes, including the majority of heat stress transcription factors and heat shock proteins, but also genes involved in photosynthesis and mitochondrial ATP production. In turn, genes involved in hormone and RNA-based regulation, such as auxin- and ethylene-related genes, or transcription factors like HsfA6b, show a differential regulation that associates with the thermotolerance pattern. Our results provide an inventory of genes likely involved in core and genotype-dependent heat stress response mechanisms with putative role in thermotolerance in tomato seedlings.

Blue Mussel (Genus Mytilus) Transcriptome Response to Simulated Climate Change in the Gulf of Maine

The biogeochemistry of the Gulf of Maine (GOM) is rapidly changing in response to the changing climate, including rising temperatures, acidification, and declining primary productivity. These impacts are projected to worsen over the next 100 y and will apply selective pressure on populations of marine calcifiers. This study investigates the transcriptome expression response to these changes in ecologically and economically important marine calcifiers, blue mussels. Wild mussels (Mytilus edulis and Mytilus trossulus) were sampled from sites spanning the GOM and exposed to two different biogeochemical water conditions: (1) present-day conditions in the GOM and (2) simulated future conditions, which included elevated temperature, increased acidity, and decreased food supply. Patterns of gene expression were measured using RNA sequencing from 24 mussel samples and contrasted between ambient and future conditions. The net calcification rate, a trait predicted to be under climate-induced stress, was measured for each individual over a 2-wk exposure period and used as a covariate along with gene expression patterns. Generalized linear models, with and without the calcification rate, were used to identify differentially expressed transcripts between ambient and future conditions. The comparison revealed transcripts that likely comprise a core stress response characterized by the induction of molecular chaperones, genes involved in aerobic metabolism, and indicators of cellular stress. Furthermore, the model contrasts revealed transcripts that may be associated with individual variation in calcification rate and suggest possible biological processes that may have downstream effects on calcification phenotypes, such as zinc-ion binding and protein degradation. Overall, these findings contribute to the understanding of blue mussel adaptive responses to imminent climate change and suggest metabolic pathways are resilient in variable environments.

Abiotic and biotic stresses induce a core transcriptome response in rice

Environmental stresses greatly limit crop yield. With the increase in extreme weather events due to climate change and the constant pressure of diseases and pests, there is an urgent need to develop crop varieties that can tolerate multiple stresses. However, our knowledge of how plants broadly respond to stress is limited. Here, we explore the rice core stress response via meta-analysis of publicly available rice transcriptome data. Our results confirm that rice universally down-regulates photosynthesis in response to both abiotic and biotic stress. Rice also generally up-regulates hormone-responsive genes during stress response, most notably genes in the abscisic acid, jasmonic acid and salicylic acid pathways. We identified several promoter motifs that are likely involved in stress-responsive regulatory mechanisms in rice. With this work, we provide a list of candidate genes to study for improving rice stress tolerance in light of environmental stresses. This work also serves as a proof of concept to show that meta-analysis of diverse transcriptome data is a valid approach to develop robust hypotheses for how plants respond to stress.

Transcriptional regulatory networks in Arabidopsis thaliana during single and combined stresses.

Differentially evolved responses to various stress conditions in plants are controlled by complex regulatory circuits of transcriptional activators, and repressors, such as transcription factors (TFs). To understand the general and condition-specific activities of the TFs and their regulatory relationships with the target genes (TGs), we have used a homogeneous stress gene expression dataset generated on ten natural ecotypes of the model plant Arabidopsis thaliana, during five single and six combined stress conditions. Knowledge-based profiles of binding sites for 25 stress-responsive TF families (187 TFs) were generated and tested for their enrichment in the regulatory regions of the associated TGs. Condition-dependent regulatory sub-networks have shed light on the differential utilization of the underlying network topology, by stress-specific regulators and multifunctional regulators. The multifunctional regulators maintain the core stress response processes while the transient regulators confer the specificity to certain conditions. Clustering patterns of transcription factor binding sites (TFBS) have reflected the combinatorial nature of transcriptional regulation, and suggested the putative role of the homotypic clusters of TFBS towards maintaining transcriptional robustness against cis-regulatory mutations to facilitate the preservation of stress response processes. The Gene Ontology enrichment analysis of the TGs reflected sequential regulation of stress response mechanisms in plants.

Core oxidative stress response in Aspergillus nidulans.

BackgroundThe b-Zip transcription factor AtfA plays a key role in regulating stress responses in the filamentous fungus Aspergillus nidulans. To identify the core regulons of AtfA, we examined genome-wide expression changes caused by various stresses in the presence/absence of AtfA using A. nidulans microarrays. We also intended to address the intriguing question regarding the existence of core environmental stress response in this important model eukaryote.ResultsExamination of the genome wide expression changes caused by five different oxidative stress conditions in wild type and the atfA null mutant has identified a significant number of stereotypically regulated genes (Core Oxidative Stress Response genes). The deletion of atfA increased the oxidative stress sensitivity of A. nidulans and affected mRNA accumulation of several genes under both unstressed and stressed conditions. The numbers of genes under the AtfA control appear to be specific to a stress-type. We also found that both oxidative and salt stresses induced expression of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of additional clusters. Moreover, certain clusters were down-regulated by the stresses tested.ConclusionOur data suggest that the observed co-regulations were most likely consequences of the overlapping physiological effects of the stressors and not of the existence of a general environmental stress response. The function of AtfA in governing various stress responses is much smaller than anticipated and/or other regulators may play a redundant or overlapping role with AtfA. Both stress inducible and stress repressive regulations of secondary metabolism seem to be frequent features in A. nidulans.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1705-z) contains supplementary material, which is available to authorized users.

A Polyketide cyclase/dehydrase and lipid transport superfamily gene of Arabidopsis and its orthologue of chickpea exhibit rapid response to wounding

In order to understand the mechanism of stress tolerance it is imperative that we identify factors that govern upstream reactions of stress signalling pathways for multiple stress tolerance. In this study, we have identified a gene, AT5G54170, from Arabidopsis and its orthologue in chickpea, which show rapid response to mechanical wounding. AT5G54170 and its orthologue in chickpea (LOC 101512743) most likely plays a role in the jasmonic acid signalling pathway and the gene is also upregulated in several other stresses making it an ideal candidate for a primary stress-responsive transcript that is involved in the core stress response. Further characterisation of this gene and its promoter, along with its chickpea homologue, could provide valuable information on jasmonic acid signalling pathway and also explore their utility towards engineering stress tolerance in plants.

A proteomic analysis of the salt, cadmium and peroxide stress responses inCandida albicansand the role of the Hog1 stress‐activated MAPK in regulating the stress‐induced proteome

Stress responses are important for the virulence of the major fungal pathogen of humans, Candida albicans. In this study we employed a 2-DE approach to examine the impact of exposure to peroxide (5 mM H(2)O(2)), salt (300 mM NaCl) or cadmium stress (0.5 mM Cd(2+)) upon the C. albicans proteome. Highly reproducible changes in the C. albicans proteome were observed in response to each stress condition. Significantly more proteins were up-regulated in response to cadmium (77) than to the salt (35) or peroxide stresses (35). These proteomic changes displayed minimal overlap with those observed in the transcriptome under equivalent conditions and, importantly, revealed functional categories that respond to stress at the protein level but not the transcript level. Six proteins were up-regulated by all three conditions: Adh1, Atp2, Cip1, Eft2, Ssa1 and Ssb1, which is consistent with the concept that a core stress response exists in C. albicans. This is the first time that a fungal core stress response has been defined at the proteomic level. We have also shown that the Hog1 stress-activated mitogen-activated protein kinase, which is activated in response to the stresses examined in this study, makes a major contribution to the C. albicans stress proteome.

An Intracellular Serpin Regulates Necrosis by Inhibiting the Induction and Sequelae of Lysosomal Injury

Extracellular serpins such as antithrombin and alpha1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine.