Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of Schizosaccharomyces pombe AAA+ ATPase Abo1

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.     

Genome-wide Identification,Phylogenetic Analysis,and Expression Profiling of CONSTANS-like (COL) Genes in Vitis vinifera

The CONSTANS (CO) gene plays an important role in the flowering of plants. However, the other precise roles of the CO gene are poorly understood. We carried out a genomic census and analysis of expression patterns for CONSTANS-like genes in Vitis vinifera (VviCOLs) to reveal the molecular characteristics of VviCOLs. Twelve VviCOLs were identified and 11 of their full-length complementary DNAs were cloned. Multiple sequence alignment suggested the VviCOLs contained B-box and CCT conserved domains. We further classified the VviCOLs into three groups according to the variability of the second B-box domain. Synteny analysis showed that eight orthologous gene pairs were identified between grapevine and Arabidopsis, suggesting that eight pairs may descend from a common evolutionary ancestor. Tissue expression analysis of COL genes in cv. Pinot Noir showed VviCOL11a and VviCOL11b were specifically expressed in flower bud, whereas VviCOL16b was only expressed in leaves. Ten VviCOLs were expressed in the developing ovule and six of them showed higher expression in the ovule of cv. Thompson Seedless than that of cv. Pinot Noir, indicating that VviCOLs were involved in the process of seed development or ovule abortion. Furthermore, nine of twelve VviCOLs were expressed in cv. Pinot Noir leaves and all of these nine genes had a response to exogenous hormone application. In summary, our findings provide a new insight into the further studies of VviCOLs, especially in terms of seed development and hormone response.

     

Mitochondrial chaperone HSP-60 regulates anti-bacterial immunity via p38 MAP kinase signaling

Mitochondria play key roles in cellular immunity. How mitochondria contribute to organismal immunity remains poorly understood. Here, we show that HSP-60/HSPD1, a major mitochondrial chaperone, boosts anti-bacterial immunity through the up-regulation of p38 MAP kinase signaling. We first identify 16 evolutionarily conserved mitochondrial components that affect the immunity of Caenorhabditis elegans against pathogenic Pseudomonas aeruginosa (PA14). Among them, the mitochondrial chaperone HSP-60 is necessary and sufficient to increase resistance to PA14. We show that HSP-60 in the intestine and neurons is crucial for the resistance to PA14. We then find that p38 MAP kinase signaling, an evolutionarily conserved anti-bacterial immune pathway, is down-regulated by genetic inhibition of hsp-60, and up-regulated by increased expression of hsp-60. Overexpression of HSPD1, the mammalian ortholog of hsp-60, increases p38 MAP kinase activity in human cells, suggesting an evolutionarily conserved mechanism. Further, cytosol-localized HSP-60 physically binds and stabilizes SEK-1/MAP kinase kinase 3, which in turn up-regulates p38 MAP kinase and increases immunity. Our study suggests that mitochondrial chaperones protect host eukaryotes from pathogenic bacteria by up-regulating cytosolic p38 MAPK signaling.     

The sulfur formation system mediating extracellular cysteine-cystine recycling in Fervidobacterium islandicum AW-1 is associated with keratin degradation

Most extremophilic anaerobes possess a sulfur formation (Suf) system for Fe–S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe–S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW-1. Comparative genomics of the order Thermotogales revealed that the feather-degrading F. islandicum AW-1 has a complete Suf-like machinery (SufCBDSU) that is highly expressed in cells grown on native feathers in the absence of elemental sulfur (S⁰). On the other hand, F. islandicum AW-1 exhibited a significant retardation in the Suf system-mediated keratin degradation in the presence of S⁰. Detailed differential expression analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Indeed, addition of SufS–SufU to cell extracts containing keratinolytic proteases accelerated keratin decomposition in vitro under reducing conditions. Remarkably, mass spectrometric analysis of extracellular and intracellular levels of amino acids suggested that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf-like machinery including the SufS–SufU complex may contribute to sulfur availability for an extracellular reducing environment as well as intracellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions.     

Erythrocyte adenosine A2B receptor prevents cognitive and auditory dysfunction by promoting hypoxic and metabolic reprogramming

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O₂) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O₂ release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte–specific ADORA2B (eAdora2b^−/−) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b^-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b^−/− mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O₂ delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B–BPGM axis is a key component for anti-aging and anti-age–related functional decline.

Hypoxia drives aging and promotes age-related functional decline in cognition and hearing. This study shows that signaling through the erythrocyte adenosine A2B receptor promotes metabolic reprogramming, leading to increased production of 2,3-bisphosphoglycerate and lowering hypoxia-induced inflammatory responses in the hippocampus, cortex and cochlea during aging.     

Biosynthesis and production of sabinene: current state and perspectives

Applied Microbiology and Biotechnology - Sabinene is an important naturally occurring bicyclic monoterpene which can be used as flavorings, perfume additives, fine chemicals, and advanced biofuels....     

Induction of gene expression in bacteria at optimal growth temperatures

Traditional temperature-sensitive systems use either heat shock (40–42 °C) or cold shock (15–23 °C) to induce gene expression at temperatures that are not the optimal temperature for host cell growth (37 °C). This impacts the overall productivity and yield by disturbing cell growth and cellular metabolism. Here, we have developed a new system which controls gene expression in Escherichia coli at more permissive temperatures. The temperature-sensitive cI857-P _L system and the classic lacI-P _lacO system were connected in series to control the gene of interest. When the culture temperature was lowered, the thermolabile cI857 repressor was activated and blocked the expression of lacI from P _L. Subsequently, the decrease of LacI derepressed the expression of gene of interest from P _lacO . Using a green fluorescent protein marker, we demonstrated that (1) gene expression was tightly regulated at 42 °C and strongly induced by lowering temperature to 25–37 °C; (2) different levels of gene expression can be induced by varying culture temperature; and (3) gene expression after induction was sustained until the end of the log phase. We then applied this system in the biosynthesis of acetoin and demonstrated that high yield and production could be achieved using temperature induction. The ability to express proteins at optimal growth temperatures without chemical inducers is advantageous for large-scale and industrial fermentations.