OxyR-Like Improves Cell Hydrogen Peroxide Tolerance by Participating in Monocyte Chemotaxis and Oxidative Phosphorylation Regulation in Magnetospirillum Gryphiswaldense MSR-1

The formation of magnetosomes inside magnetotactic bacteria is a complex process strictly controlled by the intracellular metabolic regulatory system. A series of transcriptional regulators are involved in the biosynthesis of the magnetosome, including OxyR-Like protein, which is indispensable for the maturation of magnetosomes in Magnetospirillum Gryphiswaldense MSR-1. In this study, a new function of the OxyR-Like protein that helps cells resist reactive oxygen species (ROS) was identified. A comparison of expression profile data between wild-type MSR-1 and an oxyR-Like defective mutant demonstrated that seven genes encoding chemotaxis proteins were down-regulated in the latter. On the contrary, the expression levels of numerous genes encoding proteins that are critical for cellular aerobic respiration were up-regulated. Thus, OxyR-Like enhanced the resistance of cells to ROS by increasing their environmental perception and maintaining their oxidative phosphorylation at a reasonable level to avoid the excessive production of endogenous ROS. These results increase our knowledge of the OxyR-Like regulatory network and establish a relationship between the antioxidant metabolic pathway and magnetosome biomineralization in MSR-1.

Genetic mapping and candidate gene identification of BoGL5, a gene essential for cuticular wax biosynthesis in broccoli

Background The aerial organs of most terrestrial plants are covered by cuticular waxes, which impart plants a glaucous appearance and play important roles in protecting against various biotic and abiotic stresses. Despite many glossy green (wax-defective) mutants being well characterized in model plants, little is known about the genetic basis of glossy green mutant in broccoli. Results B156 is a spontaneous broccoli mutant showing a glossy green phenotype. Detection by scanning electron microscopy (SEM) and chromatography-mass spectrometry (GC-MS) revealed that B156 is a cuticular wax-defective mutant, lacking waxes mostly longer than C28. Inheritance analysis revealed that this trait was controlled by a single recessive gene, BoGL5. Whole-genome InDel markers were developed, and a segregating F2 population was constructed to map BoGL5. Ultimately, BoGL5 was mapped to a 94.1 kb interval on C01. The BoCER2 gene, which is homologous to the Arabidopsis CER2 gene, was identified as a candidate of BoGL5 from the target interval. Sequence analyses revealed that Bocer2 in B156 harbored a G-to-T SNP mutation at the 485th nucleotide of the CDS, resulting in a W-to-L transition at the 162nd amino acid, a conserved site adjacent to an HXXXD motif of the deduced protein sequence. Expression analysis revealed that BoCER2 was significantly down-regulated in the leaves, stems, and siliques of B156 mutant than that of B3. Last, ectopic expression of BoCER2 in A. thaliana could, whereas Bocer2 could not, rescue the phenotype of cer2 mutant. Conclusions Overall, this study mapped the locus determining glossy phenotype of B156 and proved BoCER2 is functional gene involved in cuticular wax biosynthesis which would promotes the utilization of BoCER2 to enhance plant resistance to biotic and abiotic stresses, and breeding of B. oleracea cultivars with glossy traits.

Disulfide-compatible phage-assisted continuous evolution in the periplasmic space

The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution.

ppGpp Is Present in, and Regulates Sleep of, Drosophila

Discovery of molecules in living systems and demonstration of their functional roles are pivotal in furthering our understanding of the molecular basis of biology. ppGpp (guanosine-5’-diphosphate, 3’-diphosphate) has been detected in prokaryotes for more than five decades. Here we report that a genetic screen followed by chemical analysis revealed the presence of ppGpp in Drosophila.It can be detected in germ-free Drosophila and its level is controlled by an enzyme encoded by the mesh1 gene in Drosophila. Loss of function mutations in mesh1, which encoded the ppGpp degrading enzyme led to longer sleep latency and less total sleep. These phenotypes could be rescued by wild type mesh1, but not by the enzymatically defective mutant Mesh1E66A, functionally implicating ppGpp. Ectopic expression of RelA, the E. colisynthetase for ppGpp, phenocopied mesh1 knockout mutants, whereas overexpression of mesh1 resulted in the opposite phenotypes, supporting that ppGpp is both necessary and sufficient in sleep regulation. mesh1 is expressed in a specific population of neurons, and a chemoconnectomic screen followed by genetic intersection experiments implicate the pars intercerebralis (PI) as the site of ppGpp function. Our results have thus revealed that ppGpp is present in animals after long lag since its discovery in bacteria. Furthermore, we have demonstrated that ppGpp in a specific subset of neurons plays a physiological role in regulating sleep.

Arginine Methylation of hnRNPK Inhibits the DDX3-hnRNPK Interaction to Play an Anti-Apoptosis Role in Osteosarcoma Cells

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA binding protein involved in diverse cell processes; it is also a p53 coregulator that initiates apoptosis under DNA damage conditions. However, the upregulation of hnRNPK is correlated with cancer transformation, progression, and migration, whereas the regulatory role of hnRNPK in cancer malignancy remains unclear. We previously showed that arginine methylation of hnRNPK attenuated the apoptosis of U2OS osteosarcoma cells under DNA damage conditions, whereas the replacement of endogenous hnRNPK with a methylation-defective mutant inversely enhanced apoptosis. The present study further revealed that an RNA helicase, DDX3, whose C-terminus preferentially binds to the unmethylated hnRNPK and could promote such apoptotic enhancement. Moreover, C-terminus-truncated DDX3 induced significantly less apoptosis than full-length DDX3. Notably, we also identified a small molecule that docks at the ATP-binding site of DDX3, promotes the DDX3-hnRNPK interaction, and induces further apoptosis. Overall, we have shown that the arginine methylation of hnRNPK suppresses the apoptosis of U2OS cells via interfering with DDX3–hnRNPK interaction. On the other hand, DDX3–hnRNPK interaction with a proapoptotic role may serve as a target for promoting apoptosis in osteosarcoma cells.

Interplay between Non-Coding RNA Transcription, Stringent/Relaxed Phenotype and Antibiotic Production in Streptomyces ambofaciens

While in recent years the key role of non-coding RNAs (ncRNAs) in the regulation of gene expression has become increasingly evident, their interaction with the global regulatory circuits is still obscure. Here we analyzed the structure and organization of the transcriptome of Streptomyces ambofaciens, the producer of spiramycin. We identified ncRNAs including 45 small-RNAs (sRNAs) and 119 antisense-RNAs (asRNAs I) that appear transcribed from dedicated promoters. Some sRNAs and asRNAs are unprecedented in Streptomyces and were predicted to target mRNAs encoding proteins involved in transcription, translation, ribosomal structure and biogenesis, and regulation of morphological and biochemical differentiation. We then compared ncRNA expression in three strains: (i) the wild-type strain; (ii) an isogenic pirA-defective mutant with central carbon metabolism imbalance, “relaxed” phenotype, and repression of antibiotic production; and (iii) a pirA-derivative strain harboring a “stringent” RNA polymerase that suppresses pirA-associated phenotypes. Data indicated that the expression of most ncRNAs was correlated to the stringent/relaxed phenotype suggesting novel effector mechanisms of the stringent response.

Does the lack of root hairs alter root system architecture of Zea mays?

Aims Root hairs are one root trait among many which enables plants to adapt to environmental conditions. How different traits are coordinated and whether some are mutually exclusive is currently poorly understood. Comparing a root hair defective mutant with its corresponding wild-type, we explored if and how the mutant exhibited root growth adaptation strategies and how dependent this was on substrate. Methods Zea mays root hair defective mutant (rth3) and the corresponding wild-type siblings were grown under well-watered conditions on two substrates with contrasting texture and hence nutrient mobility. Root system architecture was investigated over time using repeated X-ray computed tomography. Results There was no plastic adaptation of root system architecture to the lack of root hairs, which resulted in lower uptake of nutrients especially in the substrate with high sorption capacity. The function of the root hairs for anchoring did not result in different root length density profiles between genotypes. Both maize genotypes showed a marked response to substrate. This was well reflected in the spatiotemporal development of rhizosphere volume fraction but especially in the highly significant response of root diameter to substrate, irrespective of genotype. Conclusions The most salient root plasticity trait was root diameter in response to substrate. Coping mechanisms for missing root hairs were limited to a shift in root-shoot ratio in loam. Further experiments are required, to elucidate whether observed differences can be explained by mechanical properties beyond mechanical impedance, root or microbiome ethylene production or differences in diffusion processes within the root or the rhizosphere.

Live imaging of delamination in Drosophila shows that epithelial cell motility and invasiveness are independently regulated

Delamination requires cells to undergo changes in cell-cell adhesion and in cell polarity, motility, and protrusions. This complex process must be precisely regulated during development as well as in pathogenic conditions. To determine the requirements for epithelial delamination, we analyzed the delamination of Drosophila ovary border cells, in which cells delaminate from the epithelial layer and begin to migrate collectively as is also seen in cancer metastasis. We used live imaging to examine cellular dynamics in delamination-defective mutants during the period in which delamination occurs in the wild-type ovary. We found that border cells in slow border cells (slbo), a delamination-defective mutant, lacked the properties of invasive cellular extensions but acquired motility while JAK/STAT-inhibited border cells lost both cellular properties, suggesting that the invasiveness and motility required for delamination are regulated independently. Our reconstruction experiments showed that motility is not a prerequisite for acquiring invasiveness.

Silicon Enhances Plant Resistance of Rice against Submergence Stress

Flooding is an important natural disaster limiting rice production. Silicon (Si) has been shown to have an important role in alleviating varied environmental stress. However, very few studies have investigated the effects and mechanisms of Si in alleviating flood stress in rice. In the present study, wild type rice (cv. Oochikara, WT) and Si-defective mutant (lsi1) were chosen to examine the impacts of Si application on plant growth, photosynthesis, cell structure, and antioxidant enzyme activity of rice exposed to submergence stress at tillering stage. Our results showed that Si application improved root morphological traits, and increased Si uptake and plant biomass of WT under submergence stress, but non-significantly influenced lsi1 mutant. Under submergence stress, leaf photosynthesis of WT was significantly inhibited, and Si application had no significant effects on photosynthetic rate, transpiration rate, stomatal conductance, and intercellular carbon dioxide concentration for both of WT and lsi1 mutant, but the photochemical quenching of WT was increased and the integrity of cell structure was improved. In addition, Si application significantly reduced malondialdehyde concentration and increased the activity of peroxidase and catalase in WT leaves under submergence stress. These results suggested that Si could increase rice plant resistance against submergence stress by improving root morphological traits and chloroplast ultrastructure and enhancing antioxidant defense.