Evidence against the physiological role of acetyl phosphate in the phosphorylation of the ArcA response regulator in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Arc two-component signal transduction system of Escherichia coli comprises the ArcB sensor kinase and the ArcA response regulator. Under anoxic growth conditions, ArcB autophosphorylates and transphos-phorylates ArcA, which, in turn, represses or activates its target operons. ArcA has been shown to be able to autophosphorylate in vitro at the expense of acetyl-P. Here, the in vivo effect of acetyl phosphate on the redox signal transduction by the Arc system was assessed. Our results indicate that acetyl phosphate can modulate the expression of ArcA-P target genes only in the absence of ArcB. Therefore, the acetyl phosphate dependent ArcA phosphorylation route does not seem to play a significant role under physiological conditions.     

Engineering key components in a synthetic eukaryotic signal transduction pathway

Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB‐VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB‐VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved‐signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways.     

The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis

Bacterial pathogens regulate virulence factor gene expression coordinately in response to environmental stimuli, including nutrient starvation. The phosphate (Pho) regulon plays a key role in phosphate homeostasis. It is controlled by the PhoR/PhoB two-component regulatory system. PhoR is an integral membrane signaling histidine kinase that, through an interaction with the ABC-type phosphate-specific transport (Pst) system and a protein called PhoU, somehow senses environmental inorganic phosphate (P(i)) levels. Under conditions of P(i) limitation (or in the absence of a Pst component or PhoU), PhoR activates its partner response regulator PhoB by phosphorylation, which, in turn, up- or down-regulates target genes. Single-cell profiling of PhoB activation has shown recently that Pho regulon gene expression exhibits a stochastic, "all-or-none" behavior. Recent studies have also shown that the Pho regulon plays a role in the virulence of several bacteria. Here, we present a comprehensive overview of the role of the Pho regulon in bacterial virulence. The Pho regulon is clearly not a simple regulatory circuit for controlling phosphate homeostasis; it is part of a complex network important for both bacterial virulence and stress response.     

The expressions of the SOX trio, PTHrP (parathyroid hormone-related peptide)/IHH (Indian hedgehog protein) in surgically induced osteoarthritis of the rat

This study was performed to investigate the expressions of the SOX trio, PTHrP (parathyroid hormone‐related peptide) and IHH (Indian hedgehog protein) in OA (osteoarthritis) using surgically induced rat OA model. After 12 weeks, the articular cartilage from the distal femur was harvested. The expressions of the SOX trio, PTHrP and IHH were explored at gene, protein and epigenetic levels by real‐time PCR (n=5), immunohistochemistry (n=5) and MSP (methylation‐specific PCR). The findings from OA cartilage of the right knees were compared with those from the left knees as the control. The gene expressions of SOX‐5, ?6, ?9 decreased by 58, 20 and 40%, respectively, in the OA cartilage, while their respective protein expressions increased. The PTHrP and IHH gene expressions decreased by 75 and 81%, respectively, although their protein expressions increased. Findings from MSP demonstrated increased methylation in the promoter regions of SOX‐5 and ?9 genes. This study demonstrated that increased methylation in the promoters of these genes may explain the low gene expression in the surgically induced OA model, whereas elevated protein expression is speculated to be from lag effect in the gene—protein expression.     

Joint genetic and network analyses identify loci associated with root growth under NaCl stress in Arabidopsis thaliana

Plants have evolved a series of tolerance mechanisms to saline stress, which perturbs physiological processes throughout the plant. To identify genetic mechanisms associated with salinity tolerance, we performed linkage analysis and genome‐wide association study (GWAS) on maintenance of root growth of Arabidopsis thaliana in hydroponic culture with weak and severe NaCl toxicity. The top 200 single‐nucleotide polymorphisms (SNPs) determined by GWAS could cumulatively explain approximately 70% of the variation observed at each stress level. The most significant SNPs were linked to the genes of ATP‐binding cassette B10 and vacuolar proton ATPase A2. Several known salinity tolerance genes such as potassium channel KAT1 and calcium sensor SOS3 were also linked to SNPs in the top 200. In parallel, we constructed a gene co‐expression network to independently verify that particular groups of genes work together to a common purpose. We identify molecular mechanisms to confer salt tolerance from both predictable and novel physiological sources and validate the utility of combined genetic and network analysis. Additionally, our study indicates that the genetic architecture of salt tolerance is responsive to the severity of stress. These gene datasets are a significant information resource for a following exploration of gene function.     

Chronic Ethanol Exposure Increases Cytochrome P‐450 and Decreases Activated in Blocked Unfolded Protein Response Gene Family Transcripts in Caenorhabditis elegans

Ethanol is a widely consumed and rapidly absorbed toxin. While the physiological effects of ethanol consumption are well known, the underlying biochemical and molecular changes at the gene expression level in whole animals remain obscure. We exposed the model organism Caenorhabditis elegans to 0.2 M ethanol from the embryo to L4 larva stage and assayed gene expression changes in whole animals using RNA‐Seq and quantitative real‐time PCR. We observed gene expression changes in 1122 genes (411 up, 711 down). Cytochrome P‐450 (CYP) gene family members (12 of 78) were upregulated, whereas activated in blocked unfolded protein response (ABU) (7 of 15) were downregulated. Other detoxification gene family members were also regulated including four glutathione‐S‐transferases and three flavin monooxygenases. The results presented show specific gene expression changes following chronic ethanol exposure in C. elegans that indicate both persistent upregulation of detoxification response genes and downregulation of endoplasmic reticulum stress pathway genes.     

Multinuclear Yeast Magnusiomyces (Dipodascus,Endomyces) magnusii is a Promising Isobutanol Producer

Higher alcohol isobutanol is a promising liquid fuel. During alcoholic fermentation, Saccharomyces cerevisiae produces only trace amounts of isobutanol. Screening the collection of nonconventional yeasts show that Magnusiomyces magnusii accumulates 440 mg of isobutanol per L in rich YPD medium. Here, the transformation protocol for M. magnusii is adapted based on the use of the dominant markers conferring resistance to nourseothricin or zeocin; the strong constitutive promoter TEF1 is cloned and a reporter system based on LAC4 gene from Kluyveromyces lactis coding for β‐galactosidase is constructed. In order to increase isobutanol production in M. magnusii, the heterologous gene ILV2 from S. cerevisiae is expressed in M. magnusii under control of the TEF1 promoter. The best stabilized transformants produce 620 mg of isobutanol per L in YPD medium and 760 mg L^?1 in the medium with 2‐oxoisovalerate. This suggests that M. magnusii is a promising organism for further development of a robust isobutanol producer.