Definition of the σW regulon of Bacillus subtilis in the absence of stress

Bacteria employ extracytoplasmic function (ECF) sigma factors for their responses to environmental stresses. Despite intensive research, the molecular dissection of ECF sigma factor regulons has remained a major challenge due to overlaps in the ECF sigma factor-regulated genes and the stimuli that activate the different ECF sigma factors. Here we have employed tiling arrays to single out the ECF σW regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σX, σY, and σM regulons. For this purpose, we profiled the transcriptome of a B. subtilis sigW mutant under non-stress conditions to select candidate genes that are strictly σW-regulated. Under these conditions, σW exhibits a basal level of activity. Subsequently, we verified the σW-dependency of candidate genes by comparing their transcript profiles to transcriptome data obtained with the parental B. subtilis strain 168 grown under 104 different conditions, including relevant stress conditions, such as salt shock. In addition, we investigated the transcriptomes of rasP or prsW mutant strains that lack the proteases involved in the degradation of the σW anti-sigma factor RsiW and subsequent activation of the σW-regulon. Taken together, our studies identify 89 genes as being strictly σW-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σW-dependent genes were relatively mild, which implies that σW-dependent transcription under non-stress conditions is not strictly related to RasP and PrsW. Lastly, we show that the pleiotropic phenotype of rasP mutant cells, which have defects in competence development, protein secretion and membrane protein production, is not mirrored in the transcript profile of these cells. This implies that RasP is not only important for transcriptional regulation via σW, but that this membrane protease also exerts other important post-transcriptional regulatory functions

Global Analysis of Extracytoplasmic Stress Signaling in Escherichia coli

The Bae, Cpx, Psp, Rcs, and σE pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signal-inducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response

The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

Background Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.This work was mainly supported by ERA Biotech (www.erabiotech.com). Additional support was supplied by grant SGR 2009/703 funded by the Generalitat de Catalunya (www10.gencat.net) and grants CDS2007/00036 of Consolider Ingenio program and TRA 2009/0124 of TRACE program funded by Ministerio de Ciencia e Inovación (MICINN, www.micinn.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Amphipathic DNA polymers exhibit antiviral activity against systemic Murine Cytomegalovirus infection

<p>Abstract</p> <p>Background</p> <p>Phosphorothioated oligonucleotides (PS-ONs) have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs) and exhibit potent in vitro antiviral activity against a broad spectrum of herpesviruses: HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-6A/B, and in vivo activity in a murine microbiocide model of genital HSV-2 infection. The activity of these agents against animal cytomegalovirus (CMV) infections in vitro and in vivo was therefore investigated.</p> <p>Results</p> <p>In vitro, a 40 mer degenerate AP (REP 9) inhibited both murine CMV (MCMV) and guinea pig CMV (GPCMV) with an IC₅₀of 0.045 μM and 0.16 μM, respectively, and a 40 mer poly C AP (REP 9C) inhibited MCMV with an IC₅₀of 0.05 μM. Addition of REP 9 to plaque assays during the first two hours of infection inhibited 78% of plaque formation whereas addition of REP 9 after 10 hours of infection did not significantly reduce the number of plaques, indicating that REP 9 antiviral activity against MCMV occurs at early times after infection. In a murine model of CMV infection, systemic treatment for 5 days significantly reduced virus replication in the spleens and livers of infected mice compared to saline-treated control mice. REP 9 and REP 9C were administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting 2 days prior to MCMV infection. Splenomegaly was observed in infected mice treated with REP 9 but not in control mice or in REP 9 treated, uninfected mice, consistent with mild CpG-like activity. When REP 9C (which lacks CpG motifs) was compared to REP 9, it exhibited comparable antiviral activity as REP 9 but was not associated with splenomegaly. This suggests that the direct antiviral activity of APs is the predominant therapeutic mechanism <it>in vivo</it>. Moreover, REP 9C, which is acid stable, was effective when administered orally in combination with known permeation enhancers.</p> <p>Conclusion</p> <p>These studies indicate that APs exhibit potent, well tolerated antiviral activity against CMV infection in vivo and represent a new class of broad spectrum anti-herpetic agents.</p

Preserving Charge and Oxidation State of Au(III) Ions in an Agent-Functionalized Nanocrystal Model System

Supporting functional molecules on crystal facets is an established technique in nanotechnology. To preserve the original activity of ionic metallorganic agents on a supporting template, conservation of the charge and oxidation state of, the active center is indispensable. We. present a model system of a metallorganic agent that, indeed, fulfills this design criterion on a technologically relevant metal support With potential Impact on Au(III)-porphyrin-functionalized nanoparticles for an improved anticancer-drug delivery. Employing scanning tunneling microscopy and -spectroscopy in combination with photoemission spectroscopy,we clarify at the single-molecule level the underlying mechanisms of this exceptional adsorption mode. It is based on the balance between a high-energy oxidation state and an electrostatic screening-response of the surface (image charge). Modeling with first principles methods reveals submolecular details of the metal-ligand bonding interaction and completes the study by providing an Illustrative electrostatic.. model relevant for ionic metalorganic agent molecules, in general

YaeL (EcfE) activates the sigma(E) pathway of stress response through a site-2 cleavage of anti-sigma(E), RseA.

[[sponsorship]]植物暨微生物學研究所[[note]]已出版;[SCI];有審查制度;具代表性[[note]]http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Drexel&SrcApp=hagerty_opac&KeyRecord=0890-9369&DestApp=JCR&RQ=IF_CAT_BOXPLOT[[note]]http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=RID&SrcApp=RID&DestLinkType=FullRecord&DestApp=ALL_WOS&KeyUT=00017751710001

Layered pattern receptor signaling via ethylene and endogenous elicitor peptides during Arabidopsis immunity to bacterial infection

Recognition of molecular patterns characteristic of microbes or altered-self leads to immune activation in multicellular eukaryotes. In Arabidopsis thaliana, the leucine-rich-repeat receptor kinases FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR (EFR) recognize bacterial flagellin and elongation factor EF-Tu (and their elicitor-active epitopes flg22 and elf18), respectively. Likewise, PEP1 RECEPTOR1 (PEPR1) and PEPR2 recognize the elicitor-active Pep epitopes conserved in Arabidopsis ELICITOR PEPTIDE PRECURSORs (PROPEPs). Here we reveal that loss of ETHYLENE-INSENSITIVE2 (EIN2), a master signaling regulator of the phytohormone ethylene (ET), lowers sensitivity to both elf18 and flg22 in different defense-related outputs. Remarkably, in contrast to a large decrease in FLS2 expression, EFR expression and receptor accumulation remain unaffected in ein2 plants. Genome-wide transcriptome profiling has uncovered an inventory of EIN2-dependent and EFR-regulated genes. This dataset highlights important aspects of how ET modulates EFR-triggered immunity: the potentiation of salicylate-based immunity and the repression of a jasmonate-related branch. EFR requires ET signaling components for PROPEP2 activation but not for PROPEP3 activation, pointing to both ET-dependent and -independent engagement of the PEPR pathway during EFR-triggered immunity. Moreover, PEPR activation compensates the ein2 defects for a subset of EFR-regulated genes. Accordingly, ein2 pepr1 pepr2 plants exhibit additive defects in EFR-triggered antibacterial immunity, compared with ein2 or pepr1 pepr2 plants. Our findings suggest that the PEPR pathway not only mediates ET signaling but also compensates for its absence in enhancing plant immunity

Repression of sucrose/ultraviolet-B light-induced flavonoid accumulation in microbe-associated molecular pattern-triggered immunity in Arabidopsis

Recognition of microbe-associated molecular patterns (MAMPs) leads to the generation of MAMP-triggered immunity (MTI), which restricts the invasion and propagation of potentially infectious microbes. It has been described that the perception of different bacterial and fungal MAMPs causes the repression of flavonoid induction upon light stress or sucrose application. However, the functional significance of this MTI-associated signaling output remains unknown. In Arabidopsis (Arabidopsis thaliana), FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR act as the pattern recognition receptors for the bacterial MAMP epitopes flg22 (of flagellin) and elf18 (of elongation factor [EF]-Tu), respectively. Here, we reveal that reactive oxygen species spiking and callose deposition are dispensable for the repression of flavonoid accumulation by both pattern recognition receptors. Importantly, FLS2-triggered activation of PATHOGENESIS-RELATED (PR) genes and bacterial basal defenses are enhanced in transparent testa4 plants that are devoid of flavonoids, providing evidence for a functional contribution of flavonoid repression to MTI. Moreover, we identify nine small molecules, of which eight are structurally unrelated, that derepress flavonoid accumulation in the presence of flg22. These compounds allowed us to dissect the FLS2 pathway. Remarkably, one of the identified compounds uncouples flavonoid repression and PR gene activation from the activation of reactive oxygen species, mitogen-activated protein kinases, and callose deposition, corroborating a close link between the former two outputs. Together, our data imply a model in which MAMP-induced repression of flavonoid accumulation serves a role in removing the inherent inhibitory action of flavonoids on an MTI signaling branch

Trends in psychological distress and alcoholism after The Great East Japan Earthquake of 2011

Aims: Many studies have shown that natural disasters affect mental health; however, longitudinal data on post-disaster mental health problems are scarce. The aims of our study were to investigate the trend in psychological distress and alcoholism after The Great East Japan Earthquake and tsunami in north eastern Japan, in March 2011. Methods: A longitudinal study was conducted using annual health check data for the general population, in the city of Higashi-Matsushima, which was affected by the high impact of tsunami. In 2012 and 2013, the Kessler Psychological Distress Scale and the CAGE questionnaire (for screening for alcoholism) were used to assess psychological distress and prevalence of alcoholism. Results: Of 11,855 total eligible residents, 2192 received the annual check in 2012 and 2013. The prevalence of mental illness and the mean score of alcoholism tendency increased during the follow-up period. The majority of respondents (43.8%) with baseline serious mental illness (SMI) continued to have SMI at follow-up; only 16.7% reported recovering. Older age, female sex, and severity of home damage predicted higher psychological distress, while male sex was a risk factor for alcoholism at follow-up. Conclusions: Psychological distress deteriorated 2 years after the huge natural disaster, compared with 1 year after the disaster. Long-term mental health care is needed for those affected by natural disasters, particularly those who have suffered loss. Keywords: Natural disaster, Psychological distress, Alcoholism, Longitudinal stud

Synthesis and properties of cross-linked polymers containing diarylbibenzofuranone by ADMET polymerization

Diarylbibenzofuranone (DABBF) derivatives can be reversibly cleaved to the corresponding arylbenzofuranone (ABF) radicals under mild conditions. We recently reported the synthesis and physicochemical properties of the cross-linked polyurethane containing DABBF unit. The autonomous structural transformation and the macroscopic self-healing of separated gel pieces under air at room temperature without any stimuli were accomplished by a dynamic covalent approach. Since the self-healing property was derived from DABBF units, one can expect the self-healing of DABBF-containing cross-linked polymers with various structures. We here report the acyclic diene metathesis (ADMET) polymerization of multifunctional olefin monomers containing DABBF unit. This is because a low-polarity olefin polymer can be obtained by ADMET polymerization. The DABBF-diolefin monomer was first polymerized at room temperature in the presence of Grubbs catalyst (2nd generation) in CH2Cl2. However, the yield of the obtained polymer was low (29%). Under this condition, it is considered that the ethylene molecules produced during polymerization can not be removed. The polymerization was also performed at 40 °C to give polymers in good yield (83%). Furthermore, cross-linked polymers were synthesized by ADMET polymerization of DABBF-tetraolefin monomer and copolymerization of DABBF-bifunctional olefin monomer and triolefin monomer. All cross-linked polymers were obtained in good yields. These polymers showed high swelling properties in organic solvents with relatively low polarity