RESUMO
Upon starvation, rod-shaped Myxococcus xanthus bacteria form mounds and then differentiate into round, stress-resistant spores. Little is known about the regulation of late-acting operons important for spore formation. C-signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to stimulate transcription of developmental genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA-I, exoL-P, and nfsA-H. Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro, but strikingly each promoter region was unique in terms of whether or not MrpC and/or the DNA-binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR-Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR-Cas component, which we propose produces spores suited to withstand starvation and environmental insults.
Assuntos
Proteínas de Bactérias , Sistemas CRISPR-Cas , Regulação Bacteriana da Expressão Gênica , Myxococcus xanthus , Óperon , Regiões Promotoras Genéticas , Esporos Bacterianos , Fatores de Transcrição , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Myxococcus xanthus/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Óperon/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Mutação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the ß-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF's partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/metabolismo , Cápsulas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Supressores , Ensaios de Triagem em Larga Escala , Lipopolissacarídeos/metabolismo , Complexos Multiproteicos/genética , Mutação , Fenilpropionatos/farmacologiaRESUMO
The preparation of gold nanoparticles (AuNPs) from tetrachloroauric acid in the presence of tetrahydrothiophenocucurbit[n]uril (THTmQ[n]) has been effectively achieved in a microwave reactor. The reaction was performed in the presence of an excess of the tetrahydrothiopheno function in a partial reductant role, while the remainder formed AuNP-THTmQ[n] conjugates after the reduction was completed with formic acid. An affinity for the AuNPs by the THTmQ[n] was observed in the purification of the NPs via centrifugation, removal of the supernatant and resuspension of the conjugate.
RESUMO
Upon starvation Myxococcus xanthus undergoes multicellular development. Rod-shaped cells move into mounds in which some cells differentiate into spores. Cells begin committing to sporulation at 24-30 h poststarvation, but the mechanisms governing commitment are unknown. FruA and MrpC are transcription factors that are necessary for commitment. They bind cooperatively to promoter regions and activate developmental gene transcription, including that of the dev operon. Leading up to and during the commitment period, dev mRNA increased in wild type, but not in a mutant defective in C-signaling, a short-range signaling interaction between cells that is also necessary for commitment. The C-signaling mutant exhibited ~20-fold less dev mRNA than wild type at 30 h poststarvation, despite a similar level of MrpC and only 2-fold less FruA. Boosting the FruA level twofold in the C-signaling mutant had little effect on the dev mRNA level, and dev mRNA was not less stable in the C-signaling mutant. Neither did high cooperativity of MrpC and FruA binding upstream of the dev promoter explain the data. Rather, our systematic experimental and computational analyses support a model in which C-signaling activates FruA at least ninefold posttranslationally in order to commit a cell to spore formation.
Assuntos
Proteínas de Bactérias/metabolismo , Redes Reguladoras de Genes , Myxococcus xanthus/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Metodologias Computacionais , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutação , Myxococcus xanthus/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Processamento Pós-Transcricional do RNA , Fatores de Transcrição/genéticaRESUMO
One large open reading frame (ORF) encodes 10 potyviral proteins. We compared the accumulation of cylindrical inclusion (CI) protein from the middle, coat protein (CP) from the 3'end, and Renilla luciferase (RLUC) from two distinct locations in potato virus A (PVA) RNA. 5' RLUC was expressed from an rluc gene inserted between the P1 and helper component proteinase (HCPro) cistrons, and 3' RLUC was expressed from the gene inserted between the RNA polymerase and CP cistrons. Viral protein and RNA accumulation were quantitated (i) when expressed from PVA RNA in the presence of ectopically expressed genome-linked viral protein (VPg) and auxiliary proteins and (ii) at different time points during natural infection. The rate and timing of 3' RLUC and CP accumulation were found to be different from those of 5' RLUC and CI. Ectopic expression of VPg boosted PVA RNA, 3' RLUC, and, together with HCPro, CP accumulation, whereas 5' RLUC and CI accumulation remained unaffected regardless of the increased viral RNA amount. In natural infection, the rate of the noteworthy minute early accumulation of 3' RLUC accelerated toward the end of infection. 5' RLUC accumulation, which was already pronounced at 2 days postinfection, increased moderately and stabilized to a constant level by day 5, whereas PVA RNA and CP levels continued to increase throughout the infection. We propose that these observations connect with the mechanisms by which potyvirus infection limits CP accumulation during early infection and specifically supports its accumulation late in infection, but follow-up studies are required to understand the mechanism of how this occurs.IMPORTANCE The results of this study suggest that the dynamics of potyviral protein accumulation are regulated differentially from the 3' end of viral RNA than from the rest of the genome, the significance of which would be to satisfy the needs of replication early and particle assembly late in infection.
Assuntos
Regulação Viral da Expressão Gênica , Potyvirus/crescimento & desenvolvimento , Proteínas Virais/análise , Cinética , RNA Viral/análise , Fatores de Tempo , Nicotiana/virologiaRESUMO
Proper expression of the dev operon is important for normal development of Myxococcus xanthus. When starved, these bacteria coordinate their gliding movements to build mounds that become fruiting bodies as some cells differentiate into spores. Mutations in the devTRS genes impair sporulation. Expression of the operon occurs within nascent fruiting bodies and depends in part on C signaling. Here, we report that expression of the dev operon, like that of several other C-signal-dependent genes, is subject to combinatorial control by the transcription factors MrpC2 and FruA. A DNA fragment upstream of the dev promoter was bound by a protein in an extract containing MrpC2, protecting the region spanning positions -77 to -54. Mutations in this region impaired binding of purified MrpC2 and abolished developmental expression of reporter fusions. The association of MrpC2 and/or its longer form, MrpC, with the dev promoter region depended on FruA in vivo, based on chromatin immunoprecipitation analysis, and purified FruA appeared to bind cooperatively with MrpC2 to DNA just upstream of the dev promoter in vitro. We conclude that cooperative binding of the two proteins to this promoter-proximal site is crucial for dev expression. 5' deletion analysis implied a second upstream positive regulatory site, which corresponded to a site of weak cooperative binding of MrpC2 and FruA and boosted dev expression 24 h into development. This site is unique among the C-signal-dependent genes studied so far. Deletion of this site in the M. xanthus chromosome did not impair sporulation under laboratory conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Myxococcus xanthus/metabolismo , Óperon/genética , Proteínas de Bactérias/genética , Mutação , Myxococcus xanthus/genéticaRESUMO
Multidrug -resistant tuberculosis (MDRTB) is a serious threat to mankind. India has the highest number of MDRTB cases, although majority remain undiagnosed due to inadequate diagnostic infrastructure, leading to increased community transmission and mortality. This one-year observational retrospective study highlighted the effectiveness of the National Tuberculosis Elimination Program (NTEP) for prompt detection of drug-resistant TB by GeneXpert MTB/RIF assay and revealed its associated clinico-epidemiological factors. The overall detection rates of MTB and RRTB were 20.70 % and 20.86 % respectively. The pediatric population had 7.69 % rifampicin resistance, and HIV was strongly associated with the development of TB and RRTB (P < 0.01).
RESUMO
Potyviral coat protein (CP) and helper component-proteinase (HCPro) play key roles in both the regulation of viral gene expression and the formation of viral particles. We investigated the interplay between CP and HCPro during these viral processes. While the endogenous HCPro and a heterologous viral suppressor of gene silencing both complemented HCPro-less potato virus A (PVA) expression, CP stabilization connected to particle formation could be complemented only by the cognate PVA HCPro. We found that HCPro relieves CP-mediated inhibition of PVA RNA expression likely by enabling HCPro-mediated sequestration of CPs to particles. We addressed the question about the role of replication in formation of PVA particles and gained evidence for encapsidation of non-replicating PVA RNA. The extreme instability of these particles substantiates the need for replication in the formation of stable particles. During replication, viral protein genome linked (VPg) becomes covalently attached to PVA RNA and can attract HCPro, cylindrical inclusion protein and host proteins. Based on the results of the current study and our previous findings we propose a model in which a large ribonucleoprotein complex formed around VPg at one end of PVA particles is essential for their integrity.
Assuntos
Nicotiana , Potyvirus , Doenças das Plantas , Potyvirus/genética , RNA/metabolismo , Vírion/genética , Vírion/metabolismoRESUMO
Introduction Fever is the most common presenting symptom in children and causes distress in patients and parents. Although nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used as antipyretics, they should be reserved for pain or chronic inflammatory conditions due to safety concerns. If we can safely achieve the same antipyretic effect using a higher dose (20 mg/kg) of paracetamol, NSAIDs may be avoided for treating fever. There is a paucity of literature comparing the antipyretic effect of mefenamic acid and high-dose paracetamol. We hypothesized that there would be no difference in the antipyretic effect of high-dose paracetamol and mefenamic acid. Methods In this randomized control trial, 165 febrile children were randomly allocated to one of the following three groups: standard-dose (15 mg/kg) paracetamol (SDPCM) as the control group and high-dose (20 mg/kg) paracetamol (HDPCM) and mefenamic acid (6 mg/kg) (MFN) as the intervention groups. The temperature was measured using a digital thermometer at the start of drug dosage and every 15 minutes until it reached normal. One-way between-group analysis of variance (ANOVA) was used to compare outcome measures such as time for temperature to reach normal, fall of temperature in 60 minutes, and time for the next fever. Post hoc analysis was performed to compare mean differences. Patients were monitored for adverse effects. Results Out of 165 enrolled patients, 159 were analyzed. The baseline demographic data were comparable among the groups. There was a statistically significant difference in the mean time taken for the temperature to reach normal (F-value (F) (2,156)=3.184, p<0.05) and the mean reduction in temperature at 60 minutes (F (2,156)=23.40, p<0.001) among the groups. The mean time for temperature to reach normal in the SDPCM group (97.50±26.60 minutes) was longer than that in the HDPCM (85.09±31.43 minutes) and MFN (84.90±30.42 minutes) groups. The decrease in temperature over 60 minutes was greater in the HDPCM (0.46°C±0.19°C) and MFN (0.45°C±0.11°C) groups than in the SDPCM (0.33°C±0.10°C) group. The time to the next fever spike was shorter for the SDPCM group (5.07±2.66 hours) than for the HDPCM (7.20±3.08 hours) and MFN (8.82±3.83 hours) groups. Post hoc analysis demonstrated that high-dose paracetamol and mefenamic acid had similar and faster antipyretic effects than standard-dose paracetamol. Although the duration of action was found to be longer in the mefenamic acid group, the difference was not statistically significant. There were negligible adverse effects in the groups. Conclusion Standard-dose paracetamol (15 mg/kg/dose) had a slower and shorter antipyretic effect than high-dose paracetamol (20 mg/kg/dose) and mefenamic acid (6 mg/kg/dose). A single dose of high-dose paracetamol was safe and had a similar antipyretic effect as mefenamic acid. Mefenamic acid may be avoided as an antipyretic and spared for pain and anti-inflammatory indications. Multicentered double-blind clinical trials with larger sample sizes and comparisons of other NSAIDs will be required to confirm these findings.
RESUMO
Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.
Assuntos
Basidiomycota/genética , Sistemas CRISPR-Cas/genética , Proteínas Fúngicas/genética , Polyporaceae/genética , Basidiomycota/crescimento & desenvolvimento , Edição de Genes/métodos , Lignina/genética , RNA Guia de Cinetoplastídeos/genética , Madeira/genética , Madeira/microbiologiaRESUMO
The Gram-negative bacterial cell envelope is a complex structure and its homeostasis is essential for bacterial survival. Envelope stress responses (ESRs) are signal transduction pathways that monitor the fidelity of envelope assembly during normal growth and also detect and repair envelope damage caused by external assaults, including immune factors, protein toxins, and antibiotics. In this review, we focus on three best-studied ESRs and discuss the mechanisms by which ESRs detect various perturbations of envelope assembly and integrity and regulate envelope remodeling to promote bacterial survival. We will highlight the complex relationship of ESRs with envelope biogenesis pathways and discuss some of the challenges in this field on the road to mapping the global regulatory network of envelope homeostasis.
Assuntos
Parede Celular , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , HomeostaseRESUMO
The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXLΦ) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVAVPgmut). PVAVPgmut RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3'RLUC) from the 3' end of the PVA genome. VPgmut could not support either PVA RNA stabilization or enhanced 3'RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPgmut increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXLΦ motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Interações Hospedeiro-Patógeno , Nicotiana/virologia , Potyvirus/patogenicidade , Ribonucleoproteínas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Mutação , Doenças das Plantas/virologia , Folhas de Planta/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Potyvirus/genética , Ligação Proteica , Ribonucleoproteínas/genética , Nicotiana/genética , Proteínas não Estruturais Virais/genéticaRESUMO
It is still poorly understood why α-synuclein, the intrinsically disordered protein involved in Parkinson's and other neurodegenerative diseases, is so prone to aggregation. Recent work has shown a correlation between the aggregation rate and the rate of diffusional reconfiguration by varying temperature and pH. Here we examine the effects of several point mutations in the sequence on the conformational ensemble and reconfiguration rate. We find that at lower temperatures the PD causing aggregation enhancing mutations slow down and aggregation reducing mutations drastically speed up intramolecular diffusion, as compared to the wild type sequence. However, at higher temperatures, one of three familial mutations that enhance aggregation slows intramolecular diffusion while non-natural mutations that inhibit aggregation speed up intramolecular diffusion. These results support the hypothesis that the first step of aggregation is kinetically controlled by reconfiguration in which the protein chain cannot reconfigure rapidly enough to escape oligomerization. Finally we provide physical and chemical insights into why small point mutations cause these dramatic changes in the conformational ensemble and dynamics.
Assuntos
Mutação , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genéticaRESUMO
The use of X-rays for medical diagnosis is enhancing exposure to low radiation doses. Exposure to extremely low-frequency electromagnetic or magnetic fields is also increasing. Epidemiological studies show consistent associations of childhood leukaemia with exposure to magnetic fields but any causal relationship is unclear. A limitation in assessing the consequence of such exposure is the availability of sensitive assays. The embryonic neuronal stem and progenitor cell compartments are radiosensitive tissues. Using sensitive assays, we report a statistically significant increase in DNA double-strand break (DSB) formation and apoptosis in the embryonic neuronal stem cell compartment following in utero exposure to 10-200 mGy X-rays. Both endpoints show a linear response. We also show that DSB repair is delayed following exposure to doses below 50 mGy compared with 100 mGy. Thus, we demonstrate in vivo consequences of low-dose radiation. In contrast to these impacts, we did not observe any significant induction of DSBs or apoptosis following exposure to 50 Hz magnetic fields (100 or 300 µT). We conclude that any DSB induction by treatment with magnetic fields is lower than following exposure to 10 mGy X-rays. For comparison, certain procedures involving computed tomography scanning are equivalent to 1-5 mGy X-rays.
Assuntos
Apoptose/efeitos da radiação , Encéfalo/embriologia , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Embrião de Mamíferos/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Encéfalo/metabolismo , Relação Dose-Resposta à Radiação , Embrião de Mamíferos/patologia , Feminino , Campos Magnéticos , Camundongos , Células-Tronco Neurais/patologia , Raios X/efeitos adversosRESUMO
Piriformospora indica association has been reported to increase biotic as well as abiotic stress tolerance of its host plants. We analyzed the beneficial effect of P. indica association on rice seedlings during high salt stress conditions (200 and 300 mM NaCl). The growth parameters of rice seedlings such as root and shoot lengths or fresh and dry weights were found to be enhanced in P. indica-inoculated rice seedlings as compared with non-inoculated control seedlings, irrespective of whether they are exposed to salt stress or not. However, salt-stressed seedlings performed much better in the presence of the fungus compared with non-inoculated control seedlings. The photosynthetic pigment content [chlorophyll (Chl) a, Chl b, and carotenoids] was significantly higher in P. indica-inoculated rice seedlings under high salt stress conditions as compared with salt-treated non-inoculated rice seedlings, in which these pigments were found to be decreased. Proline accumulation was also observed during P. indica colonization, which may help the inoculated plants to become salt tolerant. Taken together, P. indica rescues growth diminution of rice seedlings under salt stress.