RESUMO
Acremonium strictum Elicitor Subtilisin (AsES) is a fungal elicitor that activates innate immunity, conferring disease resistance in strawberry (Fragaria × ananassa Duch.), Arabidopsis and other plant species. The aim of the present work was to evaluate the involvement of the ethylene (ET) signalling pathway in AsES-mediated immune response in strawberry. Ethylene production and expression of the genes responsible for ET synthesis, perception and response were measured after AsES treatment. ROS (H2 O2 ) accumulation and immunity induced by AsES were studied after ET perception was blocked by 1-methylcyclopropene (1-MCP). Biochemical and molecular results showed that AsES induced a marked increase in local and systemic biosynthesis of ET, both in a biphasic manner. Blocking of ET perception by 1-MCP prior to AsES induction reduced production of ROS (H2 O2 ) and prevented AsES from eliciting defence against fungal pathogens having different lifestyles, such as Botrytis cinerea (necrotrophic) and Colletotrichum acutatum (hemibiotrophic). These findings contribute to elucidate the mode of action of the novel elicitor subtilase, AsES, specifically regarding the role of ET signalling in the activation of plant innate immunity, in addition to the multitude of processes regulated by ET in plants.
Assuntos
Etilenos , Fragaria , Transdução de Sinais , Subtilisina , Etilenos/metabolismo , Fragaria/efeitos dos fármacos , Fragaria/imunologia , Fragaria/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hypocreales/química , Transdução de Sinais/efeitos dos fármacos , Subtilisina/farmacologiaRESUMO
Different Bacillus species with PGPR (plant growth-promoting rhizobacterium) activity produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how these PGPRs recognize phytopathogens and exhibit the antifungal response. Here, we report the antagonistic interaction between Bacillus subtilis and the phytopathogenic fungus Fusarium verticillioides We demonstrate that this bacterial-fungal interaction triggers the induction of the SigB transcription factor, the master regulator of B. subtilis stress adaptation. Dual-growth experiments performed with live or dead mycelia or culture supernatants of F. verticillioides showed that SigB was activated and required for the biocontrol of fungal growth. Mutations in the different regulatory pathways of SigB activation in the isogenic background revealed that only the energy-related RsbP-dependent arm of SigB activation was responsible for specific fungal detection and triggering the antagonistic response. The activation of SigB increased the expression of the operon responsible for the production of the antimicrobial cyclic lipopeptide surfactin (the srfA operon). SigB-deficient B. subtilis cultures produced decreased amounts of surfactin, and B. subtilis cultures defective in surfactin production (ΔsrfA) were unable to control the growth of F. verticillioidesIn vivo experiments of seed germination efficiency and early plant growth inhibition in the presence of F. verticillioides confirmed the physiological importance of SigB activity for plant bioprotection.IMPORTANCE Biological control using beneficial bacteria (PGPRs) represents an attractive and environment-friendly alternative to pesticides for controlling plant diseases. Different PGPR Bacillus species produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how PGPRs recognize phytopathogens and process the antifungal response. Here, we report how B. subtilis triggers the induction of the stress-responsive sigma B transcription factor and the synthesis of the lipopeptide surfactin to fight the phytopathogen. Our findings show the participation of the stress-responsive regulon of PGPR Bacillus in the detection and biocontrol of a phytopathogenic fungus of agronomic impact.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/genética , Fungicidas Industriais/farmacologia , Fusarium/fisiologia , Fator sigma/farmacologia , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Doenças das Plantas/prevenção & controle , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/farmacologiaRESUMO
Bacterial biofilms are important in natural settings, biotechnology, and medicine. However, regulation of biofilm development and its persistence in different niches is complex and only partially understood. One key step during the biofilm life cycle is dispersal, when motile cells abandon the mature biofilm to spread out and colonize new niches. Here, we show that in the model bacterium Bacillus subtilis the general stress transcription factor SigB is essential for halting detrimental overgrowth of mature biofilm and for triggering dispersal when nutrients become limited. Specifically, SigB-deficient biofilms were larger than wild-type biofilms but exhibited accelerated cell death, significantly greater sensitivity to different stresses, and reduced dispersal. Interestingly, the signal detected by SigB to limit biofilm growth was transduced through the RsbP-dependent metabolic arm of the SigB regulatory cascade, which in turn positively controlled expression of SinR, the master regulator of biofilm formation and cell motility. This novel SigB-SinR regulatory circuit might be important in controlling the fitness of biofilms (either beneficial or harmful) in diverse environments.IMPORTANCE Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical systems. Sessile cells embedded in the self-produced extracellular matrix of the biofilm benefit from a division of labor and are protected from environmental insults. However, as the biofilm ages, cells become stressed because of overcrowding, starvation, and accumulation of waste products. How does the sessile biofilm community sense and respond to stressful conditions? Here, we show that in Bacillus subtilis, the transcription factors SigB and SinR control whether cells remain in or leave a biofilm when metabolic conditions become unfavorable. This novel SigB-SinR regulatory circuit might be important for controlling the fitness of biofilms (either beneficial or harmful) in diverse environments.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Fator sigma/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Meios de Cultura/química , Locomoção , Metabolismo , Fator sigma/deficiênciaRESUMO
Histotoxic strains of Clostridium perfringens cause human gas gangrene, a devastating infection during which potent tissue-degrading toxins are produced and secreted. Although this pathogen only grows in anaerobic-nutrient-rich habitats such as deep wounds, very little is known regarding how nutritional signals influence gas gangrene-related toxin production. We hypothesize that sugars, which have been used throughout history to prevent wound infection, may represent a nutritional signal against gas gangrene development. Here we demonstrate, for the first time, that sugars (sucrose, glucose) inhibited the production of the main protein toxins, PLC (alpha-toxin) and PFO (theta-toxin), responsible for the onset and progression of gas gangrene. Transcription analysis experiments using plc-gusA and pfoA-gusA reporter fusions as well as RT-PCR analysis of mRNA transcripts confirmed that sugar represses plc and pfoA expression. In contrast an isogenic C. perfringens strain that is defective in CcpA, the master transcription factor involved in carbon catabolite response, was completely resistant to the sugar-mediated inhibition of PLC and PFO toxin production. Furthermore, the production of PLC and PFO toxins in the ccpA mutant strain was several-fold higher than the toxin production found in the wild type strain. Therefore, CcpA is the primary or unique regulatory protein responsible for the carbon catabolite (sugar) repression of toxin production of this pathogen. The present results are analyzed in the context of the role of CcpA for the development and aggressiveness of clostridial gas gangrene and the well-known, although poorly understood, anti-infective and wound healing effects of sugars and related substances.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Clostridium perfringens/metabolismo , Regulação para Baixo , Gangrena Gasosa/microbiologia , Glucose/metabolismo , Proteínas Hemolisinas/metabolismo , Sacarose/metabolismo , Fosfolipases Tipo C/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Clostridium perfringens/enzimologia , Clostridium perfringens/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas/genética , Humanos , Fosfolipases Tipo C/genéticaRESUMO
On the basis of pharmacokinetic modeling, this study provides some insights into predicting in vivo plasma progesterone concentrations when using bovine intravaginal inserts for systemic progesterone delivery. More significantly, this contribution is the first attempt to build a simple pharmacokinetic model that links plasma progesterone concentrations with the hormone released from bovine intravaginal inserts. After evaluating three rival pharmacokinetic models and considering some phenomena involved in the intravaginal administration of progesterone, a primary pharmacokinetic model having a good data fitting capability with only two adjustable parameters is proposed to the above mentioned task. Kinetic parameters are given for lactating Holstein dairy cows with two levels of daily milk yields; and non-pregnant, non-lactating Holstein-Friesian cattle. Model predictions indicate the occurrence of a preferential distribution of the intravaginally administered progesterone dose through a first uterine pass effect.
Assuntos
Bovinos/sangue , Sincronização do Estro/métodos , Progesterona/sangue , Progesterona/farmacocinética , Administração Intravaginal , Animais , Indústria de Laticínios , Preparações de Ação Retardada , Feminino , Meia-Vida , Lactação , Modelos Biológicos , Gravidez , Progesterona/administração & dosagemRESUMO
Progesterone pharmacokinetics were analyzed for plasma hormone concentrations ranging from linear to saturated metabolism in lactating Holstein cows with differing daily milk yields. The adequacy of 2-coupled first-order (bi-exponential equation), hyperbolic (Michaelis-Menten equation), and sigmoidal (Hill equation) kinetic models to describe the experimental progesterone pharmacokinetic profiles was examined on a statistical basis. After nonlinear regression and statistical analysis of the data-fitting capability, a simple one-compartment model based on Hill equation proved to be most adequate. This model indicates an enzyme-catalyzed metabolism of progesterone involving cooperative substrate-binding sites, resulting from allosteric effects that yield a sigmoidal saturation rate curve. Kinetic parameters were estimated for 2 groups of lactating Holstein cows with different daily milk yields. We found, for the first time, a remarkable quantitative agreement of the Hill coefficient value with that reported in pharmacokinetic studies involving cytochrome P450, family 3, subfamily A (CYP3A)-mediated reactions in other mammals, humans included. It seems that positive cooperativity makes enzymes much more sensitive to plasma progesterone concentration, and their activities can undergo significant changes in a narrow range of concentration as characteristic of sigmoidal behavior. Therefore, the values of classical pharmacokinetic parameters, such as the elimination constant, half-life, and clearance rate, were found to be highly dependent on the plasma progesterone concentration.
Assuntos
Bovinos/metabolismo , Lactação/fisiologia , Progesterona/farmacocinética , Progestinas/farmacocinética , Animais , Indústria de Laticínios , Feminino , Meia-Vida , Taxa de Depuração Metabólica , Leite/metabolismo , Modelos Biológicos , Progesterona/sangue , Análise de RegressãoRESUMO
By considering two important factors involved in the codon-anticodon interactions, the hydrogen bond number and the chemical type of bases, a codon array of the genetic code table as an increasing code scale of interaction energies of amino acids in proteins was obtained. Next, in order to consecutively obtain all codons from the codon AAC, a sum operation has been introduced in the set of codons. The group obtained over the set of codons is isomorphic to the group (Z(64), +) of the integer module 64. On the Z(64)-algebra of the set of 64(N) codon sequences of length N, gene mutations are described by means of endomorphisms f:(Z(64))(N)-->(Z(64))(N). Endomorphisms and automorphisms helped us describe the gene mutation pathways. For instance, 77.7% mutations in 749 HIV protease gene sequences correspond to unique diagonal endomorphisms of the wild type strain HXB2. In particular, most of the reported mutations that confer drug resistance to the HIV protease gene correspond to diagonal automorphisms of the wild type. What is more, in the human beta-globin gene a similar situation appears where most of the single codon mutations correspond to automorphisms. Hence, in the analyses of molecular evolution process on the DNA sequence set of length N, the Z(64)-algebra will help us explain the quantitative relationships between genes.
Assuntos
Código Genético , Modelos Genéticos , Anticódon/química , Anticódon/genética , Fenômenos Químicos , Físico-Química , Códon/química , Códon/genética , Globinas/genética , Protease de HIV/genética , Humanos , Ligação de Hidrogênio , Matemática , Mutação , Recombinação Genética , TermodinâmicaRESUMO
A hallmark of sporulation of Bacillus subtilis is the formation of two distinct cells by an asymmetric septum. The developmental programme of these two cells involves the compartmentalized activities of sigmaE in the larger mother cell and of sigmaF in the smaller prespore. A potential role of de novo lipid synthesis on development was investigated by treating B. subtilis cells with cerulenin, a specific inhibitor of fatty acid biosynthesis. These experiments demonstrated that spore formation requires de novo fatty acid synthesis at the onset of sporulation. The transcription of the sporulation genes that are induced before the formation of two cell types or that are under the exclusive control of sigmaF occurred in the absence of fatty acid synthesis, as monitored by spo-lacZ fusions. However, expression of lacZ fusions to genes that required activation of sigmaE for transcription was inhibited in the absence of fatty acid synthesis. The block in sigmaE-directed gene expression in cerulenin-treated cells was caused by an inability to process pro-sigmaE to its active form. Electron microscopy revealed that these fatty acid-starved cells initiate abnormal polar septation, suggesting that de novo fatty acid synthesis may be essential to couple the activation of the mother cell transcription factors with the formation of the differentiating cells.
Assuntos
Bacillus subtilis/fisiologia , Ácidos Graxos/biossíntese , Regulação Bacteriana da Expressão Gênica , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Cerulenina/farmacologia , Ácido Graxo Sintases/metabolismo , Genes Bacterianos/genética , Microscopia Eletrônica , Regiões Promotoras Genéticas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Fator sigma/genética , Fator sigma/metabolismo , Esporos Bacterianos/fisiologia , Esporos Bacterianos/ultraestrutura , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Bacillus subtilis growing at 37 degrees C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37 degrees C is transferred to 20 degrees C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20 degrees C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20 degrees C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37 degrees C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.