RESUMO
When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N2 in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria. In this report, we obtained a mutant with a kanamycin resistance insertion in the rsh gene of Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean. This mutant was defective for type 3 secretion system induction, plant defense suppression at early root infection, and nodulation competition. Furthermore, the mutant produced smaller nodules, although with normal morphology, which led to lower plant biomass production. Soybean (Glycine max) genes GmRIC1 and GmRIC2, involved in autoregulation of nodulation, were upregulated in plants inoculated with the mutant under the N-free condition. In addition, when plants were inoculated in the presence of 10 mM NH4NO3, the mutant produced nodules containing bacteroids, and GmRIC1 and GmRIC2 were downregulated. The rsh mutant released more auxin to the culture supernatant than the wild type, which might in part explain its symbiotic behavior in the presence of combined N. These results indicate that the B. diazoefficiens stringent response integrates into the plant defense suppression and regulation of nodulation circuits in soybean, perhaps mediated by the type 3 secretion system.IMPORTANCE The symbiotic N2 fixation carried out between prokaryotic rhizobia and legume plants performs a substantial contribution to the N cycle in the biosphere. This symbiotic association is initiated when rhizobia infect and penetrate the root hairs, which is followed by the growth and development of root nodules, within which the infective rhizobia are established and protected. Thus, the nodule environment allows the expression and function of the enzyme complex that catalyzes N2 fixation. However, during early infection, the rhizobia find a harsh environment while penetrating the root hairs. To cope with this nuisance, the rhizobia mount a stress response known as the stringent response. In turn, the plant regulates nodulation in response to the presence of alternative sources of combined N in the surrounding medium. Control of these processes is crucial for a successful symbiosis, and here we show how the rhizobial stringent response may modulate plant defense suppression and the networks of regulation of nodulation.
Assuntos
Bradyrhizobium/genética , Glycine max/microbiologia , Farmacorresistência Bacteriana/genética , Fertilizantes , Resistência a Canamicina/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Nitratos , Fixação de Nitrogênio , Proteínas de Plantas/genética , Nodulação , Glycine max/genética , Simbiose , Sistemas de Secreção Tipo IIIRESUMO
Both Gram-positive and Gram-negative bacteria can take up exogenous DNA when they are in a competent state either naturally or artificially. However, the thick peptidoglycan layer in Gram-positive bacteria's cell wall is considered as a possible barrier to DNA uptake. In the present work, two transformation techniques have been evaluated in assessing the protocol's ability to introduce foreign DNA, pBBRGFP-45 plasmid which harbors kanamycin resistance and green fluorescent protein (GFP) genes into a Gram-positive bacterium, Bacillus cereus EB2. B. cereus EB2 is an endophytic bacterium, isolated from oil palm roots. A Gram-negative bacterium, Pseudomonas aeruginosa EB35 was used as a control sample for both transformation protocols. The cells were made competent using respective chemical treatment to Gram-positive and Gram-negative bacteria, and kanamycin concentration in the selective medium was also optimized. Preliminary findings using qualitative analysis of colony polymerase chain reaction (PCR)-GFP indicated that the putative positive transformants for B. cereus EB2 were acquired using the second transformation protocol. The positive transformants were then verified using molecular techniques such as observation of putative colonies on specific media under UV light, plasmid extraction, and validation analyses, followed by fluorescence microscopy. Conversely, both transformation protocols were relatively effective for introduction of plasmid DNA into P. aeruginosa EB35. Therefore, this finding demonstrated the potential of chemically prepared competent cells and the crucial step of heat-shock in foreign DNA transformation process of Gram-positive bacterium namely B. cereus was required for successful transformation.
Assuntos
Bacillus cereus/genética , Plasmídeos/genética , Transfecção/métodos , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/crescimento & desenvolvimento , Meios de Cultura/química , Meios de Cultura/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Canamicina/análise , Canamicina/farmacologia , Resistência a Canamicina/genética , Transformação BacterianaRESUMO
Finding an efficient and affordable treatment against malaria is still a challenge for medicine. Artemisinin is an effective anti-malarial drug isolated from Artemisia annua. However, the artemisinin content of A. annua is very low. We used transgenic technology to increase the artemisinin content of A. annua by overexpressing cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes. CYP71AV1 is a key enzyme in the artemisinin biosynthesis pathway, while CPR is a redox partner for CYP71AV1. Eight independent transgenic A. annua plants were obtained through Agrobacterium tumefaciens-mediated transformation, which was confirmed by PCR and Southern blot analyses. The real-time qPCR results showed that the gene cyp71av1 was highly expressed at the transcriptional level in the transgenic A. annua plants. HPLC analysis showed that the artemisinin content was increased in a number of the transgenic plants, in which both cyp71av1 and cpr were overexpressed. In one of the transgenic A. annua plants, the artemisinin content was 38% higher than in the non-transgenic plants. We conclude that overexpressing key enzymes of the biosynthesis pathway is an effective means for increasing artemisinin content in plants.
Assuntos
Artemisia annua/enzimologia , Artemisia annua/genética , Artemisininas/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Genes de Plantas/genética , NADPH-Ferri-Hemoproteína Redutase/genética , Artemisininas/química , Artemisininas/isolamento & purificação , Vias Biossintéticas/genética , Southern Blotting , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Vetores Genéticos/genética , Resistência a Canamicina/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Regeneração/genética , Transformação GenéticaRESUMO
The emergence of kanamycin resistance in a polyamine-deficient mutant of E. coli transformed with a plasmid encoding the kanamycin phosphotransferase gene has been studied. The initial inhibition of growth and protein synthesis caused by the addition of the antibiotic could be reversed earlier in polyamine-supplemented bacteria than in those depleted of the organic bases. Concomitantly, we have observed that the increase of kanamycin phosphotransferase activity evoking the antibiotic resistance was higher in bacteria cultivated in the presence of putrescine. This result seems to depend exclusively on the enhanced capacity of the translation process in bacteria grown with polyamines since the transcription of phosphotransferase gene was higher in cells subjected to polyamine starvation.
Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Expressão Gênica/efeitos dos fármacos , Resistência a Canamicina/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Poliaminas/farmacologia , Proteínas de Bactérias/biossíntese , Escherichia coli/genética , Genes Bacterianos , Canamicina Quinase , Cinética , Plasmídeos/genética , Putrescina/farmacologia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transformação GenéticaRESUMO
Enteropathogenic Escherichia coli (EPEC) can adhere to, invade and multiply in human epithelial cells. To define the elements required for bacterial invasion, we isolated from an 0111:H- EPEC a 6.6 kb plasmid that is capable of conferring to an avirulent, non-adherent E. coli K12 strain (DK1) the capacity to invade epithelial cells. With this system a dissociation was possible between bacterial invasion and adherence to epithelial cells. Bacteria containing this plasmid synthesise a protein of 32 kDa (pl 4.93) which seemed to be required for cell invasion. The results provide a new basis for strategies to prevent EPEC infections.
Assuntos
Escherichia coli/genética , Plasmídeos , Animais , Aderência Bacteriana , Mapeamento Cromossômico , Células Epiteliais , Epitélio/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Células HeLa , Humanos , Resistência a Canamicina/genética , Hibridização de Ácido Nucleico , Coelhos , Células Tumorais Cultivadas , Virulência/genéticaRESUMO
By genetic studies, it was tried to find the mechanism by which a bacterial fraction from different isolated clinical cultures resistant to 25 micrograms/ml of kanamycin can grow in media containing 500 micrograms/ml of kanamycin (at a frequency of about 10(-5)). This study was done in six clinical isolates of Escherichia coli resistant to more than three antibiotics. The results from the bacterial fraction (subpopulation) resistant to high concentrations of kanamycin in the level of resistance to aminoglycoside and non-aminoglycoside antibiotics, in the conjugation experiments, and in the percentage of resistant bacteria to 500 micrograms/ml of kanamycin when the subpopulations were subsequently cultivated in the absence of antibiotics suggest that genetic amplification occurred when one of the strains was growing in the presence of 500 micrograms/ml of kanamycin. Moreover, this strain increased its frequency of survival in high kanamycin concentrations when it was transduced by bacteriophage P1, propagated in cultures resistant to 500 micrograms/ml of kanamycin.