RESUMO
The limited host range (LHR) Agrobacterium tumefaciens strain Ag162 is an isolate with a narrow host range. Introduction of the wide host range (WHR) virA gene is essential for extending the host range to Kalanchoë daigremontiana. In this report we show that the region upstream of the ATG start codon is responsible for the LHR phenomenon and that this is probably due to the non-inducibility of the LHRvirA promoter. By comparing the characteristics of the LHR and WHR VirA receptor proteins, it was found that the LHR VirA protein is able to activate the WHR VirG protein in the presence of acetosyringone and that this acetosyringone-dependent vir-induction is enhanced by the presence of D-glucose, as in the case of WHR VirA proteins. These results indicate that the domains, acting as receptors for sugars and phenolic signals, must be conserved between the LHR and WHR VirA receptor proteins.
Assuntos
Acetofenonas/farmacologia , Agrobacterium tumefaciens/patogenicidade , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Regiões Promotoras Genéticas , Fatores de Virulência , Acetofenonas/metabolismo , Agrobacterium tumefaciens/efeitos dos fármacos , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular , Monossacarídeos/farmacologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Virulência/genéticaRESUMO
The wild-type VirA protein is known to be responsive not only to phenolic compounds but also to sugars via the ChvE protein (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA 87:6708-6712, 1990, and N. Shimoda, A. Toyoda-Yamamoto, J. Nagamine, S. Usami, M. Katayama, Y. Sakagami, and Y. Machida, Proc. Natl. Acad. Sci. USA 87:6684-6688, 1990). It is shown here that the mutant VirA(Ser-44, Arg-45) protein and the chimeric VirA-Tar protein are no longer responsive to sugars and the ChvE protein. However, whereas the chimeric VirA-Tar protein was found to be locked in a highly responsive state, the VirA(Ser-44, Arg-45) mutant protein appeared to be locked in a low responsive state. This difference turned out to be important for tumorigenicity of the host strains in virulence assays on Kalanchoë daigremontiana.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Proteínas de Membrana/genética , Receptores de Superfície Celular , Fatores de Virulência , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Células Quimiorreceptoras , Clonagem Molecular , Proteínas de Membrana/metabolismo , Fenótipo , Plantas/microbiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por RestriçãoRESUMO
The VirA protein of Agrobacterium tumefaciens is thought to be a receptor for plant phenolic compounds such as acetosyringone. Although it is not known whether the interaction between VirA and the phenolics is direct or requires other phenolic-binding proteins, it is shown in this study that the first 280 amino acids of the VirA protein are not essential for the acetosyringone mediated vir gene induction response. Considering the fact that the cytoplasmic region between the amino acids 283 and 304 is highly conserved between the different VirA proteins, and that deletion of this region abolishes VirA activity, we suggest that the acetosyringone receptor domain is located in this cytoplasmic domain of the VirA protein.
Assuntos
Acetofenonas/farmacologia , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Receptores de Superfície Celular , Fatores de Virulência , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Sequência de Bases , Células Quimiorreceptoras , Análise Mutacional de DNA , Genes Bacterianos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Relação Estrutura-Atividade , Ativação Transcricional , Virulência/genéticaRESUMO
Integration Host Factor (IHF) of E. coli can stimulate both early and repressor transcription of bacteriophage Mu. We introduced several mutations in the early promoter (Pe) and studied the effect of these mutations on the stimulation of early and repressor transcription by IHF. All mutant promoters are still positive regulated by IHF, but the level of stimulation is dependent on the strength of the promoter. The strength of the early promoter has an even greater impact on the regulation of the repressor promoter by IHF: stimulation is observed in the presence of a relatively weak Pe, whereas with a strong Pe the repressor promoter Pc is inhibited by IHF. This inhibition is most probably due to an interference of the early transcription with the opposing repressor transcription. The implication of this type of regulation for the Mu life cycle is discussed.
Assuntos
Proteínas de Bactérias/genética , Bacteriófago mu/genética , Regulação Viral da Expressão Gênica , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Sequência de Bases , Clonagem Molecular , DNA Viral/genética , Proteínas de Ligação a DNA , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , Mutação , Plasmídeos , Regiões Promotoras Genéticas/genéticaRESUMO
To find VirG proteins with altered properties, the virG gene was mutagenized. Random chemical mutagenesis of single-stranded DNA containing the Agrobacterium tumefaciens virG gene led with high frequency to the inactivation of the gene. Sequence analysis showed that 29% of the mutants contained a virG gene with one single-base-pair substitution somewhere in the open reading frame. Thirty-nine different mutations that rendered the VirG protein inactive were mapped. Besides these inactive mutants, two mutants in which the vir genes were active even in the absence of acetosyringone were found on indicator plates. A VirG protein with an N54D substitution turned out to be able to induce a virB-lacZ reporter gene to a high level even in the absence of the inducer acetosyringone. A VirG protein with an I77V substitution exhibited almost no induction in the absence of acetosyringone but showed a maximum induction level already at low concentrations of acetosyringone.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Acetofenonas/farmacologia , Agrobacterium tumefaciens/patogenicidade , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , DNA de Cadeia Simples/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutagênese , Mutagênicos , Fases de Leitura Aberta/genética , Tumores de Planta/microbiologia , Plantas/microbiologia , Mutação Puntual , Seleção Genética , Transcrição GênicaRESUMO
The induction of vir gene expression in different types of Agrobacterium strains shows different pH sensitivity profiles. The pH sensitivity pattern demonstrated by octopine Ti strains was similar to that of a supervirulent leucinopine Ti strain, whereas this was different from that shown by nopaline Ti strains and agropine Ri strains. Data are given which indicate that these differences are due to different properties of the virA genes of these wild types. An exceptional case was formed by strains with the limited-host-range plasmid pTiAG57 which showed AS-dependent vir induction only if reduced inoculum sizes were used and the temperature was 28 degrees C or below.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Rhizobium/genética , Fatores de Virulência , Acetofenonas/farmacologia , Arginina/análogos & derivados , Arginina/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Concentração de Íons de Hidrogênio , Plasmídeos/genética , Rhizobium/efeitos dos fármacos , Rhizobium/crescimento & desenvolvimento , Temperatura , Ativação TranscricionalRESUMO
The question whether sucrose (Suc) is present inside plastids has been long debated. Low Suc levels were reported to be present inside isolated chloroplasts, but these were argued to be artifacts of the isolation procedures used. We have introduced Suc-metabolizing enzymes in plastids and our experiments suggest substantial Suc entry into plastids. The enzyme levansucrase from Bacillus subtilis efficiently synthesizes fructan from Suc. Targeting of this enzyme to the plastids of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) plants leads to high-level fructan accumulation in chloroplasts and amyloplasts, respectively. Moreover, introduction of this enzyme in amyloplasts leads to an altered starch structure. Expression of the yeast invertase in potato tuber amyloplasts results in an 80% reduction of total Suc content, showing efficient hydrolysis of Suc by the plastidic invertase. These observations suggest that Suc can enter plastids efficiently and they raise questions as to its function and metabolism in this organelle.