RESUMO
Azospirillum brasilense 8-I was chromosomally labeled with green fluorescent protein (gfp) genes, using either the native promoterless gfp gene or the mutant gfpmut2 gene under the transcriptional control of the neomycin phosphate transferase (npt2) promoter inserted into Tn5 suicide plasmid vectors. One A. brasilense exconjugant, showing a steady and strong fluorescence following irradiation with 365-nm UV light was characterized in detail. This strain, A. brasilense 8-I-gfp showed increased N(2)-fixation of approximately threefold, up to a twofold increase in exopolysaccharide production, and a significant decrease in indole-3-acetic acid and poly-beta-hydroxybutyrate production over the parental strain. Sequence analysis showed that the Tn5 carrying the gfp gene was inserted in the clpX gene encoding a heat-shock protein. This data is consistent with a model in which the observed physiological changes are a consequence of pleiotropic changes that occur as a consequence of impaired heat shock (stress) protein synthesis. In summary, (i) chromosomally labelled Azospirillum brasilense was obtained carrying either native or mutant gfp genes, (ii) Pleiotropic physiological effects were caused by disruption of the clpX gene as the consequence of the insertion, (iii) a new indole-3-acetic acid-attenuated mutant of A. brasilense producing only 0.25% of the indole-3-acetic acid produced by the wild-type is presented.
Assuntos
Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/fisiologia , Mutagênese Insercional , Sequência de Aminoácidos , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Etilenos/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Hidroxibutiratos/metabolismo , Ácidos Indolacéticos/metabolismo , Dados de Sequência Molecular , Nitrogênio/metabolismo , Fixação de Nitrogênio , Poliésteres/metabolismo , Polissacarídeos Bacterianos/biossíntese , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Coloração e RotulagemRESUMO
The heat shock response in Caulobacter crescentus was previously shown to be positively regulated by the alternative sigma factor of RNA polymerase (RNAP) sigma(32), and negatively modulated by DnaK during the induction phase of the heat shock response but not during the recovery phase. In the present work we have investigated the involvement of the chaperone ClpB in the control of the heat shock response in C. crescentus. Data obtained indicated a role of ClpB in downregulation of heat shock protein (HSP) synthesis, as cells lacking this chaperone showed a prolonged shutoff phase of the heat shock response. In Escherichia coli, it has been proposed that the DnaK chaperone system switches transcription back to constitutively expressed genes through simultaneous reactivation of heat-aggregated sigma(70), as well as sequestration of sigma(32) away from RNAP. In C. crescentus, results obtained with a clpB null mutant indicate that ClpB could be involved in the reactivation of the major sigma factor sigma(73). In support of this hypothesis, we showed that transcription directed from sigma(73)-dependent promoters is not switched back in the clpB null mutant during the recovery phase. Furthermore, we observed that resolubilization of heat-aggregated sigma(73) is dependent on the presence of ClpB. Our findings also indicated that the absence of ClpB made cells more sensitive to heat shock and ethanol but not to other stresses, and unable to acquire thermotolerance.