RESUMO
Sugarcane is an economically important culture in Brazil. Endophytic bacteria live inside plants, and can provide many benefits to the plant host. We analyzed the bacterial diversity of sugarcane cultivar RB-72454 by cultivation-independent techniques. Total DNA from sugarcane stems from a commercial plantation located in Paraná State was extracted. Partial 16S rRNA genes were amplified and sequenced for library construction. Of 152 sequences obtained, 52% were similar to 16S rRNA from Pseudomonas sp, and 35.5% to Enterobacter sp. The genera Pantoea, Serratia, Citrobacter, and Klebsiella were also represented. The endophytic communities in these sugarcane samples were dominated by the families Enterobacteriaceae and Pseudomonadaceae (class Gammaproteobacteria).
Assuntos
Endófitos/genética , Enterobacteriaceae/genética , Pseudomonadaceae/genética , Saccharum/microbiologia , Técnicas de Cultura , Tipagem Molecular , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genéticaRESUMO
Several bacteria are able to degrade flavonoids either to use them as carbon sources or as a detoxification mechanism. Degradation pathways have been proposed for several bacteria, but the genes responsible are not known. We identified in the genome of the endophyte Herbaspirillum seropedicae SmR1 an operon potentially associated with the degradation of aromatic compounds. We show that this operon is involved in naringenin degradation and that its expression is induced by naringenin and chrysin, two closely related flavonoids. Mutation of fdeA, the first gene of the operon, and fdeR, its transcriptional activator, abolished the ability of H. seropedicae to degrade naringenin.
Assuntos
Flavanonas/metabolismo , Herbaspirillum/metabolismo , Proteínas de Bactérias/genética , Biotransformação , Flavonoides/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Herbaspirillum/genética , ÓperonRESUMO
DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.
Assuntos
Proteínas de Bactérias/metabolismo , Herbaspirillum/química , Recombinases Rec A/metabolismo , DNA Bacteriano , Escherichia coli/metabolismo , Ligação ProteicaRESUMO
DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.
Assuntos
Proteínas de Bactérias/metabolismo , Herbaspirillum/química , Recombinases Rec A/metabolismo , DNA Bacteriano , Escherichia coli/metabolismo , Ligação ProteicaRESUMO
Herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. In the present study, we have investigated the attachment to and internal colonization of Phaseolus vulgaris roots by the H. seropedicae wild-type strain SMR1 and by a strain of H. seropedicae expressing a red fluorescent protein (DsRed) to track the bacterium in the plant tissues. Two-day-old P. vulgaris roots were incubated at 30°C for 15 min with 6 x 10(8) CFU/mL H. seropedicae SMR1 or RAM4. Three days after inoculation, 4 x 10(4) cells of endophytic H. seropedicae SMR1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 10(6) CFU/g. The identity of the recovered bacteria was confirmed by amplification and sequencing of the 16SrRNA gene. Furthermore, confocal microscopy of P. vulgaris roots inoculated with H. seropedicae RAM4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that H. seropedicae RAM4 is capable of colonizing the roots of the dicotyledon P. vulgaris. Determination of dry weight of common bean inoculated with H. seropedicae SMR1 suggested that this bacterium has a negative effect on the growth of P. vulgaris.
Assuntos
Herbaspirillum/crescimento & desenvolvimento , Phaseolus/microbiologia , Raízes de Plantas/microbiologia , Contagem de Colônia Microbiana , Herbaspirillum/genética , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
Herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. In the present study, we have investigated the attachment to and internal colonization of Phaseolus vulgaris roots by the H. seropedicae wild-type strain SMR1 and by a strain of H. seropedicae expressing a red fluorescent protein (DsRed) to track the bacterium in the plant tissues. Two-day-old P. vulgaris roots were incubated at 30°C for 15 min with 6 x 10(8) CFU/mL H. seropedicae SMR1 or RAM4. Three days after inoculation, 4 x 10(4) cells of endophytic H. seropedicae SMR1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 10(6) CFU/g. The identity of the recovered bacteria was confirmed by amplification and sequencing of the 16SrRNA gene. Furthermore, confocal microscopy of P. vulgaris roots inoculated with H. seropedicae RAM4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that H. seropedicae RAM4 is capable of colonizing the roots of the dicotyledon P. vulgaris. Determination of dry weight of common bean inoculated with H. seropedicae SMR1 suggested that this bacterium has a negative effect on the growth of P. vulgaris.
Assuntos
Herbaspirillum/crescimento & desenvolvimento , Phaseolus/microbiologia , Raízes de Plantas/microbiologia , Contagem de Colônia Microbiana , Herbaspirillum/genética , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants.
Assuntos
Parede Celular/metabolismo , Flavanonas/farmacologia , Herbaspirillum/efeitos dos fármacos , Herbaspirillum/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Raízes de Plantas/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Zea mays/microbiologiaRESUMO
Herbaspirillum seropedicae is a nitrogen-fixing bacterium that grows well with ammonium chloride or sodium nitrate as alternative single nitrogen sources but that grows more slowly with L-alanine, L-serine, L-proline, or urea. The ntrC mutant strain DCP286A was able to utilize only ammonium or urea of these nitrogen sources. The addition of 1 mmol.L-1 ammonium chloride to the nitrogen-fixing wild-type strain inhibited nitrogenase activity rapidly and completely. Urea was a less effective inhibitor; approximately 20% of nitrogenase activity remained 40 min after the addition of 1 mmol x L-1 urea. The effect of the ntrC mutation on nitrogenase inhibition (switch-off) was studied in strain DCP286A containing the constitutively expressed gene nifA of H. seropedicae. In this strain, nitrogenase inhibition by ammonium was completely abolished, but the addition of urea produced a reduction in nitrogenase activity similar to that of the wild-type strain. The results suggest that the NtrC protein is required for assimilation of nitrate and the tested amino acids by H. seropedicae. Furthermore, NtrC is also necessary for ammonium-induced switch-off of nitrogenase but is not involved in the mechanism of nitrogenase switch-off by urea.
Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Herbaspirillum/genética , Herbaspirillum/metabolismo , Mutação , Nitrogenase/metabolismo , Ureia/metabolismo , Regulação para Baixo , Herbaspirillum/crescimento & desenvolvimento , Compostos de Amônio Quaternário/metabolismo , Fatores de Transcrição/genéticaRESUMO
Sec-independent translocation systems have been characterised in Escherichia coli and other bacteria and differ from the Sec-dependent system by transporting fully folded proteins using the transmembrane proton electrochemical gradient. Proteins transported by this system bear a twin-arginine motif (tat) in the N-terminal signal peptide and include several cofactor-containing proteins. Azotobacter chroococcum strain (MCD124) has a soluble hydrogenase, which exhibited low O(2)-dependent H(2) uptake, and a shift in the pH of the culture to a more alkaline range during growth. We show that the DNA region capable of complementing this strain contains the tatABC genes and that mutations in the tatA gene reproduced the soluble hydrogenase and the culture pH shift phenotypes. We also show that insertional mutation in the tatC gene at a position corresponding to its C-terminal region had no effect on hydrogenase activity, but induced the pH shift of the culture. Sequence and mutagenesis analyses of this genomic region suggest that these genes form an operon that does not contain a tatD-like gene. A mutation in hupZ of the main hup gene region, coding for a possible b-type cytochrome also yielded a soluble hydrogenase, but not the pH-shift phenotype.
Assuntos
Azotobacter/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Hidrogenase/genética , Azotobacter/enzimologia , Azotobacter/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Membrana Celular/metabolismo , Concentração de Íons de Hidrogênio , Hidrogenase/metabolismo , Mutagênese , Mutação , Fenótipo , Ligação ProteicaRESUMO
The NtrX protein has been identified as a transcriptional activator of genes involved in the metabolic control of alternative nitrogen sources, acting as a member of a two-component regulatory system. The in silico analysis of the NtrX amino acid sequence shows that this protein contains an N-terminal receiver domain, a central AAA+ superfamily domain and a C-terminal DNA binding domain. To over-express and purify this protein, the ntrX gene of Azospirillum brasilense lacking the first eight codons was cloned into the vector pET29a+. The NtrX protein was over-expressed as an S.Tag fusion protein induced by l-arabinose in the Escherichia coli strain BL21AI and purified by ion exchange and affinity chromatography. The ATPase activity of NtrX was measured by coupling the ATP conversion to ADP with NADH oxidation. The ATPase activity of NtrX was stimulated in the presence of A. brasilense sigma(54)/NtrC-dependent promoter of the glnBA gene. Phosphorylation by carbamyl-phosphate also stimulated ATPase, in a manner similar to the NtrC protein. Together our results suggest that NtrX is active in the phosphorylated form and that there may be a cross-talk between the NtrYX and NtrBC regulatory systems in A. brasilense.