RESUMO
Expression of the flagellar genes in Rhodobacter sphaeroides is dependent on one of the four sigma-54 factors present in this bacterium and on the enhancer binding proteins (EBPs) FleQ and FleT. These proteins, in contrast to other well-characterized EBPs, carry out activation as a hetero-oligomeric complex. To further characterize the molecular properties of this complex we mapped the binding sites or upstream activation sequences (UASs) of six different flagellar promoters. In most cases the UASs were identified at approximately 100 bp upstream from the promoter. However, the activity of the divergent promoters flhAp-flgAp, which are separated by only 53 bp, is mainly dependent on a UAS located approximately 200 bp downstream from each promoter. Interestingly, a significant amount of activation mediated by the upstream or contralateral UAS was also detected, suggesting that the architecture of this region is important for the correct regulation of these promoters. Sequence analysis of the regions carrying the potential FleQ/FleT binding sites revealed a conserved motif. In vivo footprinting experiments with the motAp promoter allowed us to identify a protected region that overlaps with this motif. These results allow us to propose a consensus sequence that represents the binding site of the FleQ/FleT activating complex.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/genética , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54/metabolismo , Rhodobacter sphaeroides/metabolismo , Transativadores/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Flagelos/química , Flagelos/metabolismo , Dados de Sequência Molecular , Ligação Proteica , RNA Polimerase Sigma 54/genética , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/genética , Transativadores/química , Transativadores/genética , Ativação TranscricionalRESUMO
The single subpolar flagellum of Rhodobacter sphaeroides shows an enlarged hook-filament junction. One of the two proteins that compose this section of the filament is HAP1(Rs) (FlgK(Rs)) it contains a central non-conserved region of 860 amino acids that makes this protein about three times larger than its homologue in Salmonella enterica serovar Typhimurium. We investigated the role of this central portion of the unusually large HAP1 protein of R. sphaeroides by monitoring the effects of serial deletions in flgK (Rs) , the gene encoding HAP1(Rs), on swimming and swarming. Two deletion mutants did not assemble functional flagella, two were paralyzed and five exhibited reduced free-swimming speeds. Some mutants produced unusual swarming patterns on soft agar without or with Ficoll 400. A segment of approximately 200-aa of the central region of HAP1(Rs) that aligns with the variable region of the flagellin sequence from other gamma- and beta-proteobacteria was also found. Therefore, it is possible that the origin of this large central domain of HAP1(Rs) could be associated with an event of horizontal transfer and subsequent duplications and/or insertions.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/genética , Sequência Conservada , Flagelos/química , Flagelos/genética , Flagelos/metabolismo , Estrutura Terciária de Proteína , Rhodobacter sphaeroides/genética , Deleção de SequênciaRESUMO
Singlet oxygen (1O2) generation in the reaction centers (RCs) of Rhodobacter sphaeroides wild type was characterized by luminescent emission in the near infrared region (time resolved transients and emission spectra) and quantified to have quantum yield of 0.03 +/- 0.005. 1O2 emission was measured as a function of temperature, ascorbate, urea and potassium ferricyanide concentrations and as a function of incubation time in H2O:D2O mixtures. 1O2 was shown to be affected by the RC dynamics and to originate from the reaction of molecular oxygen with two sources of triplets: photoactive dimer formed by singlet-triplet mixing and bacteriopheophytin formed by direct photoexcitation and intersystem crossing.