RESUMO
The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 produces specialized cells for nitrogen fixation called heterocysts. Previous work showed that the group 2 sigma factor sigE (alr4249; previously called sigF) is upregulated in differentiating heterocysts 16 h after nitrogen step-down. We now show that the sigE gene is required for normal heterocyst development and normal expression levels of several heterocyst-specific genes. Mobility shift assays showed that the transcription factor NtcA binds to sites in the upstream region of sigE and that this binding is enhanced by 2-oxoglutarate (2-OG). Deletions of the region containing the NtcA binding sites in P(sigE)-gfp reporter plasmids showed that the sites contribute to normal developmental regulation but are not essential for upregulation in heterocysts. Northern RNA blot analysis of nifH mRNA revealed delayed and reduced transcript levels during heterocyst differentiation in a sigE mutant background. Quantitative reverse transcription-PCR (qRT-PCR) analyses of the sigE mutant showed lower levels of transcripts for nifH, fdxH, and hglE2 but normal levels for hupL. We developed a P(nifHD)-gfp reporter construct that showed strong heterocyst-specific expression. Time-lapse microscopy of the P(nifHD)-gfp reporter in a sigE mutant background showed delayed development and undetectable green fluorescent protein (GFP) fluorescence. Overexpression of sigE caused accelerated heterocyst development, an increased heterocyst frequency, and premature expression of GFP fluorescence from the P(nifHD)-gfp reporter.
Assuntos
Anabaena/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma/metabolismo , Anabaena/genética , Anabaena/crescimento & desenvolvimento , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Northern Blotting , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Vídeo , Plasmídeos , Regiões Promotoras Genéticas , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator sigma/genéticaRESUMO
The conR (all0187) gene of the filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 is predicted to be part of a family of proteins that contain the LytR-CpsA-Psr domain associated with septum formation and cell wall maintenance. The conR gene was originally misannotated as a transcription regulator. Northern RNA blot analysis showed that conR expression was upregulated 8 h after nitrogen step-down. Fluorescence microscopy of a P(conR)-gfp reporter strain revealed increased GFP fluorescence in proheterocysts and heterocysts beginning 9 h after nitrogen step-down. Insertional inactivation of conR caused a septum-formation defect of vegetative cells grown in nitrate-containing medium. In nitrate-free medium, mutant filaments formed abnormally long heterocysts and were defective for diazotrophic growth. Septum formation between heterocysts and adjacent vegetative cells was abnormal, often with one or both poles of the heterocysts appearing partially open. In a conR mutant, expression of nifH was delayed after nitrogen step-down and nitrogenase activity was approximately 70â% of wild-type activity, indicating that heterocysts of the conR mutant strain are partially functional. We hypothesize that the diazotrophic growth defect is caused by an inability of the heterocysts to transport fixed nitrogen to the neighbouring vegetative cells.
Assuntos
Anabaena/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fixação de Nitrogênio/fisiologia , Anabaena/genética , Anabaena/metabolismo , Anabaena/ultraestrutura , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Eletrônica , Morfogênese , Mutagênese Insercional , Mutação , Nitratos/metabolismo , Nitratos/farmacologia , Nitrogênio/metabolismo , Fixação de Nitrogênio/genética , Análise de Sequência com Séries de Oligonucleotídeos , Compostos de Amônio Quaternário/metabolismo , Compostos de Amônio Quaternário/farmacologiaRESUMO
We used gfp transcriptional fusions to investigate the regulation of eight sigma factor genes during heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Reporter strains containing gfp fusions with the upstream regions of sigB2, sigD, sigI, and sigJ did not show developmental regulation. Time-lapse microscopy of sigC, sigE, and sigG reporter strains showed increased green fluorescent protein fluorescence in differentiating cells at 4 h, 16 h, and 9 h, respectively, after nitrogen step down.
Assuntos
Anabaena/classificação , Anabaena/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Fator sigma/genética , Regulação para CimaRESUMO
Many multicellular cyanobacteria produce specialized nitrogen-fixing heterocysts. During diazotrophic growth of the model organism Anabaena (Nostoc) sp. strain PCC 7120, a regulated developmental pattern of single heterocysts separated by about 10 to 20 photosynthetic vegetative cells is maintained along filaments. Heterocyst structure and metabolic activity function together to accommodate the oxygen-sensitive process of nitrogen fixation. This article focuses on recent research on heterocyst development, including morphogenesis, transport of molecules between cells in a filament, differential gene expression, and pattern formation.