RESUMEN
The Anabaena sp. strain PCC 7120 ntcA gene showed multiple transcripts with different 5' ends. The relative abundance of transcripts varied in response to nitrogen availability. The ntcA product, NtcA, showed binding to the promoter region of its own gene. The binding site mapped to a region between the transcription start site used under nitrogen-replete conditions and the start sites used under nitrogen-limiting conditions, suggesting that NtcA regulates its own expression.
Asunto(s)
Anabaena/genética , Proteínas Bacterianas , Proteínas de Unión al ADN/genética , Genes Bacterianos , Factores de Transcripción/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
A transcriptional-interference selection was performed to identify genes of Anabaena sp. strain PCC 7120 that encode DNA-binding proteins able to bind to the rbcL promoter. Unexpectedly, the selection yielded the previously identified sigA gene, which encodes the principal sigma factor. Protein extracts from Escherichia coli containing the sigA gene bound the rbcL promoter fragment in mobility shift assays, and competition experiments indicated binding to rbcL and glnA but not xisA or nifH upstream regions.
Asunto(s)
Anabaena/genética , Proteínas Bacterianas/genética , Clonación Molecular/métodos , ARN Polimerasas Dirigidas por ADN , Factor sigma/genética , Proteínas Bacterianas/metabolismo , Unión Competitiva , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Genes Bacterianos/genética , Vectores Genéticos/genética , Glutamato-Amoníaco Ligasa/genética , Regiones Promotoras Genéticas/genética , Ribulosa-Bifosfato Carboxilasa/genética , Factor sigma/metabolismo , Transcripción GenéticaRESUMEN
The Anabaena sp. strain PCC 7120 ntcA (bifA) gene encodes a sequence-specific DNA-binding protein, NtcA (BifA, VF1) that interacts with the upstream region of several genes, including glnA, xisA, rbcL, and nifH. We have constructed a ntcA null mutant by interrupting the gene with an omega Spr-Smr cassette. The ntcA mutant was not able to grow with nitrate or atmospheric dinitrogen as the sole nitrogen source but could be grown on medium containing ammonium. The ntcA mutant was unable to form heterocysts and did not rearrange the nifD or fdxN elements after induction on a medium lacking combined nitrogen. Northern (RNA) analysis of ntcA in the wild-type strain during nitrogen stepdown showed a peak of ntcA message at an early stage (12 h) of heterocyst induction. Complementation of the ntcA mutant with a DNA fragment containing the ntcA gene and 251 bp of upstream sequence on a shuttle vector restored a wild-type phenotype; however, a similar construction containing 87 bp of upstream sequence only partially restored the phenotype. Northern analysis of RNA samples isolated from ammonium-grown cultures of the ntcA mutant showed reduced amounts of glnA message and the absence of a 1.7-kb transcript. In the wild type, the 1.7-kb transcript represents the majority of glnA transcripts after nitrogen stepdown. The ntcA mutant showed a normal pattern of rbcLS messages under these growth conditions.
Asunto(s)
Anabaena/crecimiento & desarrollo , Proteínas de Unión al ADN/genética , Genes Bacterianos/genética , Compuestos de Nitrógeno/metabolismo , Factores de Transcripción/genética , Anabaena/genética , Proteínas Bacterianas/genética , Diferenciación Celular , Ferredoxinas/genética , Regulación Bacteriana de la Expresión Génica , Reordenamiento Génico , Glutamato-Amoníaco Ligasa/genética , Mutación , Nitratos/metabolismo , Fijación del Nitrógeno/genética , Regiones Promotoras Genéticas/genética , Compuestos de Amonio Cuaternario/metabolismo , Transcripción GenéticaRESUMEN
The DNA-binding factor BifA (previously called VF1) binds upstream of the developmentally regulated site-specific recombinase gene xisA in the cyanobacterium Anabaena sp. strain PCC 7120. Besides binding xisA, BifA also binds the glnA, rbcL, and nifH promoter regions. DNase I footprint analysis of BifA binding to glnA showed a protected region -125 to -148 bp upstream of the translation start site. The binding site is between the major glnA transcription start site used in vegetative cells (RNAII) and the major transcription start site used under nitrogen-deficient conditions (RNAI). The two BifA-binding sites on the rbcL promoter were localized to a 24-bp region from +12 to -12 nucleotides and to a 12-bp region from -43 to -54 nucleotides with respect to the transcription start site. Comparison of the BifA binding sites on the glnA, xisA, and rbcL upstream regions revealed the consensus recognition sequence TGT(N9 or 10) ACA. We have identified a second DNA-binding activity (factor 2) that interacts with rbcL and xisA upstream regions. Factor 2 can be resolved from BifA by heparin-Sepharose chromatography and was present in a bifA mutant. Analysis of partially purified vegetative cell and heterocyst extracts showed that whereas BifA was present in both cell types, factor 2 was present only in vegetative cells. DNase I footprint analysis of factor 2 binding to rbcL showed protection of a 63-bp region between positions -15 and -77 with respect to the transcription start site. The factor 2 binding site on xisA was localized to a 68-bp region that showed considerable overlap with the BifA binding sites.
Asunto(s)
Anabaena/metabolismo , Proteínas Bacterianas , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Genes Bacterianos , Genes de Plantas , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Anabaena/genética , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Desoxirribonucleasa I , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/aislamiento & purificaciónRESUMEN
Two DNA elements are excised from the chromosome during Anabaena heterocyst differentiation. We have identified the gene xisF which encodes the site-specific recombinase responsible for the excision of a 55-kb element from within the fdxN gene. The cloned xisF gene is sufficient to cause site-specific rearrangement of an artificial substrate in Escherichia coli. Inactivation of xisF in the Anabaena chromosome prevents excision of the fdxN element and growth in nitrogen-deficient medium but does not alter the development of heterocysts. Forced transcription of xisF in vegetative cells did not result in excision of the fdxN element, suggesting that other factors may be involved in cell-type specificity. The predicted XisF protein shows significant similarity to the Bacillus subtilis SpoIVCA recombinase.
Asunto(s)
Anabaena/enzimología , ADN Nucleotidiltransferasas/genética , Integrasas , Factor sigma , Factores de Transcripción , Secuencia de Aminoácidos , Anabaena/genética , Anabaena/crecimiento & desarrollo , Proteínas Bacterianas/genética , Secuencia de Bases , ADN , ADN Nucleotidiltransferasas/antagonistas & inhibidores , ADN Nucleotidiltransferasas/metabolismo , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Plásmidos , Recombinasas , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
VF1 is a DNA-binding protein from the cyanobacterium Anabaena sp. strain PCC 7120. VF1 was originally identified on the basis of its binding affinity to the upstream region of xisA, which encodes a heterocyst-specific site-specific recombinase. VF1 also binds to the glnA, rbcL, and nifH promoters in vitro, suggesting that VF1 interacts with genes expressed in both vegetative cells and heterocysts. The role of VF1 in regulating gene expression in PCC 7120 is unknown. As a step towards the goal of understanding the role of VF1 in regulating gene expression, we have cloned the bifA gene by using a genetic selection strategy. bifA encodes a protein, BifA, that has chromatographic and DNA-binding properties indistinguishable from those of VF1. The cloning strategy was based on a transcriptional interference assay in which a strong synthetic promoter, conII, interferes with the expression of an aadA gene, which provides resistance to spectinomycin and streptomycin (S. J. Elledge, P. Sugiono, L. Guarente, and R. W. Davis, Proc. Natl. Acad. Sci. USA 86:3689-3693, 1989). A selection plasmid, pAM994, which has the conII promoter negatively regulated by a VF1-binding site, was used to enrich for VF1-producing clones from an expression library containing PCC 7120 DNA fragments. Mobility shift assays were used to identify a 672-bp open reading frame that encoded VF1-like binding activity. The deduced BifA amino acid sequence shows 77% identity to NtcA, which is a global regulator involved in nitrogen control in Synechococcus sp. strain PCC 7942. Both BifA and NtcA belong to the cyclic AMP receptor protein (CRP) family of prokaryotic regulatory proteins. Genes similar to envM, hisB, and ORF60-5 were found near the bifA gene.
Asunto(s)
Anabaena/genética , Proteínas Bacterianas , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteína Receptora de AMP Cíclico/genética , Proteínas de Unión al ADN/biosíntesis , Escherichia coli/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos/genética , Selección Genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
A DNA-binding factor (VF1) partially purified from Anabaena sp. strain PCC 7120 vegetative cell extracts by heparin-Sepharose chromatography was found to have affinity for the xisA upstream region. The xisA gene is required for excision of an 11-kilobase element from the nifD gene during heterocyst differentiation. Previous studies of the xisA upstream sequences demonstrated that deletion of this region is required for the expression of xisA from heterologous promoters in vegetative cells. Mobility shift assays with a labeled 250-base-pair fragment containing the binding sites revealed three distinct DNA-protein complexes. Competition experiments showed that VF1 also bound to the upstream sequences of the rbcL and glnA genes, but the rbcL and glnA fragments showed only single complexes in mobility shift assays. The upstream region of the nifH gene formed a weak complex with VF1. DNase footprinting and deletion analysis of the xisA binding site mapped the binding to a 66-base-pair region containing three repeats of the consensus recognition sequence ACATT.