RESUMO
A 40-kb DNA region containing the major cluster of nif genes has been isolated from the Azospirillum brasilense Sp7 genome. In this region three nif operons have been identified: nifHDKorf1Y, nifENXorf3orf5fdxAnifQ and orf2nifUSVorf4. The operons containing nifENX and nifUSV genes are separated from the structural nifHDKorf1Y operon by about 5 kb and 10 kb, respectively. The present study shows the sequence analysis of the 6045-bp DNA region containing the nifENX genes. The deduced amino acid sequences from the open reading frames were compared to the nif gene products of other diazotrophic bacteria and indicate the presence of seven ORFs, all reading in the same direction as that of the nifHDKorf1Y operon. Consensus sigma54 and NifA-binding sites are present only in the promoter region upstream of the nifE gene. This promoter is activated by NifA protein and is approximately two-times less active than the nifH promoter, as indicated by the ß-galactosidase assays. This result suggests the differential expression of the nif genes and their respective products in Azospirillum
Assuntos
Azospirillum brasilense , Fixação de Nitrogênio , Óperon/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
A 40-kb DNA region containing the major cluster of nif genes has been isolated from the Azospirillum brasilense Sp7 genome. In this region three nif operons have been identified: nifHDKorf1Y, nifENXorf3orf5fdxAnifQ and orf2nifUSVorf4. The operons containing nifENX and nifUSV genes are separated from the structural nifHDKorf1Y operon by about 5 kb and 10 kb, respectively. The present study shows the sequence analysis of the 6045-bp DNA region containing the nifENX genes. The deduced amino acid sequences from the open reading frames were compared to the nif gene products of other diazotrophic bacteria and indicate the presence of seven ORFs, all reading in the same direction as that of the nifHDKorf1Y operon. Consensus sigma54 and NifA-binding sites are present only in the promoter region upstream of the nifE gene. This promoter is activated by NifA protein and is approximately two-times less active than the nifH promoter, as indicated by the beta-galactosidase assays. This result suggests the differential expression of the nif genes and their respective products in Azospirillum.
Assuntos
Azospirillum brasilense/genética , Genes Bacterianos , Óperon/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , DNA Bacteriano/análise , Dados de Sequência Molecular , Fixação de Nitrogênio/genética , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Análise de Sequência de Proteína , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense
Assuntos
Azospirillum/genética , Fixação de Nitrogênio/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Primers do DNA , Amplificação de Genes , Genoma Bacteriano , Hibridização Genética , Plasmídeos , Reação em Cadeia da Polimerase , RNA Ribossômico 16SRESUMO
Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense.
Assuntos
Azospirillum/genética , Genes Bacterianos , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Primers do DNA , Amplificação de Genes , Hibridização Genética , Dados de Sequência Molecular , Fixação de Nitrogênio/genética , Plasmídeos , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Alinhamento de SequênciaRESUMO
Disruption of an open reading frame (ORF) of 840 bp (280 amino acids; ORF280) in an Azospirillum brasilense Tn5 mutant resulted in a pleiotrophic phenotype. Besides an enhanced N(2)-fixing capacity and altered expression pattern of a nifH-gusA fusion, growth on the charged polar amino acids glutamate and arginine was severely affected. ORF280, similar to previously identified ORFs present in Bradyrhizobium japonicum (ORF277), Paracoccus denitrificans (ORF278) and Rhodobacter capsulatus (ORF277), exhibits in its C-terminus a significant similarity with the recently defined family of universal stress proteins.
Assuntos
Azospirillum brasilense/genética , Elementos de DNA Transponíveis , Fixação de Nitrogênio/genética , Nitrogenase/genética , Fases de Leitura Aberta/genética , Oxirredutases , Sequência de Aminoácidos , Azospirillum brasilense/metabolismo , Clonagem Molecular , DNA Bacteriano/análise , Glucuronidase/metabolismo , Dados de Sequência Molecular , Mutação , Fixação de Nitrogênio/fisiologia , Nitrogenase/metabolismo , Mapeamento Físico do Cromossomo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNARESUMO
NifA protein activates transcription of nitrogen fixation operons by the alternative s54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.
Assuntos
Azospirillum brasilense/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte , Fixação de Nitrogênio , Proteínas de Bactérias/metabolismoRESUMO
NifA protein activates transcription of nitrogen fixation operons by the alternative sigma 54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.
Assuntos
Azospirillum brasilense/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte , Genes Bacterianos , Fixação de Nitrogênio/genética , Proteínas de Bactérias/metabolismoRESUMO
The Azospirillum brasilense nifH promoter is positively controlled by the NifA protein bound to the upstream activator sequences (UASs). Two overlapping UASs located at -191 and -182 were identified with the consensus TGT-N10-ACA motif. The role of the two UASs of Azospirillum brasilense nifH promoter was examined by introducing base substitutions in the NifA binding sites. Both the promoter down phenotype of a mutation in UAS2 and increased activation when UAS1 was mutated reveal that the integrity of the UAS2 is required for the efficient activation of nifH promoter. This atypical NifA-binding site may represent a region interacting with two NifA dimers.
Assuntos
Azospirillum brasilense/genética , Genes Bacterianos , Nitrogenase/genética , Oxirredutases , Regiões Promotoras Genéticas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação Bacteriana da Expressão Gênica/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fixação de Nitrogênio/genética , Nitrogenase/metabolismo , Óperon/genética , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
1. The complete nucleotide sequence of the nitrogenase structural genes from Azospirillum brasilense was determined. Two additional open reading frames of 353 and 683 base pairs were detected downstream of the nifK gene, one of which shows homology to the nifY gene. 2. Structures resembling the consensus nif promoter and NifA-binding motif were found only upstream from the nifH region and an inverted repeat structure located downstream of the nifY gene may be a potential stem-and-loop transcriptional terminator. 3. The nif structural genes of Azospirillum brasilense are transcribed as a single transcription unit and organized as nifHDKorf1Y. NifH, NifD and NifK polypeptides share significant sequence identities when compared to nif structural gene products from other organisms. 4. The three polypeptides are characterized by the presence of highly conserved cysteine residues which may play a role in binding the iron-sulfur cluster.
Assuntos
Azospirillum brasilense/genética , Genes Bacterianos , Fixação de Nitrogênio/genética , Óperon , Sequência de Aminoácidos , Sequência de Bases , Códon/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The complete nucleotide sequence of the nitrogenase structural genes form Azospirillum brasilense was determined. Two additional open reading frames of 353 and 683 base pairs were detected downstream of the nifK gene. Structures resembling the consensus nif promoter and NifA-binding motif were found only upstream from the nifH region and an inverted repeat structure locate downstream of the nifY gene may be a potential stem-and-loop transcriptional terminator. The nif structural genes of Azospirillum brasiliense are transcribed as a transcription unit and organized as nifHDK orf1 Y, NifH, NifD and NifK polypeptides share significant sequence identies when compared to nif structural gene products from other organisms. The three polypeptides are characterized by the presence of highly conserved cysteine residues which may play a role in binding the iron-sulfur cluster