RESUMO
The lon gene of Escherichia coli encodes the lon (La) protease, which is associated with cellular protein degradation. A lon gene homolog from Azospirillum brasilense, a nitrogen-fixing soil bacterium which lives in association with the roots of cereal grasses, was cloned and characterized. The nucleotide sequence of the A. brasilense lon gene was determined. It contains an open reading frame that encodes a protein of 810 amino acids with a predicted molecular mass of about 90 kDa. The deduced amino acid sequence showed a high level of homology with the sequences of all the known lon gene products. An open reading frame homologous to the E. coli clpX gene was found in front of the lon gene. Transcriptional analysis showed that the lon gene of A. brasilense is induced by heat shock and that the mRNA is monocistronic. An A. brasilense mutant, with Tn5 inserted in the lon gene, was shown to be defective in iron uptake and failed to express two membrane proteins that are induced by iron starvation in the parental strain.
Assuntos
Azospirillum brasilense/genética , Proteínas de Escherichia coli , Genes Bacterianos , Proteínas de Choque Térmico/genética , Protease La , Serina Endopeptidases/genética , Proteases Dependentes de ATP , Sequência de Aminoácidos , Azospirillum brasilense/enzimologia , Sequência de Bases , Transporte Biológico , Clonagem Molecular , Primers do DNA/química , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Ferro/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Mapeamento por Restrição , Transcrição Gênica , Raios UltravioletaRESUMO
The regulatory sequences of Azospirillum brasilense Sp7 nifH gene were fused with the cam reporter gene and used for studying the factors controlling nifH transcription. A DNA sequence, downstream the ATG codon of nifH, that could be involved in the negative regulation of nifH transcription, was identified. The effect of 1 and 2 mM of ammonium on the transcription of the A. brasilense nifH gene and on the nitrogenase activity, in the presence of the Klebsiella pneumoniae NifA protein, was examined.
Assuntos
Azospirillum brasilense/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos , Fixação de Nitrogênio/genética , Oxirredutases , Fatores de Transcrição/fisiologia , Transcrição Gênica/genética , Azospirillum brasilense/metabolismo , Conjugação Genética , Klebsiella pneumoniae/genética , Nitrogenase/análise , Plasmídeos/genética , Regiões Promotoras Genéticas , Transformação BacterianaRESUMO
A 3457-base pair fragment of Azospirillum brasilense DNA which complemented mutations in the hisA and hisF genes of Escherichia coli was sequenced. The sequence analysis revealed the presence of six major contiguous open reading frames (ORF). The comparison of the predicted amino acid sequence of these ORF with those encoded by the eubacterial, archaebacterial and eukaryotic his genes sequenced thus far revealed that four of them have a significant degree of homology with the E. coli hisH, hisA, hisF and the C-terminal domain of the hisI gene products. S1 mapping experiments indicated that the putative transcription start site coincided with the AUG translational initiation codon of the hisBd gene, the first gene of the A. brasilense his operon. Downstream from the last ORF, a sequence was identified which functions as a Rho-independent transcription terminator. Comparison of amino acid sequences, gene order and organization and evolutionary aspects of the A. brasilense his cluster are discussed.
Assuntos
Azospirillum brasilense/genética , Histidina/genética , Óperon/genética , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Sequência de Bases/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Técnicas In Vitro , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Transcrição GênicaRESUMO
The DNA sequence was determined for the Azospirillum brasilense nifH gene and part of the nifD gene. The nifH gene is 885 bp long and encodes 293 amino acid residues. The region upstream of the nifH open reading frame contains a putative promoter whose sequence shows perfect homology with promoters of other diazotrophic bacteria and two putative upstream activator sequences. Experiments with the promoter-probe vector pAF300 showed that this region promotes transcription in response to the nitrogen and oxygen availability of the cell. The amino acid sequence was deduced from the DNA nucleotide sequence of nifH; the polypeptide contains the four cysteine residues highly conserved among other nifH products and an arginine residue at position 101 which could be the site of the modification occurring during the "switch-off" of nitrogenase. The codon usage appears to be very biased reflecting the high G + C content of the Azospirillum nifH gene. In a comparison of the amino acid sequence with the other 18 known nifH gene products, the A. brasilense nifH product showed the highest level of homology with fast-growing Rhizobia suggesting interesting evolutionary implications.
Assuntos
Azospirillum brasilense/genética , Genes Bacterianos , Fixação de Nitrogênio/genética , Nitrogenase/genética , Oxirredutases , Transcrição Gênica , Sequência de Aminoácidos , Azospirillum brasilense/crescimento & desenvolvimento , Sequência de Bases , Códon , DNA Fúngico/genética , Dados de Sequência Molecular , Filogenia , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxy-terminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.