RESUMO
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.