Joerger R D, Bishop P E
Department of Microbiology, North Carolina State University, Raleigh 27695-7615.
J Bacteriol. 1988 Apr;170(4):1475-87. doi: 10.1128/jb.170.4.1475-1487.1988.
A 3.8-kilobase-pair EcoRI fragment which corrects the mutations carried by the NifB- Azotobacter vinelandii strains CA30 and UW45 was cloned, and its nucleotide sequence was determined. Four complete open reading frames (ORFs) and two partial ORFs were found. The translation product of the first partial ORF is the carboxy-terminal end of a protein homologous to the nifA gene product from Klebsiella pneumoniae. A 285-base-pair sequence containing a potential nif promoter and nif regulatory sites separates this nifA gene from the first complete ORF which encodes a protein homologous to nifB gene products from K. pneumoniae and Rhizobium species. The Tn5 insertion in strain CA30 and the nif-45 mutation of strain UW45 are located within this nifB gene. The ORF downstream from nifB predicts an amino acid sequence with a cysteine residue pattern that is characteristic of ferredoxins. No similarities were found between the translation product of the third complete ORF and those of nif genes from other organisms. At the carboxy-terminal end of the predicted translation product of the fourth complete ORF, 30 of 60 amino acid residues were identical with the sequence of the nifQ gene product from K. pneumoniae. The partial ORF located at the end of the fragment encodes the N-terminal part of a potential protein with an unknown function. Northern (RNA) blot analysis indicated that transcripts from the region containing the four complete ORFs were NH4+ repressible and that the transcription products were identical in cells derepressed under conditions of Mo sufficiency or Mo deficiency or in the presence of vanadium. In contrast to the NifB- strain CA30, which is Nif- under all conditions, mutants that carry mutations affecting the C-terminal end of nifB or genes located immediately downstream from nifB, grew under all N2-fixing conditions. However, in the presence of Mo, most of the strains required 1,000 times the amount of molybdate that is sufficient for maximal growth of the wild-type strain CA under N2-fixing conditions. Growth data from strain CA37, which carries a Kanr insertion in nifQ, indicate that nifQ in A. vinelandii is not required for N2 fixation in the presence of V2O5 or under Mo-deficient conditions. Growth studies and acetylene reduction assays performed on two nifEN deletion strains showed that nifE and nifN are required for N2 fixation under Mo sufficiency, as previously observed (K. E. Brigle, M. C. Weiss, W. E. Newton, and D. R. Dean, J. Bacteriol. 169:1547-1553, 1987), but not under conditions of Mo deficiency or in the presence of 50 nM V2O5.
克隆了一个3.8千碱基对的EcoRI片段，该片段可纠正维涅兰德固氮菌NifB菌株CA30和UW45所携带的突变，并测定了其核苷酸序列。发现了四个完整的开放阅读框（ORF）和两个部分ORF。第一个部分ORF的翻译产物是与肺炎克雷伯菌nifA基因产物同源的蛋白质的羧基末端。一个包含潜在nif启动子和nif调控位点的285碱基对序列将这个nifA基因与第一个完整的ORF分开，该ORF编码一种与肺炎克雷伯菌和根瘤菌属的nifB基因产物同源的蛋白质。菌株CA30中的Tn5插入和菌株UW45的nif - 45突变位于这个nifB基因内。nifB下游的ORF预测的氨基酸序列具有铁氧化还原蛋白特有的半胱氨酸残基模式。第三个完整ORF的翻译产物与其他生物体的nif基因的翻译产物之间未发现相似性。在第四个完整ORF预测的翻译产物的羧基末端，60个氨基酸残基中有30个与肺炎克雷伯菌nifQ基因产物的序列相同。位于片段末端的部分ORF编码一种功能未知的潜在蛋白质的N末端部分。Northern（RNA）印迹分析表明，来自包含四个完整ORF区域的转录本是NH4 + 可阻遏的，并且转录产物在钼充足或钼缺乏条件下或在钒存在下解除阻遏的细胞中是相同的。与在所有条件下均为固氮阴性的NifB菌株CA30不同，携带影响nifB羧基末端或nifB下游紧邻基因的突变的突变体在所有固氮条件下均能生长。然而，在钼存在的情况下，大多数菌株所需的钼酸盐量是固氮条件下野生型菌株CA最大生长所需量的1000倍。携带nifQ中Kanr插入的菌株CA37的生长数据表明，在存在V2O5或钼缺乏条件下，维涅兰德固氮菌中的nifQ对于固氮不是必需的。对两个nifEN缺失菌株进行的生长研究和乙炔还原测定表明，如先前观察到的（K. E. Brigle、M. C. Weiss、W. E. Newton和D. R. Dean，《细菌学杂志》169:1547 - 1553，1987），在钼充足的条件下固氮需要nifE和nifN，但在钼缺乏条件下或存在50 nM V2O5时则不需要。
