Chien Y T, Zinder S H
Section of Microbiology, Cornell University, Ithaca, New York 14853, USA.
J Bacteriol. 1996 Jan;178(1):143-8. doi: 10.1128/jb.178.1.143-148.1996.
Determination of the nucleotide sequence of the nitrogenase structural genes (nifHDK2) from Methanosarcina barkeri 227 was completed in this study by cloning and sequencing a 2.7-kb BamHI fragment containing the 3' end of nifK2 and 1,390 bp of the nifE2-homologous genes. Open reading frame nifK2 is 1,371 bp long including the stop codon TAA and encodes a polypeptide of 456 amino acids. Phylogenetic analysis of the deduced amino acid sequences of the nifK2 and nifE2 gene products from M. barkeri showed that both genes cluster most closely with the corresponding nif-1 gene products from Clostridium pasteurianum, consistent with our previous analyses of nifH2 and nifD2. The nifE gene product is known to be homologous to that of nifD, and our analysis shows that the branching pattern for the nifE proteins resembles that for the nifD product (with the exception of vnfE from Azotobacter vinelandii), suggesting that a gene duplication occurred before the divergence of nitrogenases. Primer extension showed that nifH2 had a single transcription start site located 34 nucleotides upstream of the ATG translation start site for nifH2, and a sequence resembling the archaeal consensus promoter sequence [TTTA(A/T)ATA] was found 32 nucleotides upstream from that transcription start site. A tract of four T's, previously identified as a transcription termination site in archaea, was found immediately downstream of the nifK2 gene, and a potential promoter was located upstream of the nifE2 gene. Hybridization with nifH2 and nifDK2 probes with M. barkeri RNA revealed a 4.6-kb transcript from N2-grown cells, large enough to harbor nifHDK genes and their internal open reading frames, while no transcript was detected from NH4(+)-grown cells. These results support a model in which the nitrogenase structural genes in M. barkeri are cotranscribed in a single NH4(+)-repressed operon.
本研究通过克隆和测序一个2.7 kb的BamHI片段完成了巴氏甲烷八叠球菌227固氮酶结构基因（nifHDK2）核苷酸序列的测定，该片段包含nifK2的3'端和1390 bp的nifE2同源基因。开放阅读框nifK2长1371 bp，包括终止密码子TAA，编码一个456个氨基酸的多肽。对巴氏甲烷八叠球菌nifK2和nifE2基因产物推导的氨基酸序列进行系统发育分析表明，这两个基因与巴氏梭菌相应的nif-1基因产物聚类关系最为密切，这与我们之前对nifH2和nifD2的分析一致。已知nifE基因产物与nifD的同源，我们的分析表明nifE蛋白的分支模式类似于nifD产物（除了棕色固氮菌的vnfE），这表明在固氮酶分化之前发生了基因复制。引物延伸显示nifH2有一个单一的转录起始位点，位于nifH2的ATG翻译起始位点上游34个核苷酸处，并且在该转录起始位点上游32个核苷酸处发现了一个类似于古菌共有启动子序列[TTTA(A/T)ATA]的序列。在nifK2基因的紧邻下游发现了一段四个T的序列，之前被鉴定为古菌中的转录终止位点，并且在nifE2基因的上游定位了一个潜在的启动子。用巴氏甲烷八叠球菌RNA与nifH2和nifDK2探针杂交显示，来自N2培养细胞的转录本为4.6 kb，大到足以包含nifHDK基因及其内部开放阅读框，而在NH4(+)-培养细胞中未检测到转录本。这些结果支持了一个模型，即巴氏甲烷八叠球菌中的固氮酶结构基因在一个受NH4(+)-抑制的单一操纵子中共同转录。
