Anthamatten D, Scherb B, Hennecke H
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
J Bacteriol. 1992 Apr;174(7):2111-20. doi: 10.1128/jb.174.7.2111-2120.1992.
We describe the cloning, sequencing, regulation, and mutational analysis of a Bradyrhizobium japonicum fixK-like gene whose product belongs to the family of Fnr-Crp-related regulatory proteins. The predicted 237-amino-acid FixK protein was found to share between 28 and 38% sequence identity with the Escherichia coli Fnr protein, other bacterial Fnr-like proteins (FnrN, Anr, and HlyX), and two rhizobial FixK proteins. The B. japonicum fixK-like gene, when expressed from a lac promoter, could functionally complement an fnr mutant strain of E. coli and activate transcription from an fnr-dependent promoter in the E. coli background; this activation was sixfold higher in anaerobic cultures than in aerobically grown cells, a finding that suggested oxygen sensitivity of the FixK protein and was consistent with the presence of a cysteine-rich, putatively oxygen-responsive domain at its N-terminal end. Similar to the situation in Rhizobium meliloti, expression of the fixK gene in B. japonicum was shown to be induced at low O2 tension and this induction was dependent on the two-component regulatory system FixLJ. Despite this dependency, however, a B. japonicum fixK mutant did not have the phenotypic characteristics of B. japonicum fixL and fixJ mutants: the fixK mutant was neither Fix- in symbiosis with soybean plants nor defective in anaerobic respiration with nitrate as the terminal electron acceptor. Also, the fixK mutant was unaffected in the expression of one of the two B. japonicum sigma 54 genes, rpoN1, which was previously shown to be controlled by the fixLJ genes. When fixK was introduced into the B. japonicum fixJ mutant and expressed therein from a constitutive promoter (i.e., uncoupling it from regulation by FixJ), the FixK protein thus synthesized fully restored anaerobic nitrate respiration in that strain. We interpret this to mean that the B. japonicum wild type has two homologs of fixLJ-regulated fixK genes which can functionally substitute for each other.
我们描述了一种慢生根瘤菌（Bradyrhizobium japonicum）中类fixK基因的克隆、测序、调控及突变分析，该基因的产物属于Fnr-Crp相关调控蛋白家族。预测的237个氨基酸的FixK蛋白与大肠杆菌Fnr蛋白、其他细菌的Fnr样蛋白（FnrN、Anr和HlyX）以及两种根瘤菌的FixK蛋白的序列同一性在28%至38%之间。当慢生根瘤菌类fixK基因从lac启动子表达时，它可以在功能上互补大肠杆菌的fnr突变株，并激活大肠杆菌背景中fnr依赖性启动子的转录；这种激活在厌氧培养物中比在有氧生长的细胞中高六倍，这一发现表明FixK蛋白对氧敏感，并且与其N端存在富含半胱氨酸的、推测为氧响应结构域一致。与苜蓿根瘤菌（Rhizobium meliloti）的情况类似，慢生根瘤菌中fixK基因的表达在低氧张力下被诱导，并且这种诱导依赖于双组分调控系统FixLJ。然而，尽管存在这种依赖性，慢生根瘤菌fixK突变体并不具有慢生根瘤菌fixL和fixJ突变体的表型特征：fixK突变体在与大豆植物共生时既不是固氮缺陷型，也不是以硝酸盐作为末端电子受体的厌氧呼吸缺陷型。此外，fixK突变体对慢生根瘤菌的两个sigma 54基因之一rpoN1的表达没有影响，rpoN1先前已被证明受fixLJ基因控制。当将fixK导入慢生根瘤菌fixJ突变体并从组成型启动子在其中表达时（即使其与FixJ的调控脱钩），由此合成的FixK蛋白完全恢复了该菌株中的厌氧硝酸盐呼吸。我们将此解释为慢生根瘤菌野生型有两个受fixLJ调控的fixK基因同源物，它们在功能上可以相互替代。
