Hernando Y, Palacios J M, Imperial J, Ruiz-Argüeso T
Laboratorio de Microbiología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Spain.
J Bacteriol. 1995 Oct;177(19):5661-9. doi: 10.1128/jb.177.19.5661-5669.1995.
Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5. Expression of the P5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK- mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN- mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P(5b) was reduced to ca. 50% of the wild-type level in the FnrN(-) mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum by. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.
豌豆根瘤菌生物型豌豆UPM791产生的豌豆（Pisum sativum L.）类菌体合成一种膜结合的（NiFe）氢化酶，该酶可氧化根瘤中固氮过程产生的H2。活性酶的合成需要结构基因hupSL的产物以及至少15个其他基因的一系列辅助蛋白，包括可能参与镍代谢的基因簇hypABFCDE。与仅在共生中表达的hupSL基因不同，hypBFCDE操纵子在营养细胞中也会因培养基中低pO2而被激活。在微需氧细胞和类菌体中，hypBFCDE操纵子的转录从启动子P5b开始，转录起始位点位于hypB的ATG起始密码子上游190 bp处，在hypA的编码序列内。转录起始位点5b之前有一个Fnr框（厌氧框），5'-TTGAgccatgTCAA-3'，中心位于-39.5位置。P5b启动子在异源苜蓿根瘤菌细菌宿主中的表达取决于活性fixK基因的存在。通过补充苜蓿根瘤菌FixK-突变体，从豌豆根瘤菌生物型豌豆UPM791基因文库中分离出一个2.6 kb的EcoRI片段。对该DNA片段进行测序鉴定出一个fnrN基因，盒式插入诱变表明豌豆根瘤菌生物型豌豆fnrN能够替代苜蓿根瘤菌fixK基因，以激活豌豆根瘤菌生物型豌豆hypBFCDE操纵子和苜蓿根瘤菌fix基因。然而，豌豆根瘤菌生物型豌豆基因组FnrN-突变体的类菌体表现出野生型水平的氢化酶活性。在FnrN(-)突变体中，P(5b)的微需氧表达降至野生型水平的约50%。这些结果表明hyp基因表达不受fnrN基因诱变的影响，并表明豌豆根瘤菌生物型豌豆中存在第二个类似fnr的基因。用fnrN内部探针进行的Southern印迹分析揭示了存在与fnrN具有同源性的第二个基因组区域。
