Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHAO
Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHAO
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DOI：
10.1128/jb.180.12.3187-3196.1998
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发表时间：
1998-06-01
影响因子：
3.2
通讯作者：
Haas, D
中科院分区：
文献类型：
--
作者：
Laville, J;Blumer, C;Haas, D
荧光假单胞菌（Pseudomonasflorescens）是在微需氧条件下从甘氨酸中产生次级代谢产物氰化氢（HCN）的细菌。编码HCN合酶的连续结构基因hcnABC在大肠杆菌中从T7启动子表达，导致在该细菌中产生HCN。hcnABC基因的核苷酸序列的分析显示每个HCN合酶亚基与参与氢转移的已知酶相似，即，甲酸脱氢酶（对于HcnA）或氨基酸氧化酶（对于HcnB和HcnC）。这些相似性以及HcnB和HcnC中黄素腺嘌呤二核苷酸-或NAD（P）-结合基序的存在表明HCN合酶可能在从甘氨酸到HCN和CO2的反应中充当脱氢酶。hcnA启动子通过引物延伸定位;-40序列（TTGGC... ATCAA）类似于共有FNR（富马酸和硝酸还原酶调节剂）结合序列（TTGAT）。ATCAA）。从荧光假单胞菌（P. florescens）中克隆了编码FNR样蛋白ANR（anaerobic regulator）的基因，并进行了序列测定，其ANR与铜绿假单胞菌（P. aeruginosa）的ANR和棕色固氮菌（Azotobacter vinelandii）的CydR最相似。荧光假单胞菌的anr突变体（CHA 21）产生很少的HCN并且不能表达hcnA-lacZ翻译融合体，而在野生型菌株CHA 0中，微需氧条件强烈地有利于hcnA-lacZ融合体的表达。突变体CHA 21以及hcn缺失突变体在无菌条件下抑制烟草黑根腐病（一种由Thielaviopsis basicola引起的疾病）的能力受损，这种效应在水分饱和的人工土壤中最为明显，与野生型菌株CHA 0相比，anr突变体丧失了约30%的病害抑制能力。这些结果表明，厌氧调节剂ANR所需的氰化物的合成在严格的好氧菌株CHA 0，并建议ANR介导的氰有助于抑制黑根腐烂。
The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas florescens from glycine, essentially under microaerophilic conditions, The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium, Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC), These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase, in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC....ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT....ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. florescens CHA0 and sequenced, ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion, Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions, This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.