Laville J, Blumer C, Von Schroetter C, Gaia V, Défago G, Keel C, Haas D
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
J Bacteriol. 1998 Jun;180(12):3187-96. doi: 10.1128/JB.180.12.3187-3196.1998.
The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens 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. fluorescens 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.
次生代谢产物氰化氢（HCN）主要在微需氧条件下由荧光假单胞菌从甘氨酸合成。对荧光假单胞菌CHA0中HCN合成的遗传基础进行了研究。编码HCN合酶的相邻结构基因hcnABC在大肠杆菌中由T7启动子表达，导致该细菌产生HCN。对hcnABC基因的核苷酸序列分析表明，每个HCN合酶亚基与已知的参与氢转移的酶相似，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合酶在从甘氨酸到HCN和CO2的反应中可能作为脱氢酶起作用。通过引物延伸确定了hcnA启动子的位置；-40序列（TTGGC...ATCAA）类似于共有FNR（延胡索酸和硝酸盐还原酶调节因子）结合序列（TTGAT...ATCAA）。从荧光假单胞菌CHA0中克隆并测序了编码FNR样蛋白ANR（厌氧调节因子）的基因。CHA0菌株的ANR与铜绿假单胞菌的ANR和棕色固氮菌的CydR最相似。荧光假单胞菌的anr突变体（CHA21）产生的HCN很少，并且无法表达hcnA-lacZ翻译融合体，而在野生型菌株CHA0中，微需氧条件强烈促进hcnA-lacZ融合体的表达。在无菌条件下，突变体CHA21以及hcn缺失突变体抑制烟草黑根腐病（由烟草炭疽病菌引起的一种疾病）的能力受损。在水饱和的人工土壤中，这种影响最为明显，与野生型菌株CHA0相比，anr突变体的病害抑制能力丧失了约30%。这些结果表明，严格需氧的CHA0菌株中氰化物合成需要厌氧调节因子ANR，并且表明ANR介导的氰化物生成有助于抑制黑根腐病。
