反硝化假单胞菌中参与辅酶B12生物合成的基因的克隆与分析。
Cloning and analysis of genes involved in coenzyme B12 biosynthesis in Pseudomonas denitrificans.
作者信息
Cameron B, Briggs K, Pridmore S, Brefort G, Crouzet J
机构信息
Génética S. A., Joinville-le-Pont, France.
出版信息
J Bacteriol. 1989 Jan;171(1):547-57. doi: 10.1128/jb.171.1.547-557.1989.
Abstract
Cobalamin synthesis probably requires 20 to 30 different enzymatic steps. Pseudomonas putida and Agrobacterium tumefaciens mutants deficient in cobalamin synthesis (Cob have been isolated. In P. putida, Cob mutants were identified as being unable to use ethanolamine as a source of nitrogen in the absence of added cobalamin (deamination of ethanolamine requires coenzyme B12 as a cofactor). In A. tumefaciens, Cob mutants were simply screened for their reduced cobalamin synthesis. A genomic library of Pseudomonas denitrificans was constructed on a mobilizable wide-host-range vector. Eleven plasmids from this library were able to complement most of these mutants. By complementation and restriction mapping analysis, four genomic loci of P. denitrificans were found to be responsible for complementation of the Cob mutants. By subcloning fragments from the four genomic loci, we identified at least 14 different genes involved in cobalamin synthesis.
摘要
钴胺素的合成可能需要20到30个不同的酶促步骤。已分离出钴胺素合成缺陷的恶臭假单胞菌和根癌土壤杆菌突变体（Cob）。在恶臭假单胞菌中，Cob突变体被鉴定为在不添加钴胺素的情况下无法将乙醇胺用作氮源（乙醇胺的脱氨需要辅酶B12作为辅因子）。在根癌土壤杆菌中，Cob突变体通过其降低的钴胺素合成进行简单筛选。利用可移动的广宿主范围载体构建了反硝化假单胞菌的基因组文库。该文库中的11个质粒能够互补大多数这些突变体。通过互补和限制性图谱分析，发现反硝化假单胞菌的四个基因组位点负责Cob突变体的互补。通过从这四个基因组位点亚克隆片段，我们鉴定出至少14个参与钴胺素合成的不同基因。
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