Insertional gene inactivation in a phototrophic sulphur bacterium: APS-reductase-deficient mutants of Chromatium vinosum
Insertional gene inactivation in a phototrophic sulphur bacterium: APS-reductase-deficient mutants of Chromatium vinosum
复制标题
DOI：
10.1099/13500872-142-12-3363
复制
发表时间：
1996-12-01
期刊：
影响因子：
2.8
通讯作者：
Dahl, C
中科院分区：
文献类型：
--
作者：
Dahl, C
在色素科紫色硫磺细菌中，通过形成腺基硫酸盐的中间产物亚硫酸盐氧化是一个具有很好的酶学特性的过程。相反，通过亚硫酸盐酶：受体氧化还原酶的另一种直接氧化途径的作用还没有解决。从紫色硫磺细菌的代表菌株D(DSM 180(T))克隆了腺基硫酸盐形成酶腺苷-5‘-磷酸(APS)还原酶基因，并通过插入卡那霉素Omega盒构建了这些基因的突变。突变基因通过接合转移到pSUP系列自杀载体上，并通过双同源重组传递到染色体上。对获得的重组子进行Southern杂交和聚合酶链式反应分析，证实了紫硫杆菌中的第一个插入基因失活。酶学研究表明，突变株中不存在APS还原酶。进一步的表型鉴定表明，在光自养生长条件下，APS还原酶缺乏对细胞亚硫酸盐氧化能力没有显着影响。在野生型和突变型菌株中，钼酸盐的特异性拮抗剂钨酸盐在硫化物氧化过程中导致介质中亚硫酸盐的积累，并强烈抑制以亚硫酸盐为光合电子供体的生长，这表明黄曲霉的主要亚硫酸盐氧化酶可能是一种钼酶，可能是亚硫酸盐：受体氧化还原酶。本研究中所选基因的特异性失活为进一步分析光养硫磺细菌的硫代谢和其他代谢途径提供了有力的遗传工具。
In purple sulphur bacteria of the family Chromatiaceae sulphite oxidation via intermediary formation of adenylylsulphate is an enzymologically well characterized process. In contrast, the role of an alternative direct oxidation pathway via the enzyme sulphite:acceptor oxidoreductase has not been resolved. This paper reports the cloning of the genes encoding the adenylylsulphate-forming enzyme adenosine-5'-phosphosulphate (APS) reductase from Chromatium vinosum strain D (DSM 180(T)), a representative of the purple sulphur bacteria, and the construction of mutations in these genes by insertion of a kanamycin Omega cartridge. The mutated genes were transferred to C. vinosum on suicide vectors of the pSUP series by conjugation and delivered to the chromosome by double homologous recombination. Southern hybridization and PCR analyses of the recombinants obtained verified the first insertional gene inactivation in purple sulphur bacteria. Enzymological studies demonstrated the absence of APS reductase from the mutants. Further phenotypic characterization showed no significant effect of APS reductase deficiency on the sulphite-oxidizing ability of the cells under photolithoautotrophic growth conditions. In the wild-type as well as in mutant strains, tungstate, the specific antagonist of molybdate, led to the intermediary accumulation of sulphite in the medium during sulphide oxidation and strongly inhibited growth with sulphite as photosynthetic electron donor; this indicates that a molybdoenzyme, probably sulphite:acceptor oxidoreductase, is the main sulphite-oxidizing enzyme in C. vinosum. Specific inactivation of selected genes as developed for C. vinosum in this study provides a powerful genetic tool for further analysis of sulphur metabolism and other metabolic pathways in phototrophic sulphur bacteria.