A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i
A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i
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一种在脱氮副球菌基因组中引入未标记突变的方法：构建周质细胞色素 c550、c551i 和 c553i 编码基因中具有多个突变的菌株
DOI：
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发表时间：
1991
影响因子：
3.2
通讯作者：
A. H. Stouthamer
中科院分区：
文献类型：
--
作者：
Rob J. M. Van;C. Wansell;W. Reijnders;N. Harms;J. Ras;L. Oltmann;A. H. Stouthamer
构建了一种新的自杀载体pRVS 1，用于在副球菌染色体上定点导入未标记的突变。该载体衍生自自杀载体pGRPd 1，其配备有编码β-半乳糖苷酶的lacZ基因。报告基因是一个成功的筛选标记，用于区分质粒整合株和同源重组后丢失质粒的突变株。自杀载体pGRPd 1和pRVS 1被用于基因置换技术，用于构建分别编码周质细胞色素c550、c551 i和c553 i的cycA、moxG和cycB基因中具有多个突变的突变株。Southern分析的DNA和蛋白质分析的结果单，双，和三重突变株证实了突变的正确性。野生型和突变型菌株都能够在琥珀酸和氯化胆碱上生长。此外，所有菌株生长在甲胺上，并显示野生型水平的甲胺脱氢酶活性。然而，cycA突变株在甲胺底物上显示出降低的最大比生长速率。野生型菌株、cycA和cycB突变菌株以及cycA cycB双突变菌株能够在甲醇上生长，并显示出野生型水平的甲醇脱氢酶活性。moxG突变菌株不能在甲醇上生长，并且具有低水平的甲醇脱氢酶活性。cycA突变株在甲醇上的最大比生长速率与野生型菌株相当。这些数据表明，在明确定义的电子传递途径的可溶性细胞色素c的参与。
A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans. The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase. The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities. cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.
从脱氮副球菌中分离泛醇氧化酶并解析成细胞色素 bc1 和细胞色素 c-aa3 复合物。
DOI：
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发表时间：
1985
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Berry,EA;Trumpower,BL
通讯作者：
Trumpower,BL
脱氮副球菌的两种诱导型周质 c 型细胞色素的表征。
DOI：
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发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Husain,M;Davidson,VL
通讯作者：
Davidson,VL