Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-octulosonate for synthesis of lipopolysaccharide
Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-octulosonate for synthesis of lipopolysaccharide
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利用外源 3-脱氧-D-甘露酸辛酸合成脂多糖的鼠伤寒沙门氏菌菌株的分离
DOI：
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发表时间：
1987
影响因子：
3.2
通讯作者：
Edward M. Devine
中科院分区：
文献类型：
--
作者：
R. Goldman;Edward M. Devine
对鼠伤寒沙门氏菌LT2的自发突变株进行了积累外源3-脱氧-D-甘露糖(KDO)能力的研究。含有编码温度敏感的KDO-8-磷酸合成酶基因(KdsA)的细菌在42摄氏度的限制性温度下培养在含有5 mM KDO的培养基上。由于含有kdsA损伤的细菌由于内毒素(LPS)合成受到抑制和类脂A前体的积累而无法在42℃下生长，因此该方法允许直接、阳性地选择能够利用外源KDO合成内毒素的突变体。自发突变体的选择频率约为10(-6)，需要外源KDO在42℃下生长。在20 mM KDO存在下，42℃下的生长速度几乎是正常的，并与20 mM以下的KDO浓度成正比。外源KDO也抑制了类脂A前体的积累。KDO累积的表观Km为23 mm，最大转运速率为每10(8)个细胞每分钟505pmolKDO。细菌将外源性[~3H]KDO完全掺入到脂多糖中，由于内源性KDO的残留合成，比活性稀释不到10%。这种能够积累外源KDO的突变在局部诱变后直接筛选kdsA基因的新突变是非常有用的。共分离到5个kdsA基因突变，其中4个经精细结构分析确定为新等位基因。在体内引入标记的(~3H，~(13)C和~(14)C)KDO的能力应该简化和扩展对革兰氏阴性细菌这一关键代谢途径的分析。
Spontaneous mutants of Salmonella typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperature-sensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42 degrees C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grow at 42 degrees C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10(-6), required exogenous KDO for growth at 42 degrees C. The growth rate at 42 degrees C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent Km for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of KDO per min per 10(8) cells. Bacteria incorporated exogenous [3H]KDO exclusively into LPS, with less than 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenous KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (3H, 13C, and 14C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria.
DOI：
10.1073/pnas.80.5.1159
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
MULFORD, CA;OSBORN, MJ
通讯作者：
OSBORN, MJ
大肠杆菌 YrbI 是 3-脱氧-D-甘露酸辛酸 8-磷酸磷酸酶。
DOI：
10.1074/jbc.m301983200
发表时间：
2003
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Wu,Jing;Woodard,RonaldW
通讯作者：
Woodard,RonaldW