Defects in D-alanyl-lipoteichoic acid synthesis in Streptococcus mutans results in acid sensitivity
Defects in D-alanyl-lipoteichoic acid synthesis in Streptococcus mutans results in acid sensitivity
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DOI：
10.1128/jb.182.21.6055-6065.2000
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发表时间：
2000-11-01
影响因子：
3.2
通讯作者：
Hamilton, IR
中科院分区：
文献类型：
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作者：
Boyd, DA;Cvitkovitch, DG;Hamilton, IR
在致龋微生物变形链球菌中，低pH诱导酸耐受性反应（ATR）。为了鉴定包含ATR的酸调节蛋白，使用温敏质粒pGh 9：ISS 1的转座子诱变来产生能够在中性pH下生长但不能在pH 5.0的培养基中生长的克隆。一个突变体（IS 1A）的序列分析表明，转座产生了一个6.3 kb的缺失，其中一端是在dlt操纵子的dltB编码四个蛋白质（DltA-DltD）参与D-丙氨酰脂磷壁酸的合成。失活的dltC基因，编码D-丙氨酰载体蛋白（DCP），导致酸敏感的突变体，BH 97 LC的产生。与野生型菌株LT 11相比，突变体的倍增时间延长了3倍，生长产量降低了33%。此外，它不能在低于pH 6.5时开始生长，并且未适应的细胞不能在pH 3.5缓冲的培养基中暴露3小时后存活，而在相同的时间段内杀死野生型需要pH 3.0。此外，在BH 97 LC中ATR的诱导，如通过在杀死不适应细胞的pH下的存活者的数目所测量的，比野生型所表现出的低3至4个数量级。虽然两种菌株的LTA含有相似的甘油磷酸残基的平均数，透化细胞的BH 97 LC没有纳入D-[C-14]丙氨酸到这个两亲物。这种缺陷与Dcp缺乏有关。从突变体纯化的LTA的化学分析证实了D-丙氨酸酯的存在。电子显微照片显示BH 97 LC的特征在于不相等的极帽，并且缺乏野生型细胞表面上存在的纤维细胞外基质。质子渗透性测定表明，突变体是更渗透质子比野生型。这一观察结果提示了S.变异体
In the cariogenic organism, Streptococcus mutans, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:ISS1 was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in dltB of the dlt operon encoding four proteins (DltA-DltD) involved in the synthesis of D-alanyl-lipoteichoic acid. Inactivation of the dltC gene, encoding the D-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a similar average number of glycerolphosphate residues, permeabilized cells of BH97LC did not incorporate D-[C-14]alanine into this amphiphile. This defect was correlated with the deficiency of Dcp. Chemical analysis of the LTA purified from the mutant confirmed the absence of D-alanine-esters. Electron micrographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild-type cells. Proton permeability assays revealed that the mutant was more permeable to protons than the wild type. This observation suggests a mechanism for the loss of the characteristic acid tolerance response in S. mutans.