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首页> 外文期刊>Journal of bacteriology >Cloning and Functional Characterization of an NAD+-Dependent DNA Ligase from Staphylococcus aureus
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Cloning and Functional Characterization of an NAD+-Dependent DNA Ligase from Staphylococcus aureus

机译：金黄色葡萄球菌NAD +依赖的DNA连接酶的克隆和功能表征

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A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD+-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD+-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of 32P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD+-dependent DNA ligase fromB. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.
机译：通过分离编码 S的互补质粒克隆，鉴定了DNA连接酶中有条件缺陷的金黄色葡萄球菌突变体。金黄色葡萄球菌 A基因。假定的 S的直向同源物。在嗜热脂肪芽孢杆菌和其他革兰氏阳性细菌的基因组中可以鉴定出金黄色葡萄球菌NAD + 依赖性DNA连接酶，并证实存在四个保守的氨基酸基序，包括基序I，带有赖氨酸112的KXDG，被认为是提出的腺苷酸化位点。野生型和温度敏感型 S的 ligA 基因的DNA序列比较。金黄色葡萄球菌NT64菌株鉴定出一个单碱基改变，预计会导致氨基酸取代E46G。 S。克隆并在大肠杆菌中过表达金黄色葡萄球菌ligA 基因，并纯化该酶至近乎同质。通过测量退火与DNA互补链退火的 32 P标记的30-mer和29-mer寡核苷酸的连接，可以用纯化的酶证明NAD + 依赖性DNA连接酶的活性。 。纯化的 S的蛋白水解作用有限。嗜热菌蛋白酶产生的金黄色葡萄球菌DNA连接酶的产物的表观分子量为40、22和21 kDa。纯化片段并通过N端测序和质量分析表征。发现N末端片段（40kDa）被完全腺苷酸化。来自残基1-35的片段在 E中表达为His-tagged融合体。大肠杆菌并纯化用于功能分析。用烟酰胺单核苷酸进行腺苷酸化后，纯化的片段可以自腺苷酸化，但缺乏可检测的DNA结合活性。缺少DNA连接酶的最后76个氨基酸的21-kDa和22-kDa C端片段没有腺苷酸化活性或DNA结合活性。 S的完整30-kDa C末端。金黄色葡萄球菌的LigA蛋白在 E中表达。大肠杆菌确实显示了DNA结合活性。这些观察结果表明，与来自 B的NAD + 依赖性DNA连接酶的情况一样。嗜热脂肪族， S中存在两个独立的功能域。金黄色葡萄球菌DNA连接酶，由独立的腺苷酸化和DNA结合活性组成。它们还证明了连接酶的极端C末端在DNA结合中的作用。由于有大量证据表明DNA连接酶对于细菌存活至关重要，因此它在重要的人类病原体 S中被发现。金黄色葡萄球菌表明其作为鉴定新型抗生素的广谱抗菌靶标的潜力。

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