The Fluorescent D-Amino Acid NADA as a Tool to Study the Conditional Activity of Transpeptidases in Escherichia coli.
The Fluorescent D-Amino Acid NADA as a Tool to Study the Conditional Activity of Transpeptidases in Escherichia coli.
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DOI：
10.3389/fmicb.2018.02101
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发表时间：
2018
影响因子：
5.2
通讯作者：
den Blaauwen T
中科院分区：
文献类型：
--
作者：
Montón Silva A;Otten C;Biboy J;Breukink E;VanNieuwenhze M;Vollmer W;den Blaauwen T
负责合成肽聚糖（PG）层的酶构成了一大类抗生素的基本靶标。β-内酰胺类抗生素家族可抑制青霉素结合蛋白（PBPs）的dd -转肽酶（TPase）活性，而其碳青霉烯类亚群也可阻断ld -TPase的TPase活性。D-Ala荧光探针，如NADA，可能是通过大肠杆菌中的tpase结合到PG中，并可用于研究其功能所需的条件。在大肠杆菌的所有ld - tpase中，只有LdtD能够在指数生长中结合NADA。LdtD的过量产生导致NADA在lpob激活的PBP1b和C类PBP5存在的情况下特别插入到中间细胞。通过NADA实验，我们可以证实LpoB在中细胞激活PBP1b，而CpoB在体内调节PBP1b的活性。LdtD的过量产生能够部分补偿氮曲南对细胞分裂特异性B类PBP3的抑制作用。我们发现，当PBP1b和PBP5缺失时，A类PBP1c和C类PBP6b分别与ldd合作纳入NADA。此外，我们证明了LdtD在pH 7.0时具有活性，而LdtE和LdtF在pH 5.0生长的细胞中更活跃，并且它们似乎协同合作。NADA分析被证明是一种有用的工具，用于分析参与PG合成的蛋白质的体内活性，我们的结果提供了额外的证据，证明ld - tpase在不同条件下参与PG的维持。
The enzymes responsible for the synthesis of the peptidoglycan (PG) layer constitute a fundamental target for a large group of antibiotics. The family of β-lactam antibiotics inhibits the DD-transpeptidase (TPase) activity of the penicillin binding proteins (PBPs), whereas its subgroup of carbapenems can also block the TPase activity of the LD-TPases. D-Ala fluorescent probes, such as NADA, are incorporated into the PG presumably by TPases in Escherichia coli and can be used to study conditions that are required for their function. Of all LD-TPases of E. coli, only LdtD was able to incorporate NADA during exponential growth. Overproduction of LdtD caused NADA to be especially inserted at mid cell in the presence of LpoB-activated PBP1b and the class C PBP5. Using the NADA assay, we could confirm that LpoB activates PBP1b at mid cell and that CpoB regulates the activity of PBP1b in vivo. Overproduction of LdtD was able to partly compensate for the inhibition of the cell division specific class B PBP3 by aztreonam. We showed that class A PBP1c and the class C PBP6b cooperated with LdtD for NADA incorporation when PBP1b and PBP5 were absent, respectively. Besides, we proved that LdtD is active at pH 7.0 whereas LdtE and LdtF are more active in cells growing at pH 5.0 and they seem to cooperate synergistically. The NADA assay proved to be a useful tool for the analysis of the in vivo activities of the proteins involved in PG synthesis and our results provide additional evidence that the LD-TPases are involved in PG maintenance at different conditions.