ANALYSIS OF THE SODIUM DODECYL SULFATE-STABLE PEPTIDOGLYCAN AUTOLYSINS OF SELECT GRAM-NEGATIVE PATHOGENS BY USING RENATURING POLYACRYLAMIDE-GEL ELECTROPHORESIS
ANALYSIS OF THE SODIUM DODECYL SULFATE-STABLE PEPTIDOGLYCAN AUTOLYSINS OF SELECT GRAM-NEGATIVE PATHOGENS BY USING RENATURING POLYACRYLAMIDE-GEL ELECTROPHORESIS
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DOI：
10.1128/jb.176.17.5225-5232.1994
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发表时间：
1994-09-01
影响因子：
3.2
通讯作者：
CLARKE, AJ
中科院分区：
文献类型：
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作者：
BERNADSKY, G;BEVERIDGE, TJ;CLARKE, AJ
首次检测到来自铜绿假单胞菌 PAO1、大肠杆菌 W7、肺炎克雷伯菌 CWK2 和奇异变形杆菌 19 超声处理培养物的细胞组分中的肽聚糖自溶素，并通过酶谱分析进行部分表征。将来自铜绿假单胞菌 PAO1 的纯化胞壁质以 0.05%（重量/体积）的浓度掺入十二烷基硫酸钠 (SDS)-聚丙烯酰胺凝胶中，作为分离的自溶素的底物。在每种匀浆培养物中至少检测到 11 个不同强度的自溶素条带，M(r) 范围在 17,000 至 122,000 之间。四种细菌的一些自溶素具有相似的 M(r)。酶谱分析表明，重金属 Hg2+ 和 CU2+（浓度分别为 1 和 10 mM）、高离子强度以及已知影响脂多糖堆积的试剂可抑制多种大肠杆菌自溶素。 M(r) 为 65,000 的自溶素的活性也被青霉素 G 削弱，而庆大霉素则增强。通过使用双重测定进行初步筛选，以确定青霉素结合蛋白 (PBP) 和自溶素之间的关系，其中放射性标记的青霉素 V 带通过自溶素酶谱可视化。与来自大肠杆菌和铜绿假单胞菌的 PBP 3、4、5 和 6 相对应的放射性标记条带；来自奇异变形杆菌的 PBP 3、4 和 6；来自肺炎克雷伯菌的 PBP 6 和 PBP 6 降解了凝胶中的胞壁质球囊，并被推测具有自溶活性，但不能排除两种不同的酶（每种酶具有其中一种活性）在 SDS-聚丙烯酰胺凝胶中共迁移的可能性。一些放射性标记带的 M(r) 为
For the first time, peptidoglycan autolysins from cellular fractions derived from sonicated cultures of Pseudomonas aeruginosa PAO1, Escherichia coli W7, Klebsiella pneumoniae CWK2, and Proteus mirabilis 19 were detected and partially characterized by zymogram analysis. Purified murein sacculi from P. aeruginosa PAO1 were incorporated into a sodium dodecyl sulfate (SDS)-polyacrylamide gel at a concentration of 0.05% (wt/vol) to serve as a substrate for the separated autolysins. At least 11 autolysin bands of various intensities with M(r)s ranging between 17,000 and 122,000 were detected in each of the homogenated cultures. Some of the autolysins of the four bacteria had similar M(r)s. The zymogram analysis was used to show that a number of the autolysins from E. coli were inhibited by the heavy metals Hg2+ and CU2+, at 1 and 10 mM, respectively, high ionic strengths, and reagents known to affect the packing of lipopolysaccharides. The activity of an autolysin with an M(r) of 65,000 was also impaired by penicillin G, whereas it was enhanced by gentamicin. A preliminary screen to determine the relationship between penicillin-binding proteins (PBPs) and autolysins was carried out by using a dual assay in which radiolabelled penicillin V bands were visualized an an autolysin zymogram. Radiolabeled bands corresponding to PBPs 3, 4, 5, and 6 from E. coli and P. aeruginosa; PBPs 3, 4, and 6 from Proteus mirabilis; and PBP 6 from K. pneumoniae degraded the murein sacculi in the gels and were presumed to have autolytic activity, although the possibility of two distinct enzymes, each with one of the activities, comigrating in the SDS-polyacrylamide gels could not be excluded. Some radiolabelled bands possessed an M(r) of