Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus
Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus
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DOI：
10.1046/j.1365-2958.1997.2911657.x
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发表时间：
1997-03-01
影响因子：
3.6
通讯作者：
Gotz, F
中科院分区：
文献类型：
--
作者：
Thumm, G;Gotz, F
溶葡萄球菌素是一种由模拟葡萄球菌解葡萄球菌 ATCC1362 产生的胞外甘氨酰甘氨酸内肽酶，通过水解肽聚糖的多甘氨酸肽间桥来裂解葡萄球菌细胞，对溶葡萄球菌素基因 (lss) 的序列进行了重新分析，并对纯化的溶葡萄球菌素的氨基末端进行了测序。溶葡萄球菌素原和成熟溶葡萄球菌素的研究表明，溶葡萄球菌素被组织为 493 个氨基酸 (aa) 的前蛋白原，具有由 36 个氨基酸组成的信号肽、211 个氨基酸的前肽，其中 195 个氨基酸以 13 个氨基酸长度的 15 个串联重复排列，以及 246 个氨基酸的成熟蛋白，溶葡萄球菌素在培养物上清液中加工通过胞外半胱氨酸蛋白酶对S. simulans biovar staphylolyticus进行裂解，虽然前溶葡萄球菌素具有裂解活性，但成熟的溶葡萄球菌素的活性高出约4.5倍，在肉葡萄球菌中Iss和Iss的受控表达以及前原肽区域的缺失表明前肽的串联重复对于蛋白质输出或Lss的激活不是必需的，但保持Lss处于不太活跃的状态，细胞内表达的前溶葡萄球菌素和成熟的溶葡萄球菌素在无细胞提取物中发挥葡萄球菌分解活性，但不影响相应克隆的生长。我们表征了与Iss方向相反的溶葡萄球菌素免疫因子基因（lif）。 Lif 在 S. carnosus 中的表达导致肽聚糖肽间桥的丝氨酸/甘氨酸比率从 2% 增加至 35%，这表明溶葡萄球菌素免疫依赖于丝氨酸掺入肽间桥。如果除了lif之外，还共表达Iss，则丝氨酸/甘氨酸比率进一步增加至58%，表明Lss选择最佳的丝氨酸掺入，Lif显示出与FemA和Feme蛋白的相似性，它们参与葡萄球菌肽聚糖的甘氨酸间肽桥的生物合成。与Lif相反，S. carnosus中FemA和FemB的产生不引起溶葡萄球菌素免疫，位于 Iss 下游的推定 tRNASer 基因对溶葡萄球菌素免疫没有可识别的影响，Iss 和 lif 两侧有插入序列，表明 S. simulans biovar staphylolyticus 通过水平基因转移接收了 lif 和 lss。
Lysostaphin is an extracellular glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus ATCC1362 that lyses staphylococcal cells by hydrolysing the polyglycine interpeptide bridges of the peptidoglycan, Renewed analysis of the sequence of the lysostaphin gene (lss), and the sequencing of the amino-terminus of purified prolysostaphin and of mature lysostaphin revealed that lysostaphin is organized as a preproprotein of 493 amino acids (aa), with a signal peptide consisting of 36 aa, a propeptide of 211 aa from which 195 aa are organized in 15 tandem repeats of 13 aa length, and a mature protein of 246 aa, Prolysostaphin is processed in the culture supernatant of S. simulans biovar staphylolyticus by an extracellular cysteine protease, Although prolysostaphin was staphylolytically active, the mature lysostaphin was about 4.5-fold more active, The controlled expression in Staphylococcus carnosus of Iss and Iss with deletions in the prepropeptide region indicated that the tandem repeats of the propeptide are not necessary for protein export or activation of Lss, but keep Lss in a less active state, Intracellularly expressed pro- and mature lysostaphin exert staphylolytic activity in cell-free extracts, but do not affect growth of the corresponding clones, We characterized a lysostaphin immunity factor gene (lif) which is located in the opposite direction to Iss. The expression of lif in S. carnosus led to an increase in the serine/glycine ratio of the interpeptide bridges of peptidoglycan from 2 to 35%, suggesting that lysostaphin immunity depends on serine incorporation into the interpeptide bridge. If, in addition to lif, Iss is coexpressed the serine/glycine ratio is further increased to 58%, suggesting that Lss selects for optimal serine incorporation, Lif shows similarity to FemA and Feme proteins, which are involved in the biosynthesis of the glycine interpeptide bridge of staphylococcal peptidoglycan, In contrast to that of Lif, the production of FemA and FemB in S. carnosus does not cause lysostaphin immunity, The putative tRNASer gene located downstream of Iss had no recognizable influence on lysostaphin immunity, Iss and lif are flanked by insertion sequences, suggesting that S. simulans biovar staphylolyticus received lif and lss by horizontal gene transfer.