PDF(Pubmed)
Abstract：
Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 μM) include PBPs (PBP1a, KD = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, KD = 0.09 μM; RlpA, KD = 0.4 μM); a type VI secretion system effector (Tse5, KD = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.
摘要：
肽聚糖是细菌细胞壁的主要成分。其作为聚合物大厦的完整性对细菌生存至关重要，因此，它是抗生素的突出目标。肽聚糖是动态交联聚合物,其经历恒定的生物合成和周转。铜绿假单胞菌的可溶性裂解转糖基酶（Slt）是参与这种动态周转的周质酶。在活细菌中使用琥珀密码子抑制方法，我们将荧光发色团掺入Slt的结构中。荧光显微镜显示，Slt填充了周质空间的长度，并集中在子细胞中的分隔位点。该浓度在细胞分离后持续存在。琥珀密码子抑制方法也用于掺入光亲和氨基酸以捕获伴侣蛋白。基于质谱的蛋白质组学在体内鉴定了Slt的12个伴侣。这些蛋白质组学实验用体外下拉分析补充。确定了另外20个合作伙伴。我们克隆了基因并纯化至同质性22个鉴定的伴侣。生物物理表征证实所有都是真正的Slt粘合剂。Slt的蛋白质伴侣的身份跨越不同的周质蛋白质家族，包括已知存在于分裂体中的几种蛋白质。值得注意的周质伴侣（KD<0.5μM）包括PBPs（PBP1a，KD=0.07μM；PBP5=0.4μM）；其他裂解转糖基转移酶（SltB2，KD=0.09μM；RlpA，KD=0.4μM）；VI型分泌系统效应子（Tse5，KD=0.3μM）；和用于藻酸盐生物合成的调节蛋白酶（AlgO，KD<0.4μM)。鉴于其相互作用的功能广度，Slt被概念化为周质内的中心蛋白。
