PDF(Pubmed)
Abstract：
The 11 lytic transglycosylases of Pseudomonas aeruginosa have overlapping activities in the turnover of the cell-wall peptidoglycan. Rare lipoprotein A (RlpA) is distinct among the 11 by its use of only peptidoglycan lacking peptide stems. The spatial localization of RlpA and its interactome within P. aeruginosa are unknown. We employed suppression of introduced amber codons at sites in the rlpA gene for the introduction of the unnatural-amino-acids Νζ -[(2-azidoethoxy)carbonyl]-l-lysine (compound 1) and Nζ -[[[3-(3-methyl-3H-diazirin-3-yl)propyl]amino]carbonyl]-l-lysine (compound 2). In live P. aeruginosa, full-length RlpA incorporating compound 1 into its sequence was fluorescently tagged using strained-promoted alkyne-azide cycloaddition and examined by fluorescence microscopy. RlpA is present at low levels along the sidewall length of the bacterium, and at higher levels at the nascent septa of replicating bacteria. In intact P. aeruginosa, UV photolysis of full-length RlpA having compound 2 within its sequence generated a transient reactive carbene, which engaged in photoaffinity capture of neighboring proteins. Thirteen proteins were identified. Three of these proteins-PBP1a, PBP5, and MreB-are members of the bacterial divisome. The use of the complementary methodologies of non-canonical amino-acid incorporation, photoaffinity proximity analysis, and fluorescent microscopy confirm a dominant septal location for the RlpA enzyme of P. aeruginosa, as a divisome-associated activity. This accomplishment adds to the emerging recognition of the value of these methodologies for identification of the intracellular localization of bacterial proteins.
摘要：
铜绿假单胞菌的11种裂解性转糖基酶在细胞壁肽聚糖的周转中具有重叠的活性。稀有脂蛋白A（RlpA）在11个中有所不同，因为它仅使用缺乏肽茎的肽聚糖。RlpA及其相互作用组在铜绿假单胞菌内的空间定位是未知的。我们在rlpA基因中的位点处抑制引入的琥珀色密码子，以引入非天然氨基酸Nζ-[（2-叠氮基乙氧基）羰基]-1-赖氨酸（化合物1）和Nζ-[[[3-（3-甲基-3H-二氮杂嘧啶-3-基）丙基]氨基]羰基]-1-赖氨酸（化合物2）。在活的铜绿假单胞菌中，使用应变促进的炔-叠氮化物环加成对将化合物1掺入其序列中的全长RlpA进行荧光标记,并通过荧光显微镜检查。RlpA沿着细菌的侧壁长度以低水平存在，在复制细菌的新生隔层中含量较高。在完整的铜绿假单胞菌中，在其序列中具有化合物2的全长RlpA的UV光解产生了瞬时反应性卡宾，参与邻近蛋白质的光亲和捕获。鉴定出13种蛋白质。这些蛋白质中的三种-PBP1a，PBP5和MreB-是细菌分裂体的成员。使用非规范氨基酸掺入的互补方法，光亲和邻近分析，和荧光显微镜证实了铜绿假单胞菌RlpA酶的主要间隔位置，作为与分裂相关的活动。这一成就增加了对这些方法用于鉴定细菌蛋白质的细胞内定位的价值的新兴认识。本文受版权保护。保留所有权利。
