期刊
PROTEIN SCIENCE
卷 32, 期 10, 页码 -出版社
WILEY
DOI: 10.1002/pro.4781
关键词
amber-codon suppression; cell division; divisome; fluorescence microscopy; noncanonical-amino-acid incorporation; protein photoaffinity; protein-protein interactions; lytic transglycosylase
This study reveals the localization and interactome of RlpA enzyme in Pseudomonas aeruginosa during cell division. Through the incorporation of non-canonical amino acids and photoaffinity capture, it is confirmed that RlpA enzyme mainly exists in the bacterial divisome. These methods provide new means for the localization study of bacterial proteins.
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 Nu(zeta)-[(2-azidoethoxy)carbonyl]-l-lysine (compound 1) and N-zeta-[[[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.
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