Amber-codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase.

Luis F Avila-Cobian, Hidekazu Hoshino, Mark E Horsman, Van T Nguyen, Yuanyuan Qian, Rhona Feltzer, Choon Kim, Daniel D Hu, Matthew M Champion, Jed F Fisher, Shahriar Mobashery

Protein science : a publication of the Protein Society 2023 Oct

filter terms:

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. © 2023 The Protein Society.

Citation

Luis F Avila-Cobian, Hidekazu Hoshino, Mark E Horsman, Van T Nguyen, Yuanyuan Qian, Rhona Feltzer, Choon Kim, Daniel D Hu, Matthew M Champion, Jed F Fisher, Shahriar Mobashery. Amber-codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase. Protein science : a publication of the Protein Society. 2023 Oct;32(10):e4781