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

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.

Interactors and effects of overexpressing YlxR/RpnM, a conserved RNA binding protein in cyanobacteria

“…Rare lipoproteins such as RepA are part of the divisome and elongasome complexes [51] and function as lytic transglycosylases, enzymes that are critical for cell wall remodeling during cell division [40]. RlpA (in Pseudomonas ) is present in higher amounts at the nascent septa of replicating bacteria while it is otherwise found at lower levels along the sidewall length of the bacterium [52]. Therefore, the needed higher levels required for cell division may result from resumption of repA translation when YlxR binding is released and possibly captured by RNase P RNA ( Figure 6 ).…”

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein

“…Genetic-code expansion is a powerful tool for site-specific amber-codon suppression for the incorporation of an unnatural amino acid in the sequence of a protein of interest within a live cell (Avila-Cobian et al, 2023;Hadar et al, 2021;Wan et al, 2014;Zheng et al, 2020). We repurposed the previously reported pHH254 plasmid (Avila-Cobian et al, 2023) by placement of the gene for Slt (pLFA240 plasmid; Section 5 and Figure S1a).…”

“…Genetic-code expansion is a powerful tool for site-specific amber-codon suppression for the incorporation of an unnatural amino acid in the sequence of a protein of interest within a live cell (Avila-Cobian et al, 2023;Hadar et al, 2021;Wan et al, 2014;Zheng et al, 2020). We repurposed the previously reported pHH254 plasmid (Avila-Cobian et al, 2023) by placement of the gene for Slt (pLFA240 plasmid; Section 5 and Figure S1a). We selected six suitable sites-Lys-38, Lys-75, Lys-122, Lys-180, Arg-399, and Arg-446-for the incorporation of unnatural amino acids, based on our analysis of the three-dimensional structure of Slt (PDB: 5OHU) (Lee et al, 2018).…”

“…Six variant pLFA240 plasmids (Tables S1-S3) were obtained by amber-stop-codon insertion at these positions of the slt gene. We incorporated the azidecontaining unnatural amino acid 1 for microscopic spatial localization of Slt in bacteria and the diazirine-containing unnatural amino acid 2 for photoaffinity capture of proximal proteins (Avila-Cobian et al, 2023). A C-terminal FLAG-tag was incorporated into the Slt sequence for purification of the protein and for Western-blot analysis.…”

“…The methodology for in vivo site-specific photoaffinity capture of interacting partner proteins is as outlined (Avila-Cobian et al, 2023;Zheng et al, 2020). Conceptually, irradiation of Slt-2 in live P. aeruginosa generates a reactive carbene species, which covalently captures the partner protein.…”