Protein complex formation during denitrification by Pseudomonas aeruginosa

Abstract

The most efficient means of generating cellular energy is through aerobic respiration. Under anaerobic conditions, several prokaryotes can replace oxygen by nitrate as final electron acceptor. During denitrification, nitrate is reduced via nitrite, NO and N₂ O to molecular nitrogen (N₂ ) by four membrane-localized reductases with the simultaneous formation of an ion gradient for ATP synthesis. These four multisubunit enzyme complexes are coupled in four electron transport chains to electron donating primary dehydrogenases and intermediate electron transfer proteins. Many components require membrane transport and insertion, complex assembly and cofactor incorporation. All these processes are mediated by fine-tuned stable and transient protein-protein interactions. Recently, an interactomic approach was used to determine the exact protein-protein interactions involved in the assembly of the denitrification apparatus of Pseudomonas aeruginosa. Both subunits of the NO reductase NorBC, combined with the flavoprotein NosR, serve as a membrane-localized assembly platform for the attachment of the nitrate reductase NarGHI, the periplasmic nitrite reductase NirS via its maturation factor NirF and the N₂ O reductase NosZ through NosR. A nitrate transporter (NarK2), the corresponding regulatory system NarXL, various nitrite (NirEJMNQ) and N₂ O reductase (NosFL) maturation proteins are also part of the complex. Primary dehydrogenases, ATP synthase, most enzymes of the TCA cycle, and the SEC protein export system, as well as a number of other proteins, were found to interact with the denitrification complex. Finally, a protein complex composed of the flagella protein FliC, nitrite reductase NirS and the chaperone DnaK required for flagella formation was found in the periplasm of P. aeruginosa. This work demonstrated that the interactomic approach allows for the identification and characterization of stable and transient protein-protein complexes and interactions involved in the assembly and function of multi-enzyme complexes.

Figure 1

General interactomic workflow. The illustration depicts step by step the protein–protein interaction elucidation pathway via affinity chromatography copurification coupled with mass spectrometry. A. Shows: (1) Induction of the bacterial host for bait–protein production by the use of anaerobic growth condition in the presence of nitrate; (2) the in vivo cross‐linking by the addition of diffusible cross‐linkers; (3) the cell fractionation and separation of in‐ and soluble fraction by centrifugation; and in (4) the affinity tag‐based purification of the formed and cross‐linked protein complexes, their visualization on an SDS–PAGE gel, the mass spectrometry analyses of corresponding peptides and the computational identification of bait interaction partner candidates. B. shows the downstream processing of cross‐linked peptides. There are four possible varieties of resulting peptides after trypsin digestion: non‐cross‐linked peptides, interpeptides (cross‐links between diverse peptides of the same protein), intrapeptides (cross‐links within the same peptide of a certain protein) and trans‐peptides (cross‐links between peptides of different proteins). The LC‐MS/MS analysis is depicted in detail. Abun., Abundant proteins that unspecifically attach to the bait–prey complex (identified by the analysis of their protein abundance prior to affinity purification enrichment. Thus, proteins enriched after purification are considered as potential interaction partners); PPI, protein–protein interactions; Cont., contaminant with affinity to the column material (detected with a control strain lacking the expression plasmid); MS, mass spectrometry; PG, peptidoglycan; C, cytoplasm; IM, inner membrane; OM, outer membrane.

Figure 2

Illustration of the detected denitrification supercomplexes. A. The protein interaction partners for NirS, NosR, NorC and NorB that are related to denitrification or flagellum assembly (FliC and DnaK) are shown. The four reduction steps ${\text{NO}}_3^{-}$ → ${\text{NO}}_2^{-}$; ${\text{NO}}_2^{-}$ → NO; NO → N₂O and N₂O → N₂ are represented. The flagellum structure is drafted based on previous works on Salmonella enterica and E. coli. B. The respirasome of Pseudomonas aeruginosa encompassing, among others, primary dehydrogenases, respiratory chain complexes I‐V, ubiquinol, cytochrome c and the SecAYEG translocon. The electron flow is specified with black arrows. The proton translocation associated with ATP generation is also depicted. NAM, N‐acetylglucosamine; NAG, N‐acetylmuramic acid; LPS, lipopolysaccharide; OM, outer membrane; PG, peptidoglycan; P, periplasm; IM, inner membrane; C, cytoplasm; type III SS, type III secretion system; TCA enzymes, enzymes involved in the tricarboxylic acid (TCA) cycle.

Figure 3

Visualization of impaired flagellar formation in the Pseudomonas aeruginosa nirS mutant and determination of the interacting domains of the NirS and FliC proteins by LC‐MS/MS. A. The flagellar formation in the wild type and nirS strains (grown under anaerobic conditions and 20 mM arginine) is shown by fluorescence microscopy. Antibodies against DnaK and FliC were raised and employed for detection. Goat anti‐rabbit Alexa 488 (for FliC) or goat anti‐rabbit Alexa 568 (for DnaK) were used as fluorescently labelled secondary antibodies. DAPI dye was utilized for DNA staining. B. The interacting domains between NirS and FliC elucidated by mass spectrometry by determination of the cross‐linked peptides are shown for NirS: AAEQYQGAASAVDPTHVVR (white), CAGCHGVLRK (blue) and GQQYLEALITYGTPLGMPNWGSSGELSK (orange). The haem d ₁ (green) and haem c (red) are also highlighted in the NirS structure.