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Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

Summary: 3‐Deoxy‐d‐manno‐oct‐2‐ulosonic acid (Kdo) is an essential component of lipopolysaccharides (LPS) in the Gram‐negative bacterial outer membrane. Metabolic labeling of Escherichia coli LPS with 8‐azido‐3,8‐dideoxy‐d‐manno‐oct‐2‐ulosonic acid (Kdo‐N3) has been reported but is inefficient. For optimization, it is important to understand how exogenous Kdo‐N3 enters the cytoplasm. Based on similarities between Kdo and sialic acids, we proposed and verified that the sialic acid transporter NanT imports exogenous Kdo‐N3 into E. coli. We demonstrated that E. coli ΔnanT were not labeled with Kdo‐N3, while expression of NanT in the ΔnanT mutant restored Kdo‐N3 incorporation. Induced NanT expression in a strain lacking Kdo biosynthesis led to higher exogenous Kdo incorporation and restoration of full‐length core‐LPS, suggesting that NanT also transports Kdo. While Kdo‐N3 incorporation was observed in strains having NanT, it was not detected in Pseudomonas aeruginosa and Acinetobacter baumannii, which lack nanT. However, heterologous expression of E. coli NanT in P. aeruginosa enabled Kdo‐N3 incorporation and labeling, though this led to abnormal morphology and growth arrest. NanT seems to define which bacteria can be labeled with Kdo‐N3, provides opportunities to enhance Kdo‐N3 labeling efficiency and spectrum, and raises the possibility of Kdo biosynthetic bypass where exogenous Kdo is present, perhaps even in vivo. Lipopolysaccharides (LPS) form the outside of the Gram‐negative bacterial outer membrane. The uptake and incorporation of a Kdo sugar analog, Kdo‐N3, into Escherichia coli LPS is inefficient. Our work identifies the sialic acid sugar transporter NanT as the uptake mechanism of exogenous Kdo and Kdo‐N3. This insight will help to enhance Kdo‐N3 incorporation, to understand which bacterial strains can be labeled with Kdo‐N3, and to enable LPS labeling in bacteria which lack the transporter. [ABSTRACT FROM AUTHOR]

LpxD, acyl-ACP-dependent N -acyltransferase, is the third enzyme of lipid A biosynthesis in Gram-negative bacteria. A recent probe-based screen identified several compounds, including 6359-0284 (compound 1 ), that inhibit the enzymatic activity of Escherichia coli ( E. coli ) LpxD. Here, we use these inhibitors to chemically validate LpxD as an attractive antibacterial target. We first found that compound 1 was oxidized in solution to the more stable aromatized tetrahydro-pyrazolo-quinolinone compound 1o . From the Escherichia coli strain deficient in efflux, we isolated a mutant that was less susceptible to compound 1o and had an lpxD missense mutation (Gly268Cys), supporting the cellular on-target activity. Using surface plasma resonance, we showed direct binding to E. coli LpxD for compound 1o and other reported LpxD inhibitors in vitro . Furthermore, we determined eight cocrystal structures of E. coli LpxD/inhibitor complexes. These costructures pinpointed the 4'-phosphopantetheine binding site as the common ligand binding hotspot, where hydrogen bonds to Gly269 and/or Gly287 were important for inhibitor binding. In addition, the LpxD/compound 1o costructure rationalized the reduced activity of compound 1o in the LpxD Gly268Cys mutant. Moreover, we obtained the LpxD structure in complex with a previously reported LpxA/LpxD dual targeting peptide inhibitor, RJPXD33, providing structural rationale for the unique dual targeting properties of this peptide. Given that the active site residues of LpxD are conserved in multidrug resistant Enterobacteriaceae, this work paves the way for future LpxD drug discovery efforts combating these Gram-negative pathogens.