Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.

Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.

Iron is a key nutrient for almost all living organisms. Paradoxically, it is poorly soluble and 3 consequently poorly bioavailable. Bacteria have thus developed multiple strategies to access 4 this metal. One of the most common consists of the use of siderophores, small compounds that chelate ferric iron with very high affinity. Many bacteria are able to produce their own 6 siderophores or use those produced by other microorganisms (exosiderophores) in a piracy 7 strategy. Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and 8 is also able to use a large panel of exosiderophores. We investigated the ability of P. aeruginosa 9 to use nocardamine (NOCA) and ferrioxamine B (DFOB) as exosiderophores under iron-10 limited planktonic growth conditions. Proteomic and RT-qPCR approaches showed induction 11 of the transcription and expression of the outer membrane transporter FoxA in the presence of 12 NOCA or DFO in the bacterial environment. Expression of the proteins of the heme-or 13 pyoverdine-and pyochelin-dependent iron uptake pathways was not affected by the presence 14 of these two tris-hydroxamate siderophores. 55 Fe uptake assays using foxA mutants showed 15 ferri-NOCA to be exclusively transported by FoxA, whereas ferri-DFOB was transported by 16 FoxA and at least one other unidentified transporter. The crystal structure of FoxA complexed 17 with NOCA-Fe revealed very similar siderophore binding sites between NOCA-Fe and DFOB-18 Fe. We discuss iron uptake by hydroxamate exosiderophores in P. aeruginosa cells in the light 19 of these results. 20 Page 2 of 40 ACS Paragon Plus Environment ACS Chemical Biology 1 detected the presence of ferri-exosiderophores, these transcriptional regulators activate the 2 transcription of the corresponding TBDT for iron acquisition. 9,22,29-32 3 Here, we investigated the ability of P. aeruginosa to use nocardamine (NOCA, Figure 1A) as 4 an exosiderophore. NOCA, also called desferrioxamine E, is a cyclic tris-hydroxamate 5 siderophore related to DFOB, with a higher affinity for iron: 10 32 M-1 for NOCA and 10 30 M-1 6 for DFOB 33 (Figure 1). NOCA is synthesized by various actinomycetes, such as Streptomyces, Nocardia, and Micromonospora, and bacteria, such as Streptomyces griseus, 35 Pseudomonas 8 stutzeri, 36 and Enterobacter agglomerans. 37 We show that the presence of NOCA in the P. 9 aeruginosa environment strongly induces the transcription and expression of foxA with the 10 same efficiency as DFOB. We demonstrate, using 55 Fe, that NOCA-55 Fe is exclusively 11 transported by the TBDT FoxA, whereas DFOB uses, in addition to FoxA, at least one other 12 transporter. We also determined the crystal structure of FoxA complexed with ferri-NOCA, 13 revealing a siderophore-binding site very similar to that of DFOB.

Many microbes and fungi acquire the essential ion Fe3+ through the synthesis and secretion of high-affinity chelators termed siderophores. In Gram-negative bacteria, these ferric-siderophore complexes are actively taken up using highly specific TonB-dependent transporters (TBDTs) located in the outer bacterial membrane (OM). However, the detailed mechanism of how the inner-membrane protein TonB connects to the transporters in the OM as well as the interplay between siderophore- and TonB-binding to the transporter is still poorly understood. Here, we present three crystal structures of the TBDT FoxA from Pseudomonas aeruginosa (containing a signalling domain) in complex with the siderophore ferrioxamine B and TonB and combine them with a detailed analysis of binding constants. The structures show that both siderophore and TonB-binding is required to form a translocation-competent state of the FoxA transporter in a two-step TonB-binding mechanism. The complex structure also indicates how TonB-binding influences the orientation of the signalling domain.

Escherichia coli possesses a number of specific K(+) influx and efflux systems that maintain an appropriate intracellular K(+) concentration. Although regulatory mechanisms have been identified for a number of these transport systems, the exact mechanism through which K(+) concentration is sensed in the cell remains unknown. In this work we show that Kbp (K(+) binding protein, formerly YgaU), a soluble 16-kDa cytoplasmic protein from Escherichia coli, is a highly specific K(+) binding protein and is required for normal growth in the presence of high levels of external K(+). Kbp binds a single potassium ion with high specificity over Na(+) and other metal ions found in biological systems, although, in common with K(+) transporters, it also binds Rb(+) and Cs(+). Dissection of the K(+) binding determinants of Kbp suggests a mechanism through which Kbp is able to sense changes in K(+) concentration over the relevant range of intracellular K(+) concentrations.