Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.

In Pseudomonas aeruginosa, the transition between planktonic and biofilm lifestyles is modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP) in response to environmental conditions. Here, we used gene deletions to investigate how the environmental stimulus nitric oxide (NO) is linked to biofilm dispersal, focusing on biofilm dispersal phenotype from proteins containing putative c-di-GMP turnover and Per-Arnt-Sim (PAS) sensory domains. We document opposed physiological roles for the genes ΔrbdA and Δpa2072 that encode proteins with identical domain structure: while ΔrbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in Δpa2072, and biofilm formation was inhibited, compared to wild type. A second pair of genes, ΔfimX and ΔdipA, were selected on the basis of predicted impaired c-di-GMP turnover function: ΔfimX showed increased, ΔdipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for ΔfimX and reduced swimming for ΔdipA. For all four deletion mutants we find that NO-induced biomass reduction correlates with increased NO-driven swarming, underlining a significant role for this motility in biofilm dispersal. Hence P. aeruginosa is able to differentiate c-di-GMP output using structurally highly related proteins that can contain degenerate c-di-GMP turnover domains. Pseudomonas aeruginosa is a gram-negative bacterium known for its environmental versatility. As an opportunistic pathogen, P. aeruginosa causes disease, particularly in immune compromised individuals, and is a major source of morbidity and mortality in cystic fibrosis (CF) patients with chronic colonisation in lungs and airways 1. The ability of P. aeruginosa to form biofilms within CF patients elicits increased antibiotic tolerance, which makes treatment of infections problematic in clinical settings 2. P. aeruginosa biofilm formation and dispersal are known to correlate with intracellular concentrations of the secondary messenger, cyclic dimeric-GMP (c-di-GMP) 3,4. The production and degradation of c-di-GMP relies on two enzymatic activities. Diguanylate cyclases (DGCs) synthesise c-di-GMP from two GTP molecules, while phosphodiesterases (PDEs) hydrolyse the secondary messenger to linear pGpG 3. P. aeruginosa PAO1 encodes 17 different proteins with a DGC domain, 8 with a PDE domain, and 16 that contain both of these domains, with the DGC N-terminal to the PDE domain 5. The transition between planktonic and biofilm lifestyles is accompanied by extracellular polymeric substance (EPS) production and motility changes. Extensive studies have characterised the link between flagella, pili and biofilm morphologies 6-8. The main motility types in P. aeruginosa PAO1 are flagella mediated swimming and pili mediated twitching. Further, the complex swarming motility is required for dispersal, which relies on flagella, pili and surfactants, and involves multicellular group movement on a surface 9,10. The reg...

The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation.