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Enzymes with industrial potential often face limitations due to stability and longevity constraints. Thermostable quorum quenching lactonases are appealing biotechnology tools for controlling microbial pathogenicity and biofilm formation via the interference of quorum sensing. However, the effective formulation of these enzymes remains a challenge. Here, we evaluate the resistance and activity of two thermostable quorum quenching lactonase enzymes (SsoPox and GcL) across diverse formulations relevant to industrial applications. We systematically tested these enzymes with 16 different crop adjuvants (including oils, an anti-foaming agent, surfactants, deposition aids, a water conditioner, and a sticking agent) over a 210-day period, demonstrating broad compatibility except with oil-based adjuvants. Additionally, both enzymes maintained their activity when incorporated into five different coating bases (acrylic, silicone, polyurethane, epoxy, and latex) with activity levels varying according to polymer type. Further investigation of enzymatic acrylic coating characterized the effects of salt water and temperature on enzyme activity levels. Functionalized coatings maintained remarkable stability over 250 days in both wet and dry conditions. These findings establish a practical demonstration and framework for integrating quorum quenching lactonases into industrial materials and formulations, significantly advancing their potential for ‘real-world’ applications for microbial control across multiple sectors.

Numerous bacteria utilize molecular communication systems referred to as quorum sensing (QS) to synchronize the expression of certain genes regulating, among other aspects, the expression of virulence factors and the synthesis of biofilm. To achieve this process, bacteria use signaling molecules, known as autoinducers (AIs), as chemical messengers to share information. Naturally occurring strategies that interfere with bacterial signaling have been extensively studied in recent years, examining their potential to control bacteria. To interfere with QS, bacteria use quorum sensing inhibitors (QSIs) to block the action of AIs and quorum quenching (QQ) enzymes to degrade signaling molecules. Recent studies have shown that these strategies are promising routes to decrease bacterial pathogenicity and decrease biofilms, potentially enhancing bacterial susceptibility to antimicrobial agents including antibiotics and bacteriophages. The efficacy of QSIs and QQ enzymes has been demonstrated in various animal models and are now considered in the development of new medical devices against bacterial infections, including dressings, and catheters for enlarging the therapeutic arsenal against bacteria.

Peptide backbone α- N -methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides. Borosin natural product pathways are the only known ribosomally encoded and posttranslationally modified peptides (RiPPs) pathways to incorporate backbone α- N -methylations on translated peptides. Here we report the discovery of type IV borosin natural product pathways (termed ‘split borosins’), featuring an iteratively acting α- N -methyltransferase and separate precursor peptide substrate from the metal-respiring bacterium Shewanella oneidensis . A series of enzyme-precursor complexes reveal multiple conformational states for both α- N -methyltransferase and substrate. Along with mutational and kinetic analyses, our results give rare context into potential strategies for iterative maturation of RiPPs. Borosins are ribosomally encoded and posttranslationally modified peptide (RiPP) natural products featuring amide-backbone α- N -methylation. Here, the authors report the discovery and characterization of type IV borosin ‘split’ pathways encoding distinct, separate α- N -methyltransferases and precursor peptide substrates.

Bacteria communicate through the accumulation of autoinducer (AI) molecules that regulate gene expression at critical densities in a process called quorum sensing (QS). Extensive work using simple systems and single strains of bacteria have revealed a role for QS in the regulation of virulence factors and biofilm formation; however, less is known about QS dynamics among communities, especially in vivo. In this review, we summarize the diversity of QS signals as well as their ability to influence “non-target” behaviors among species that have receptors but not synthases for those signals. We highlight host-microbe interactions facilitated by QS and describe cross-talk between QS and the mammalian endocrine and immune systems, as well as host surveillance of QS. Further, we describe emerging evidence for the role of QS in non-infectious, chronic, microbially associated diseases including inflammatory bowel diseases and cancers. Finally, we describe potential therapeutic approaches that involve leveraging QS signals as well as quorum quenching approaches to block signaling in vivo to mitigate deleterious consequences to the host. Ultimately, QS offers a previously underexplored target that may be leveraged for precision modification of the microbiota without deleterious bactericidal consequences.

Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.

Quorum quenching lactonases are enzymes that are capable of disrupting bacterial signaling based on acyl homoserine lactones (AHL) via their enzymatic degradation. In particular, lactonases have therefore been demonstrated to inhibit bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. Here we characterized biochemically and structurally a novel representative from the metallo-β-lactamase superfamily, named AaL that was isolated from the thermoacidophilic bacterium Alicyclobacillus acidoterrestris . AaL is a potent quorum quenching enzyme as demonstrated by its ability to inhibit the biofilm formation of Acinetobacter baumannii . Kinetic studies demonstrate that AaL is both a proficient and a broad spectrum enzyme, being capable of hydrolyzing a wide range of lactones with high rates (k cat /K M  > 10 5  M −1 .s −1 ). Additionally, AaL exhibits unusually low K M values, ranging from 10 to 80 µM. Analysis of AaL structures bound to phosphate, glycerol, and C6-AHL reveals a unique hydrophobic patch (W26, F87 and I237), involved in substrate binding, possibly accounting for the enzyme’s high specificity. Identifying the specificity determinants will aid the development of highly specific quorum quenching enzymes as potential therapeutics.

Microbial colonization can be detrimental to the integrity of metal surfaces and lead to microbiologically influenced corrosion (MIC). Biocorrosion is a serious problem for aquatic and marine industries in the world. In Minnesota (USA), where this study was conducted, biocorrosion severely affects the maritime transportation industry. The anticorrosion activity of a variety of compounds, including chemical (magnesium peroxide) and biological (surfactin, capsaicin, and gramicidin) molecules were investigated as coating additives. We also evaluated a previously engineered, extremely stable, non-biocidal enzyme known to interfere in bacterial signaling, SsoPox (a quorum quenching lactonase). Experimental steel coupons were submerged in water from the Duluth Superior Harbor (DSH) for 8 weeks in the laboratory. Biocorrosion was evaluated by counting the number and the coverage of corrosion tubercles on coupons and also by ESEM imaging of the coupon surface. Three experimental coating additives significantly reduced the formation of corrosion tubercles: surfactin, magnesium peroxide and the quorum quenching lactonase by 31%, 36% and 50%, respectively. DNA sequence analysis of the V4 region of the bacterial 16S rRNA gene revealed that these decreases in corrosion were associated with significant changes in the composition of bacterial communities on the steel surfaces. These results demonstrate the potential of highly stable quorum quenching lactonases to provide a reliable, cost-effective method to treat steel structures and prevent biocorrosion.

A new member of the Phosphotriesterase-Like Lactonases (PLL) family from the hyperthermophilic archeon Sulfolobus islandicus (SisLac) has been characterized. SisLac is a native lactonase that exhibits a high promiscuous phosphotriesterase activity. SisLac thus represents a promising target for engineering studies, exhibiting both detoxification and bacterial quorum quenching abilities, including human pathogens such as Pseudomonas aeruginosa. Here, we describe the substrate specificity of SisLac, providing extensive kinetic studies performed with various phosphotriesters, esters, N-acyl-homoserine lactones (AHLs) and other lactones as substrates. Moreover, we solved the X-ray structure of SisLac and structural comparisons with the closely related SsoPox structure highlighted differences in the surface salt bridge network and the dimerization interface. SisLac and SsoPox being close homologues (91% sequence identity), we undertook a mutational study to decipher these structural differences and their putative consequences on the stability and the catalytic properties of these proteins. We show that SisLac is a very proficient lactonase against aroma lactones and AHLs as substrates. Hence, data herein emphasize the potential role of SisLac as quorum quenching agent in Sulfolobus. Moreover, despite the very high sequence homology with SsoPox, we highlight key epistatic substitutions that influence the enzyme stability and activity.

Ultrahigh-resolution X-ray crystallography study of a phosphate-binding protein from Pseudomonas fluorescens yields insight into how phosphate ions essential for life are discriminated from the arsenate ions inimical to it, even in arsenate-rich environments. Short bond answer to arsenate/phosphate discrimination riddle How do bacteria discriminate between phosphate, which is essential to life, and the chemically similar arsenate, a compound highly toxic to most organisms? In this manuscript, the authors characterize the biochemistry of several of the periplasmic phosphate-binding proteins (PBPs) that mediate phosphate uptake into bacterial cells. The proteins discriminate phosphate from arsenate by factors of between 500 and 4,000, depending on how prevalent arsenate is in the habitat. Comparison of ultra-high-resolution structures of PBPs from arsenate-sensitive and arsenate-resistant bacteria suggests that arsenate, which has a radius just 4% larger than that of phosphate, distorts an unusually short high-energy hydrogen bond that is a key component of the recognition site. Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical p K a values 1 , 2 , similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3 ). Phosphate is indispensable and arsenate is toxic, but this extensive similarity raises the question whether arsenate may substitute for phosphate in certain niches 4 , 5 . However, whether it is used or excluded, discriminating phosphate from arsenate is a paramount challenge. Enzymes that utilize phosphate, for example, have the same binding mode and kinetic parameters as arsenate, and the latter’s presence therefore decouples metabolism 6 , 7 . Can proteins discriminate between these two anions, and how would they do so? In particular, cellular phosphate uptake systems face a challenge in arsenate-rich environments. Here we describe a molecular mechanism for this process. We examined the periplasmic phosphate-binding proteins (PBPs) of the ABC-type transport system that mediates phosphate uptake into bacterial cells, including two PBPs from the arsenate-rich Mono Lake Halomonas strain GFAJ-1. All PBPs tested are capable of discriminating phosphate over arsenate at least 500-fold. The exception is one of the PBPs of GFAJ-1 that shows roughly 4,500-fold discrimination and its gene is highly expressed under phosphate-limiting conditions. Sub-ångström-resolution structures of Pseudomonas fluorescens PBP with both arsenate and phosphate show a unique mode of binding that mediates discrimination. An extensive network of dipole–anion interactions 8 , 9 , and of repulsive interactions, results in the 4% larger arsenate distorting a unique low-barrier hydrogen bond. These features enable the phosphate transport system to bind phosphate selectively over arsenate (at least 10 3 excess) even in highly arsenate-rich environments.