Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity.

Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver of the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of lactonase SsoPox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being >1000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, and some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops and changes to their conformations, as revealed by a suite of crystal structures. This suggests that a specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining the enzyme activity profile.

Catalytic Redundancies and Conformational Plasticity Drives Selectivity and Promiscuity in Quorum Quenching Lactonases.

Several enzymes from the metallo-ß-lactamase-like family of lactonases (MLLs) degrade N- acyl-L-homoserine lactones (AHLs). In doing so, they play a role in a microbial communication system, quorum sensing, which contributes to pathogenicity and biofilm formation. There is currently great interest in designing quorum quenching ( QQ ) enzymes that can interfere with this communication and be used in a range of industrial and biomedical applications. However, tailoring these enzymes for specific targets requires a thorough understanding of their mechanisms and the physicochemical properties that determine their substrate specificities. We present here a detailed biochemical, computational, and structural study of the MLL GcL, which is highly proficient, thermostable, and has broad substrate specificity. Strikingly, we show that GcL does not only accept a broad range of substrates but is also capable of utilizing different reaction mechanisms that are differentially used in function of the substrate structure or the remodeling of the active site via mutations. Comparison of GcL to other lactonases such as AiiA and AaL demonstrates similar mechanistic promiscuity, suggesting this is a shared feature across lactonases in this enzyme family. Mechanistic promiscuity has previously been observed in the lactonase/paraoxonase PON1, as well as with protein tyrosine phosphatases that operate via a dual general-acid mechanism. The apparent prevalence of this phenomenon is significant from both a biochemical and an engineering perspective: in addition to optimizing for specific substrates, it is possible to optimize for specific mechanisms, opening new doors not just for the design of novel quorum quenching enzymes, but also of other mechanistically promiscuous enzymes.

N-acyl homoserine lactone signaling modulates bacterial community associated with human dental plaque.

N-acyl homoserine lactones (AHLs) are small diffusible signaling molecules that mediate a cell density-dependent bacterial communication system known as quorum sensing (QS). AHL-mediated QS regulates gene expression to control many critical bacterial behaviors including biofilm formation, pathogenicity, and antimicrobial resistance. Dental plaque is a complex multispecies oral biofilm formed by successive colonization of the tooth surface by groups of commensal, symbiotic, and pathogenic bacteria, which can contribute to tooth decay and periodontal diseases. While the existence and roles of AHL-mediated QS in oral microbiota have been debated, recent evidence indicates that AHLs play significant roles in oral biofilm development and community dysbiosis. The underlying mechanisms, however, remain poorly characterized. To better understand the importance of AHL signaling in dental plaque formation, we manipulated AHL signaling by adding AHL lactonases or exogenous AHL signaling molecules. We find that AHLs can be detected in dental plaque grown under 5% CO2 conditions, but not when grown under anaerobic conditions, and yet anaerobic cultures are still responsive to AHLs. QS signal disruption using lactonases leads to changes in microbial population structures in both planktonic and biofilm states, changes that are dependent on the substrate preference of the used lactonase but mainly result in the increase in the abundance of commensal and pioneer colonizer species. Remarkably, the opposite manipulation, that is the addition of exogenous AHLs increases the abundance of late colonizer bacterial species. Hence, this work highlights the importance of AHL-mediated QS in dental plaque communities, its potential different roles in anaerobic and aerobic parts of dental plaque, and underscores the potential of QS interference in the control of periodontal diseases.

Engineering quorum quenching acylases with improved kinetic and biochemical properties.

Many Gram-negative bacteria use N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm-related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to generate these improved properties using the Protein One-Stop Shop Server. The engineering of acylases is complicated by low-throughput enzymatic assays. Alleviating this challenge, we report a time-course kinetic assay for AHL acylases that monitors the real-time production of homoserine lactone. Using the assay, we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2°C, which translated into high resistance against organic solvents and increased compatibility with material coatings. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl-enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases.

Dinickel enzyme evolved to metabolize the pharmaceutical metformin and its implications for wastewater and human microbiomes.

Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug's direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmA and mfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 × 103 M-1s-1 and KM for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.

Field testing of an enzymatic quorum quencher coating additive to reduce biocorrosion of steel.

Microbial colonization can be detrimental to the integrity of metal surfaces and lead to microbiologically influenced corrosion. Biocorrosion is a serious problem for aquatic and marine industries in the world and severely affects the maritime transportation industry by destroying port infrastructure and increasing fuel usage and the time and cost required for maintenance of transport vessels. Here, we evaluate the potential of a stable quorum quenching lactonase enzyme to reduce biocorrosion in the field. Over the course of 21 months, steel samples coated with lactonase-containing acrylic paint were submerged at two different sites and depths in the Duluth-Superior Harbor (Lake Superior, MN, USA) and benchmarked against controls, including the biological biocide surfactin. In this experiment, the lactonase treatment outperformed the surfactin biocide treatment and significantly reduced the number of corrosion tubercles (37%; P < 0.01) and the corroded surface area (39%; P < 0.01) as compared to the acrylic-coated control coupons. In an attempt to evaluate the effects of signal disruption of surface microbial communities and the reasons for lower corrosion levels, 16S rRNA sequencing was performed and community populations were analyzed. Interestingly, surface communities were similar between all treatments, and only minor changes could be observed. Among these changes, several groups, including sulfate-reducing bacteria (SRB), appeared to correlate with corrosion levels, and more specifically, SRB abundance levels were lower on lactonase-treated steel coupons. We surmise that these minute community changes may have large impacts on corrosion rates. Overall, these results highlight the potential use of stable quorum quenching lactonases as an eco-friendly antifouling coating additive. IMPORTANCE Biocorrosion severely affects the maritime transportation industry by destroying port infrastructure and increasing fuel usage and the time and cost required to maintain transport vessels. Current solutions are partly satisfactory, and the antifouling coating still largely depends on biocide-containing products that are harmful to the environment. The importance of microbial signaling in biofouling and biocorrosion is not elucidated. We here take advantage of a highly stable lactonase that can interfere with N-acyl homoserine lactone-based quorum sensing and remain active in a coating base. The observed results show that an enzyme-containing coating can reduce biocorrosion over 21 months in the field. It also reveals subtle changes in the abundance of surface microbes, including sulfate-reducing bacteria. This work may contribute to pave the way for strategies pertaining to surface microbiome changes to reduce biocorrosion.

Engineering Quorum Quenching Acylases with Improved Kinetic and Biochemical Properties.

Many Gram-negative bacteria respond to N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as acylases, chemically degrade AHL signals, prevent signal reception by bacteria, and inhibit undesirable traits related to biofilm. These capabilities make these enzymes appealing candidates for controlling microbes. Yet, enzyme candidates with high activity levels, high substrate specificity for specific interference, and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to obtain improved acylase variants. The engineering of acylase is complicated by low-throughput enzymatic assays. To alleviate this challenge, we report a time-course kinetic assay for AHL acylase that tracks the real-time production of homoserine lactone. Using the protein one-stop shop server (PROSS), we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2 °C, which translated into high resistance against organic solvents and increased compatibility with material coatings. We also generated mutants of MacQ with considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. In fact, the variants presented here exhibit unique combinations of stability and activity levels. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl-enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases.

Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity.

Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver for the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of the lactonase SsoPox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being > 1,000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, ~1,000,000-fold and beyond, since some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops, as revealed by a suite of crystal structures. This suggests that specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining an enzyme activity profile.

Structure and Functional Properties of Bacterial Communities in Surface Sediments of the Recently Declared Nutrient-Saturated Lake Villarrica in Southern Chile.

Lake Villarrica, one of Chile's main freshwater water bodies, was recently declared a nutrient-saturated lake due to increased phosphorus (P) and nitrogen (N) levels. Although a decontamination plan based on environmental parameters is being established, it does not consider microbial parameters. Here, we conducted high-throughput DNA sequencing and quantitative polymerase chain reaction (qPCR) analyses to reveal the structure and functional properties of bacterial communities in surface sediments collected from sites with contrasting anthropogenic pressures in Lake Villarrica. Alpha diversity revealed an elevated bacterial richness and diversity in the more anthropogenized sediments. The phylum Proteobacteria, Bacteroidetes, Acidobacteria, and Actinobacteria dominated the community. The principal coordinate analysis (PCoA) and redundancy analysis (RDA) showed significant differences in bacterial communities of sampling sites. Predicted functional analysis showed that N cycling functions (e.g., nitrification and denitrification) were significant. The microbial co-occurrence networks analysis suggested Chitinophagaceae, Caldilineaceae, Planctomycetaceae, and Phycisphaerae families as keystone taxa. Bacterial functional genes related to P (phoC, phoD, and phoX) and N (nifH and nosZ) cycling were detected in all samples by qPCR. In addition, an RDA related to N and P cycling revealed that physicochemical properties and functional genes were positively correlated with several nitrite-oxidizing, ammonia-oxidizing, and N-fixing bacterial genera. Finally, denitrifying gene (nosZ) was the most significant factor influencing the topological characteristics of co-occurrence networks and bacterial interactions. Our results represent one of a few approaches to elucidate the structure and role of bacterial communities in Chilean lake sediments, which might be helpful in conservation and decontamination plans.

Evidence for Complex Interplay between Quorum Sensing and Antibiotic Resistance in Pseudomonas aeruginosa.

Quorum sensing (QS) is a cell-density-dependent, intercellular communication system mediated by small diffusible signaling molecules. QS regulates a range of bacterial behaviors, including biofilm formation, virulence, drug resistance mechanisms, and antibiotic tolerance. Enzymes capable of degrading signaling molecules can interfere in QS-a process termed as quorum quenching (QQ). Remarkably, previous work reported some cases where enzymatic interference in QS was synergistic to antibiotics against Pseudomonas aeruginosa. The premise of combination therapy is attractive to fight against multidrug-resistant bacteria, yet comprehensive studies are lacking. Here, we evaluate the effects of QS signal disruption on the antibiotic resistance profile of P. aeruginosa by testing 222 antibiotics and antibacterial compounds from 15 different classes. We found compelling evidence that QS signal disruption does indeed affect antibiotic resistance (40% of all tested compounds; 89/222), albeit not always synergistically (not synergistic for 19% of compounds; 43/222). For some tested antibiotics, such as sulfathiazole and trimethoprim, we were able to relate the changes in resistance caused by QS signal disruption to the modulation of the expression of key genes of the folate biosynthetic pathway. Moreover, using a P. aeruginosa-based Caenorhabditis elegans killing model, we confirmed that enzymatic QQ modulates the effects of antibiotics on P. aeruginosa's pathogenicity in vivo. Altogether, these results show that signal disruption has profound and complex effects on the antibiotic resistance profile of P. aeruginosa. This work suggests that combination therapy including QQ and antibiotics should be discussed not globally but, rather, in case-by-case studies. IMPORTANCE Quorum sensing (QS) is a cell-density-dependent communication system used by a wide range of bacteria to coordinate behaviors. Strategies pertaining to the interference in QS are appealing approaches to control microbial behaviors that depend on QS, including virulence and biofilms. Interference in QS was previously reported to be synergistic with antibiotics, yet no systematic assessment exists. Here, we evaluate the potential of combination treatments using the model opportunistic human pathogen Pseudomonas aeruginosa PA14. In this model, collected data demonstrate that QS largely modulates the antibiotic resistance profile of PA14 (for more than 40% of the tested drugs). However, the outcome of combination treatments is synergistic for only 19% of them. This research demonstrates the complex relationship between QS and antibiotic resistance and suggests that combination therapy including QS inhibitors and antibiotics should be discussed not globally but, rather, in case-by-case studies.

Applying molecular and phenotypic screening assays to identify efficient quorum quenching lactonases.

Quorum sensing (QS) is a molecular communication system used by microorganisms to adopt behaviors in a cell density-dependent manner. Lactonase enzymes, able to hydrolyze the signal molecules acyl-homoserine lactones (AHL) can counteract QS-mediated virulence in Gram-negative bacteria. Optimizing lactonases activity or specificity for AHL through enzyme engineering approaches is thus highly attractive to increase protective effect. However, only a limited number of screening methods have been developed to handle and evaluate AHL-degrading enzyme libraries. Here, a series of screening procedures were developed to identify improved lactonases using two previously reported enzymes as benchmarks, namely SsoPox and GcL. Specifically, molecular screenings using six different AHL and based on two reporter strains; i.e., Chromobacterium violaceum CV026 and Pseudomonas putida KS35, are reported. In addition, three phenotype-based screenings aiming to evaluate the ability of enzymes to quench a particular QS-related behavior are reported, using C. violaceum, Pseudomonas aeruginosa and Vibrio harveyi as pathogenic type strains. These assays were used to screen a small-sized library and allowed for the identification of various improved variants. To confirm that these variants were real "hits", four of them were produced and purified. Their kinetic parameters against AHL substrates were found to be increased by 2-44.5 -fold as compared to the starting enzyme. Moreover, their increased activity was confirmed by measuring their ability to quench QS in different bacterial systems. These new assays will facilitate the screening of enzyme libraries and will pave the way for the development of proficient engineered QS-disrupting enzymes.

Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis.

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.

Correction: Evaluation of biological and enzymatic quorum quencher coating additives to reduce biocorrosion of steel.

[This corrects the article DOI: 10.1371/journal.pone.0217059.].

Corrigendum: Signal Disruption Leads to Changes in Bacterial Community Population.

[This corrects the article DOI: 10.3389/fmicb.2019.00611.].

Discovery of an ultraspecific triuret hydrolase (TrtA) establishes the triuret biodegradation pathway.

Triuret (carbonyldiurea) is an impurity found in industrial urea fertilizer (<0.1% w/w) that is applied, worldwide, around 300 million pounds each year on agricultural lands. In addition to anthropogenic sources, endogenous triuret has been identified in amoeba and human urine, the latter being diagnostic for hypokalemia. The present study is the first to describe the metabolic breakdown of triuret, which funnels into biuret metabolism. We identified the gene responsible for triuret decomposition (trtA) in bacterial genomes, clustered with biuH, which encodes biuret hydrolase and has close protein sequence homology. TrtA is a member of the isochorismatase-like hydrolase (IHL) protein family, similarly to BiuH, and has a catalytic efficiency (kcat/KM) of 6 x 105 M-1s-1, a KM for triuret of 20 µM, and exquisite substrate specificity. Indeed, TrtA has four orders of magnitude less activity with biuret. Crystal structures of TrtA in apo and holo form were solved and compared with the BiuH structure. The high substrate selectivity was found to be conveyed by second shell residues around each active site. Mutagenesis of residues conserved in TrtA to the alternate consensus found in BiuHs revealed residues critical to triuret hydrolase activity but no single mutant evolved more biuret activity, and likely a combination of mutations is required to interconvert between TrtA, BiuH functions. TrtA-mediated triuret metabolism is relatively rare in recorded genomes (1-2%), but is largely found in plant-associated, nodulating, and endophytic bacteria. This study suggests functions for triuret hydrolase in certain eukaryotic intermediary processes and prokaryotic intermediary or biodegradative metabolism.

Quorum quenching enzymes and their effects on virulence, biofilm, and microbiomes: a review of recent advances.

INTRODUCTION: Numerous bacterial behaviors are regulated by a cell-density dependent mechanism known as Quorum Sensing (QS). QS relies on communication between bacterial cells using diffusible signaling molecules known as autoinducers. QS regulates physiological processes such as metabolism, virulence, and biofilm formation. Quorum Quenching (QQ) is the inhibition of QS using chemical or enzymatic means to counteract behaviors regulated by QS. AREAS COVERED: We examine the main, diverse QS mechanisms present in bacterial species, with a special emphasis on AHL-mediated QS. We also discuss key in vitro and in vivo systems in which interference in QS was investigated. Additionally, we highlight promising developments, such as the substrate preference of the used enzymatic quencher, in the application of interference in QS to counter bacterial virulence. EXPERT OPINION: Enabled via the recent isolation of highly stable quorum quenching enzymes and/or molecular engineering efforts, the effects of the interference in QS were recently evaluated outside of the traditional model of single species culture. Signal disruption in complex microbial communities was shown to result in the disruption of complex microbial behaviors, and changes in population structures. These new findings, and future studies, may result in significant changes in the traditional views about QS.

Engineering acyl-homoserine lactone-interfering enzymes toward bacterial control.

Enzymes able to degrade or modify acyl-homoserine lactones (AHLs) have drawn considerable interest for their ability to interfere with the bacterial communication process referred to as quorum sensing. Many proteobacteria use AHL to coordinate virulence and biofilm formation in a cell density-dependent manner; thus, AHL-interfering enzymes constitute new promising antimicrobial candidates. Among these, lactonases and acylases have been particularly studied. These enzymes have been isolated from various bacterial, archaeal, or eukaryotic organisms and have been evaluated for their ability to control several pathogens. Engineering studies on these enzymes were carried out and successfully modulated their capacity to interact with specific AHL, increase their catalytic activity and stability, or enhance their biotechnological potential. In this review, special attention is paid to the screening, engineering, and applications of AHL-modifying enzymes. Prospects and future opportunities are also discussed with a view to developing potent candidates for bacterial control.

Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa.

The human opportunistic pathogen Pseudomonas aeruginosa orchestrates the expression of many genes in a cell density-dependent manner by using quorum sensing (QS). Two acyl-homoserine lactones (AHLs) are involved in QS circuits and contribute to the regulation of virulence factors production, biofilm formation, and antimicrobial sensitivity. Disrupting QS, a strategy referred to as quorum quenching (QQ) can be achieved using exogenous AHL-degrading lactonases. However, the importance of enzyme specificity on quenching efficacy has been poorly investigated. Here, we used two lactonases both targeting the signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12 HSL) and butyryl-homoserine lactone (C4 HSL) albeit with different efficacies on C4 HSL. Interestingly, both lactonases similarly decreased AHL concentrations and comparably impacted the expression of AHL-based QS genes. However, strong variations were observed in Pseudomonas Quinolone Signal (PQS) regulation depending on the lactonase used. Both lactonases were also found to decrease virulence factors production and biofilm formation in vitro, albeit with different efficiencies. Unexpectedly, only the lactonase with lower efficacy on C4 HSL was able to inhibit P. aeruginosa pathogenicity in vivo in an amoeba infection model. Similarly, proteomic analysis revealed large variations in protein levels involved in antibiotic resistance, biofilm formation, virulence and diverse cellular mechanisms depending on the chosen lactonase. This global analysis provides evidences that QQ enzyme specificity has a significant impact on the modulation of QS-associated behavior in P. aeruginosa PA14.

Evaluation of biological and enzymatic quorum quencher coating additives to reduce biocorrosion of steel.

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.