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Quorum sensing signals form complexes with Ag and Cu cations Eric McGivney, Kayleigh Elizabeth Jones, Bandrea Weber, Ann Margaret Valentine, Jeanne M. Vanbriesen, and Kelvin B. Gregory ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b01000 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Quorum sensing signals form complexes with Ag+ and Cu2+ cations

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Eric McGivneya,b, Kayleigh Elizabeth Jonesc, Bandrea Webera, Ann Margaret Valentinec, Jeanne

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M. VanBriesena,b, Kelvin B. Gregorya,b*

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Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh,

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Pennsylvania, USAa; Center for the Environmental Implications of NanoTechnology (CEINT),

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Durham, North Carolina, USAb; Department of Chemistry, Temple University, Philadelphia,

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Pennsylvania, USAc

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*Address correspondence to Kelvin B. Gregory: [email protected]

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Abstract: Quorum sensing (QS) regulates important bacterial behaviors such as virulent protein

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production and biofilm formation. QS requires that molecular signals are exchanged between

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cells, extracellularly, where environmental conditions influence signal stability. In this work, we

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present a novel complexation between metal cations (Ag+ and Cu2+) and a QS autoinducer signal,

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N-hexanoyl-L-homoserine lactone (HHL). The molecular interactions were investigated using

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mass spectrometery, attenuated total reflectance—Fourier transform infrared spectroscopy, and

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computational simulations. Results show that HHL forms predominantly 1:1 complexes with Ag+

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(Kd= 3.41 X 10-4 M) or Cu2+ (Kd= 1.40 X 10-5 M), with the coordination chemistry occuring on

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the oxygen moeities. In vivo experiments with Chromobacterium violaceum CV026 show that

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sublethal concentrations of Ag+ and Cu2+ decreased HHL-regulated QS activity. Furthermore,

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when Ag+ was preincubated with HHL, Ag+ toxicity to CV026 decreased by an order of

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magnitude, suggesting HHL:metal complexes alters the bioavailability of the individual

26

constituents.


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INTRODUCTION Quorum sensing (QS) in bacteria is responsible for the regulation of a remarkably wide

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range of coordinated behavior including biofilm formation, pathogenicity, and CRISPR-Cas

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adaptive immunology.1–3 During QS, individual bacterial cells detect their local population

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density and coordinate gene expression in response to endogenously synthesized chemical

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signals known as autoinducers (AI). A commonly studied class of AI in gram-negative bacteria

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are acyl-homoserine lactones (AHL).4 AHLs are synthesized internally via a member of the LuxI

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protein family, and diffuse between the cytoplasm and external environment. Once a threshold

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concentration of AHL is reached in a cell’s local microenvironment, a LuxR receptor-protein

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complex, activated by AHL binding, regulates gene expression. Thus, activites regulated by QS

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are directly dependent upon the concentration and structure of AHLs outside of the cell,

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understanding environmental conditions that affect AHL stability have important implications in

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natural, engineered, and human health systems.

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Several studies have idetified environmental factors that influence stability of AHLs

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(reviewed by 5,6): The pH of a system can predict the hydrolysis of AHLs7; Common soil

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mineralis adsorb AHLs8; Various organisms produce lactonases which can degrade AHLs9;

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Reactive oxygen species, such as hydroxyl radicals, can oxidize AHLs10,11. One environmental

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factor on AHL stability that remains unexamined is the prescence of metals. Bacteria often exist

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in metal-rich environments (e.g., soils, water distribution systems, hospital surfaces).12–14 Some

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metals, such as copper, are essential to bacteria, while others have no biological role, such as

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silver. However, even the essential metals can be toxic at high concentrations.

48 49

Recently, metal-based (nano)materials have been used to reduce QS in bacteria.15–20. Silver has garnered the most attention. Naik, et al., found that AgCl coated TiO2 NPs inhibited



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QS by interfering with AHL activity and inhibited the production of antioxidant pigments in

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Chromobacterium violaceum.18 Singh, et al., also showed that mycogenic AgNPs reduced

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violacein synthesis in C. violaceum and attenuated biofilm formation and virulence factors in

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Pseudomonas aeruginosa via a down regulation of transcriptional activity of AHL synthase and

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receptor enzymes.17 Mohanty, et al., found reduced levels of AHL in cultures of P. syringae when

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exposed to silver NPs.16 How the evidence of using metals to inhibit QS is strong, but none of

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the studies, that we are aware of, have studied the interaction between released ions and AHLs,

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and elucidating a mechanism for metallic nanomaterial-QS interactions requires separating the

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effect of a nanoparticle due to its physical properties (e.g., size, crystalline structure, specific

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surface area, conductivity, etc.) from the effect of the solubilized ions released from the particle.

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Distinguishing between metal-ion effects and nanoparticle effects is of particular concern in QS-

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inhibition studies because previous research has identified that AHL degradation products or

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synthetic precursors have metal chelation potential and thus may alter soluble metal

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concentrations.21,22 However, The potential for the unhydrolyzed native AHLs to participate in

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metal complexation reactions has not, to the best of our knowledge, been considered.

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Understanding these interactions is crucial, as cations occur naturally in the environment, and are

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purposefully utilized in healthcare and engineering systems for their antibacterial and anti QS

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properties, e.g., copper and silver.13,23

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The purpose of this work was to reveal the interaction between a QS signaling molecule,

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N-hexanoyl homoserine lactone (HHL) and two commonly released cations used in antimicrobial

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and anti-QS applications, silver and copper. We hypothesized that HHL and cations could form

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complexes, as similarly structure organic molecules have been demonstrated to previously.21 We


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further hypothesized that complexation between free AHL and free Ag+ or Cu2+ could limit the

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bioavailability of each constituent, diminishing signal reception and metal toxicity.

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RESULTS AND DISCUSSION The interactions between a well-studied AHL, N-hexanoyl-L-homoserine lactone (HHL),

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and two commonly used antibacterial metals, Ag+ and Cu2+ was examined first. Mixtures of 1:1

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(molar ratio) of HHL:Ag+ and HHL:Cu2+ were prepared in an aqueous buffer and analyzed on a

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tandem mass spectrometer to identify the mass-to-charge ratio (m/z) of the resulting compounds

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(Figure 1, panels a and b). Free HHL (m/z 200 [MW+H]+) was identified in each sample. In the

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HHL:Ag+ mixture, a 1:1 and a 1:2 Ag:HHL complex were identified, herewithin refered to as 1

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and 2, respectively. 1 is identified by the m/z peaks at 306 and 308, arising from the natural

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isotopes of Ag; 107Ag (51.83% natural abundance) and 109Ag (48.16% natural abundance),

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respectively. 2 is identified by the m/z peaks at 505 and 507, which correspond to the additive

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molecular weight of two HHL molecules plus 107Ag or 109Ag, respectively. Of the total

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HHLx(Ag+) complexes formed, 89.80% was 1 and 10.20% was 2.

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The mixtures of 1:1 HHL:Cu2+ exhibited similar complexation behavior as silver, where

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an apparent 1:1 HHL:copper complex, herewithin 3, is identified by the m/z peaks at 262 and

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264, corresponding to the natural abundance of

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(30.83% natural abundance), respectively. Similarly, a 2:1 HHL:copper complex, herewithin 4, is

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identified by the m/z peaks at 461 and 463. Interestingly, 3 and 4 were expected to carry an

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overall net charge of +2, which would result in an m/z of 131 and 231. The observed peaks at

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m/z= 262, 264, 461, and 463 suggest that Cu2+ has been reduced to Cu+. One explanation is that

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the ESI-MS ionization process induced a redox reaction of Cu2+, as previous studies reported that

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Cu (69.17% natural abundance) and 65Cu



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Cu2+ reduction to Cu+ can occur in ESI-MS ionization due to charge transfer between copper

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complexes and solvent molecules in the gas phase of the chamber or in an aqueous sample

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matrix.24–26 Of the total HHLx(Cu2+) complexes formed, 89.17% was 3 and 10.83% was 4. None

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of the peaks attributed to 1, 2, 3, or 4 were present in the MS/MS scan of HHL control, Ag(NO3)

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control, or Cu(NO3)2 control (Supporting Figure S1). Conversely, all unlabeled peaks (in Figure

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1, panels a and b) were present in the controls.

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Kauffmann et al. revealed that tetramic acid, an AHL degradation product from

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Pseudomonas aeruginosa, complexed Fe3+.21 When we monitored HHL:Fe3+ mixtures at 1:1,

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2:1, and 3:1 ratios via MS/MS, we did not identify any peaks that were characteristic to the Fe

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isotope pattern in any of the samples (Supporting Figure S1). According to chemical speciation

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simulations, >99% of Fe3+ would be insoluble iron(III) oxide-hydroxide (Supporting Table S4).

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Other studies have found that AHL degradation products complex various metal ions, and

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Kauffmann et al. suggests that these degradation products may be “primodial siderophores”.21,27

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However, to the best of our knowledge, this is the first report of metal complexation with an

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unhydrolyzed, condensed, AHL signaling molecule.

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To elucidate the structure of the HHL:metal complexes, attenuated total reflectance-

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Fourier transformed infrared (ATR-FTIR) spectra were gathered in order to identify the shifts in

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HHL bond vibrations after association with Ag+ or Cu2+. The spectra of the aqueous HHL

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solution was used as a control and subtracted from the spectra of the experimental aqueous

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solutions, 1:1 mixtures of HHL:Ag+ or HHL:Cu2+, so that any shifts or changes were attributed

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to the complexation of HHL to Ag+ or Cu2+ (Figure 1, panels c and d; raw spectra for HHL,

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HHL:Ag+, and HHL:Cu2+ are presented in Supporting Figure S2). The FTIR spectra for the 1:1


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HHL:Ag+ and HHL:Cu2+ mixtures are similar, exhibiting a broad peak at 3500 cm-1, with sharper

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peaks at 2990-2890 cm-1 and 1690 cm-1, which were assigned to the stretching vibrations of v(O-

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H)/v(N-H), -v(C-H), and -v(C=O), respectively. Simulated coordination complexation occurred

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with the carbonyl oxygen moieties of HHL, which are believed to form hydrogen bonds with the

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LuxR-type cognate receptor, required for QS activated gene expression (Figure 1, panels e, f, g,

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and h, XYZ coordinates can be found in Supporting Information).28 The estimated bond lengths

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and bond angles for 1, 2, 3, and 4 four complexes are presented in Supporting Table S1. While

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gas phase calculations do not account for aqueous behavior with neighboring solvents, the

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coordination with the oxygen moieties can be accounted for twofold—first, the charge of the

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cations would favor and suggest binding to the O over the amine N; secondly, computational

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modeling was attempted to minimize the geometry with preferential binding to the amine N and

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the metal and a structure would not converge. While it is feasible that the binding in solution

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would be different than in the gas phase, previous reports21,27 suggest binding to these oxygen

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groups in the AHL degradation product, tetramic acid.

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HHL is the endogenous signaling molecule produced by C. violaceum ATCC 31532. In

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response to HHL signals, 31532 produces an antioxidant pigment called violacein. C. violaceum

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CV026 is a QS-negative mutant strain of 31532 that is unable to produce its own HHL, however,

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CV026 can synthesize violacein in response to the exogenous addition of HHL.29 We tested

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HHL-metal mixtures for their ability to induce QS in CV026. HHL (0.75 µM, Supporting Figure

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S3) was incubated with sublethal concentrations of Ag+ or Cu2+ (Table 1) before cultures of C.

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violaceum CV026 were innoculated into each well. After 24 hours of incubation at 30°C,

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violacein production was detected spectroscopically and normalized to cell concentration (Figure

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2, panel a). Over the initial metal exposure concentrations (6.0 x 10-8–1.0 x 10-3 mM Ag+ and 8.5



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x 10-5–1.0 mM Cu2+), we did not observe a decrease of cell growth suggestive of toxicity, but

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there was a significant decrease in violacein production in a dose-dependent manner. It should be

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noted that wild type C. violcaeum (31532) constitutively produces HHL, so it is possible that

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HHL signal accumulation eventually exceeds cation concentration required to repress QS

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activity. When the dose response experiments were repeated in the absence of HHL, C.

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violaceum CV026 was more sensitive to Ag+ (Figure 2, panel b, and Table 1). However, the

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absence of HHL did not significantly alter the toxicity of Cu2+. Previously, Braud, et al., found

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that the extracellular binding of metals by siderophores reduced metal toxicity in Pseudomonas

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aeruginosa.30

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151

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The formation of 1, 2, 3, and 4, which would result in a loss of both free HHL and free

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cation from solution, might explain the observed decrease in violacein production and the

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observed increase in IC50 value for Ag+ when HHL is present. Previous cell-free studies revealed

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that copper had no impact on the metallo-enzymes that synthesize violacein (silver was not

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tested).31 Other factors that are known to influence violacein production (i.e., temperature,

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agitation, pH, nutrients, and absorbance peak-shift due to metals) were controlled in the

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experimental system here (Supporting Figure S4).31–33 Recently, Glišić et al., studied the impacts

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of Cu(II) ion on QS systems and saw a reduction in violacein production in Chromobacterium

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violaceum CV026 and biofilm formation in Pseudomonas aeruginosa PAO1.34 Their proposed

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mode of inhibition was modulation of AHL production, while AHL metal interactions were not

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discussed.


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The removal of free HHL after incubation with increasing concentrations of Ag+ or Cu2+

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was quantified by measuring HHL loss via liquid chromatography-MS/MS (Figure 2, panel c).

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Using an initial HHL concentration of 1 mM, we observed a nearly 40% decrease in free HHL

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ligand after a 30-minute incubation with 100 mM Ag+ or Cu2+ compared to a no-metal control.

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The detected loss of free HHL from solution is assumed to occur due to metal complexation, as

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there was no correllation to an increase in the hydrolysis product (Supporting Figures S5 and

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S6). The measurement of a apparent dissociation constants, Kd, in ultra-pure water were

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estimated using equation 1,

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[ுு௅൫ெ௘ ೙శ ൯]

[ுு௅ ൫ெ௘ ೙శ ൯]

మ ݂ଵ:ଵ [ெ௘ ೙శ][ுு௅] + ݂ଶ:ଵ [ெ௘ ೙శ = 1/‫ܭ‬ௗ ][ுு௅]మ

(Equation 1.)

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by using the known molar metal concentration, [Men+], the fraction of 1:1, f1:1, and 2:1, f2:1,

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HHL:Men+ complexes, the measured molar HHL concentration, [HHL], and assuming all HHL

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loss was due to metal complexation in 1:1, [HHL(Men+)], and 2:1, [HHL2(Men+)]. Using these

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values, HHL was deduced to have a stronger affinity for Cu2+ (Kd= 1.40 X 10-5 M) than Ag+ (Kd=

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3.41 X 10-4 M).

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The current work demonstrates that HHL, a QS autoinducer of the AHL-class, forms

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complexes with Ag+ and Cu2+ cations, respectively, likely forming a coordination complex at the

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carbonyl oxygen groups of the HHL molecule. Ag+ toxicity was reduced in C. violaceum CV026

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when HHL was added to the system, possibly due to HHL(Ag+) complexation reducing free Ag+

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concentration. This suggests that QS signals may increase bacterial metal tolerance. When Ag+ or

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Cu2+ was preincubated with HHL, QS-induced violacein production was limited. The mechanism

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of QS-induced violacein production involves many steps, from the synthesis of signaling

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molecules, to the synthesis of antioxidant pigment, where the addition of reactive metal ions may

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interfere. Thus, the nature of the interference cannot be conclusively determined. Future



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experimental studies should explore the binding capability of cation–HHL complexes to LuxR

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proteins. Furthermore, it is important to note here that the biological assays which tested metal

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toxicity and QS activity (Figure 2, panels a and b) were tested in LB media, an undefined

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nutrient rich media used to enduce QS in CV026. Due to the complexity of LB media, the

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determination of metal speciation is extremely complex, and the copper and silver cations are

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competing for other organic ligands in addition to HHL. Previous studies looking at copper

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speciatoin in complex growth media (pH 7–7.2) find that 95%, Cayman Chemicals, Ann Arbor, MI), Silver nitrate (Acros Organics, Morris

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Plains, NJ), Copper(II) nitrate (Acros Organics, Morris Plains, NJ), ferric chloride (>99%,

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Sigma-Aldrich, St. Louis, MO), ammonium acetate (Fischer Scientific, Pittsburgh, PA),

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acetonitrile (HPLC grade, EMD, Gibbstown, NJ), Millipore 18 MΩcm Milli-Q water (Millipore,

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Bedford, MA, USA), formic acid (Alfa Aesar, Ward Hill, MA), Luria-Bertani Broth (Miller,

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LBDifco BD), kayamycin sulfate (Amresco, Solon, OH), Dimethyl sulfoxide (>99.9%, Alpha

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Aesar, Ward Hill, MA). Chromobacterium violaceum CV026 was acquired through the

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Colección Española de Cultivos Tipo (CECT 5999). Instruments used include Thermo

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Scientitific Spectronic 2000; Agilent Technologies Cary Series UV-Vis-NIR Spectrophotometer;

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and Agilent 6430 Triple Quad LC/MS, Agilent 1100 Series.

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HHL—metal complexation studies. Stock solutions of silver nitrate (10 mM), copper(II) nitrate

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(10 mM), ferric chloride (10 mM), and HHL (10 mM) were prepared in ammonium acetate

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buffer (20 mM, pH 7). None of the stock solutions contained any precipitates. Stock solutions

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were diluted to 5 mM to create 1:1 molar ratios of HHL:Ag+ and HHL:Cu2+ separately. In an

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attempt to detect HHL:Fe complexes, 1:1, 2:1, and 3:1 ratios were tested. The mixtures were

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immediately vortexed, filtered (0.45 µm) and analyzed on liquid chromatography—mass

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spectrometry (LC-MS, Supporting Tables S2 and S3). Stock solutions prior to mixing were also

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analyzed by LC-MS. Speciation simulations were carried out using Visual MINTEQ (Supporting

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Table S4).36

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Mixtures were then analyzed using attenuated total reflection Fourier transform infrared

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spectroscopy (ATR-FTIR). ATR-FTIR spectra were obtained using a PerkinElmer Frontier FT-IR

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spectrometer equipped with a germanium ATR crystal. Scans were performed from 4000 cm-1 to



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700 cm-1 at a rate of 0.2 cm s-1. The traces are the average of 4 scans with a 4 cm-1 resolution.

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Spectra were generated using PerkinElmer Spectrum software. The source was an MIR and the

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detector was an MIR TGS (1500-370) cm-1 with a OptKBr beamsplitter.

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To further assess the structure of the formed complexes, gas-phase calculations were performed

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at the density functional theory (DFT) level using Gaussian 03 through the server WebMO.37

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Geometry optimization was performed using the hybrid B3LYP gradient corrected correlation

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function, and two different basis sets were employed. The basis set of 6-31G(d) was used on all

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the atoms for HHL(Cu2+) and HHL2(Cu2+). The silver complexes were a bit more complicated—

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The basis set used for these complexes was 3-21G(d) due to the limitations of the server with

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calculations of heavier metal ions. However, structures could be calculated for HHL(Ag+) and

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HHL2(Ag+) complexes.

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Stock solutions of silver nitrate, copper(II) nitrate, and HHL (10 mM) were prepared in

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ammonium acetate buffer (20 mM, pH 7). Stock solutions were diluted to create 1:100, 1:50, 1:5,

245

and 1:1 molar ratios of HHL:Ag+ and HHL:Cu2+ separately. The mixtures were immediately

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vortexed and analyzed by liquid chromatography-MS to monitor HHL m/z 200 [MW+H]+ and

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the degradation product, hydrolyzed HHL m/z 218 [MW+H2O+H]+ (Supporting Figure S6). The

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total abundance counts for the hydrolyzed HHL were too low to quantify differences compared

249

to controls. Abundance counts of hydrolyzed HHL were 105 counts for

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unhydrolyzed HHL. Thus, loss of HHL due to hydrolysis was not considered to be a significant

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factor.

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Chromobacterium violaceum biological assay. Freezer stock cultures of Chromobacterium

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violaceum CV026 (CECT 5999) were inoculated in LB broth supplemented with 50 µg mL-1

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kanamycin and were grown overnight aerobically (200 rpm, 30°C, pH 7). Cultures were then


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diluted to OD600=0.001 before experimentation. 96-well plates were used to monitor cell growth

256

and violacein production at various Ag+ and Cu2+ exposure concentrations, delivered as Ag(NO3)

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or Cu(NO3)2, respectively. Individual wells contained 190 µL of LB broth (50 µg mL-1

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kanamycin, pH 7). Experimental wells contained a fixed amount of 0.75 µM HHL and were

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incubated with various concentrations of either Ag(NO3) or Cu(NO3)2 before being innoculated

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with 10 µL of CV026 culture. As suggested by the name, No–HHL control wells did not contain

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any HHL. The 96-well plates were then scanned on a multi-well plate reader to gather an initial

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OD600 measurement. Plates were then wrapped in aluminum foil and placed on an orbital plate

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shaker at 200 rpm for 24 hours at 30°C. The plates were then scanned again at OD600 to

264

determine growth, which was normalized to the initial OD600 reading.

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Violacein production after 24 hours of growth was determined as described previously.38 The 96-

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well plates were placed in an oven at 60°C without a cover until the well contents were

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completely evaporated (at least 6 hours). Once dry, 200 µL of DMSO was added to each well.

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The plates were then covered, wrapped in aluminum foil, and placed on an orbital shaker at 200

269

rpm overnight at 30°C to dissolve the violacein. Violacein production was then detected by

270

measuring OD580 on a multi-well plate reader. To test if the presence of Ag+ or Cu2+ would shift

271

the absorbance of violacein, cultures of CV026 + HHL (without Ag+ or Cu2+ supplementation)

272

were grown as described above for 24 hours and violacein was extracted using DMSO

273

supplemented with either 0.1 mM of Ag(NO3) or 1.0 mM Cu(NO3)2. No peak shift was observed

274

(Supporting Figure S4).

275

MS condition. Stock solutions of silver nitrate (10 mM), copper(II) nitrate (10 mM),

276

ferric chloride (10 mM), and HHL (10 mM) were prepared in ammonium acetate buffer (20 mM,

277

pH 7). None of the stock solutions contained any precipitates. Stock solutions were diluted to 5



278

mM to create 1:1 molar ratios of HHL:Ag+ and HHL:Cu2+ separately. In an attempt to detect

279

HHL:Fe complexes, 1:1, 2:1, and 3:1 ratios were tested. The mixtures were immediately

280

vortexed and analyzed by MS (Agilent 6430 Triple Quad LC/MS, Agilent 1100 Series). Stock

281

solutions prior to mixing were also analyzed by MS (Supporting Figure S1).

Page 18 of 26

282

LC-MS. Stock solutions of silver nitrate, copper(II) nitrate, and HHL (10 mM) were

283

prepared in ammonium acetate buffer (20 mM, pH 7). Stock solutions were diluted to create

284

1:100, 1:50, 1:5, and 1:1 molar ratios of HHL:Ag+ and HHL:Cu2+ separately. The mixtures were

285

immediately vortexed and analyzed by liquid chromatography-MS (Supporting Table S3) to

286

monitor HHL m/z 200 [MW+H]+ and hydrolyzed HHL m/z 218 [MW+H2O+H]+. The total

287

abundance counts for the hydrolyzed HHL were too low to quantify differences compared to

288

controls. Abundance counts of hydrolyzed HHL were 105 counts for

289

unhydrolyzed HHL. Thus, loss of HHL due to hydrolysis was not considered to be a significant

290

factor. However, it does appear that the presence of Cu2+ may promote hydrolysis. This would

291

agree with previous findings that have shown divalent cations can catalyze hydrolysis of organic

292

compounds.39

293

ATR-FTIR. Stock solutions of silver nitrate (10 mM), copper(II) nitrate (10 mM) and

294

HHL (10 mM) were prepared in ammonium acetate buffer (20 mM, pH 7). Stock solutions were

295

mixed to create 1:1 molar ratios of HHL:Ag+ (5 mM:5 mM) and HHL:Cu2+ (5 mM:5 mM)

296

separately. The samples were then analyzed using attenuated total reflection Fourier transform

297

infrared spectroscopy (ATR-FTIR). ATR-FTIR spectra were obtained using a PerkinElmer

298

Frontier FT-IR spectrometer equipped with a germanium ATR crystal. Scans were performed

299

from 4000 cm-1 to 700 cm-1 at a rate of 0.2 cm s-1. The traces are the average of 4 scans with a 4


Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


300

cm-1 resolution. Spectra were generated using PerkinElmer Spectrum software. The source was

301

an MIR, the detector an MIR TGS (1500-370) cm-1 with a OptKBr beamsplitter.

302 303

ACKNOWLEDGMENTS

304

We acknowledge the NSF and EPA funding under NSF Cooperative Agreement EF-1266252,

305

Center for the Environmental Implications of NanoTechnology (CEINT), the Integrative

306

Graduate Education and Research Traineeship in Nanotechnology Environmental Effects and

307

Policy fellowship program under grant no. DGE0966227, and the Bertucci Fellowship granted

308

by the College of Engineering at Carnegie Mellon University. Furthermore, we thank K. Perkins

309

for his help in ATR-FTIR analysis.

310

SUPPORTING INFORMATION

311

The Supporting Information is available free of charge via the Internet. It contains

312

experimental details, XYZ coordinates for modeled compounds, and results to support the main

313

figures/conclusions of the manuscript.

314 315 316

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Figure 1. Positive ion MS/MS spectra of (a) HHL:Ag+ and (b) HHL:Cu2+. ATR-FTIR

432

spectra of (c) HHL:Ag+ and (d) HHL:Cu2+ mixture minus cation-free HHL spectra on KBr tablet.

433

Optimized structure of (e) HHL(Ag+), (f) HHL2(Ag+), (g) HHL(Cu2+), (h) HHL2(Cu2+) using

434

B3LYP 3-21G(d) and 6-31G(d) for silver and copper complexes, respectively.

435 436

Figure 2 a) Violacein production (OD580) normalized to cell growth (OD600) of

437

Chromobacterium violaceum CV026 exposed to Ag+ or Cu2+, delivered as AgNO3 or Cu(NO3)2,

438

respectively. Connecting lines are provided to aid interpretation. b) Dose-response curves fit

439

using the Gompertz equation 40 (solid lines are without HHL and dotted lines are with HHL ).

440

The X-axis of (a) and (b) represent the log molar concentration of Ag+ of Cu2+ added as AgNO3,

441

or Cu(NO3)2, respectively, and does not represent the free concentration at equilibrium. c) Loss

442

of free HHL as a function of relative molar concentrations of Ag+ or Cu2+. Connecting lines are

443

provided as a visualization guide. On all graphs, symbols represent the average, error bars

444

represent the standard error of the mean (SEM), and significance asterisks are the results of

445

unpaired t-tests versus the CV026 + HHL Control: *P

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Quorum Sensing Signals Form Complexes with Ag ... - ACS Publications

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