Imaging Bacterial Interspecies Chemical Interactions by Surface

Imaging Bacterial Interspecies Chemical Interactions by Surface-Enhanced Raman Scattering Gustavo Bodelón,*,† Verónica Montes-García,† Celina Costas,† Ignacio Pérez-Juste,† Jorge Pérez-Juste,† Isabel Pastoriza-Santos,† and Luis M. Liz-Marzán*,†,‡,§,∥ †

Departamento de Química Física and Biomedical Research Center (CINBIO), Universidade de Vigo, 36310 Vigo, Spain Bionanoplasmonics Laboratory, CIC biomaGUNE, Paseo de Miramón 182, 20014 Donostia-San Sebastián, Spain § Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain ∥ Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), 20014 Donostia-San Sebastián, Spain ‡

S Supporting Information *

ABSTRACT: Microbes produce bioactive chemical compounds to influence the physiology and growth of their neighbors, and our understanding of their biological activities may be enhanced by our ability to visualize such molecules in vivo. We demonstrate here the application of surface-enhanced Raman scattering spectroscopy for simultaneous detection of quorum-sensing-regulated pyocyanin and violacein, produced respectively by Pseudomonas aeruginosa and Chromobacterium violaceum bacterial colonies, grown as a coculture on agar-based plasmonic substrates. Our plasmonic approach allowed us to visualize the expression and spatial distribution of the microbial metabolites in the coculture taking place as a result of interspecies chemical interactions. By combining surface-enhanced Raman scattering spectroscopy with analysis of gene expression we provide insight into the chemical interplay occurring between the interacting bacterial species. This highly sensitive, costeffective, and easy to implement approach allows spatiotemporal imaging of cellular metabolites in live microbial colonies grown on agar with no need for sample preparation, thereby providing a powerful tool for the analysis of microbial chemotypes. KEYWORDS: SERS, SERRS, plasmonic, imaging, metabolite, bacteria, quorum sensing

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important sources of drugs and lead compounds used in medicine and agriculture.8,9 Quorum sensing is a cell-to-cell chemical communication process that allows bacteria to coordinate gene expression in response to cell density and changes in the environment.10,11 In a canonical quorum-sensing system LuxI-type enzymes synthesize N-acyl homoserine lactones (AHL) as signals, which freely diffuse in and out of the cell. At a threshold concentration, they bind and activate cognate LuxR-type transcriptional receptors, which in turn regulate the expression of a wide range of cell activities and phenotypes, with a preferential influence on extracellular traits such as secreted SMs.4,12

acteria rarely grow as isolated species but rather coexist in mixed microbial communities (e.g., microbiotas), which contribute to sustain life across our planet and greatly influence human health.1,2 Microbial populations are highly complex, as exemplified by the microbial mats, the rhizosphere, or the microbes residing in the gastrointestinal tract, which include hundreds of discrete genera living together, frequently at high population densities.3 Within such densely populated environments, microbes compete for nutrients and space, engaging in intraspecies and interspecies synergistic and antagonistic interactions.4,5 These relationships are often mediated by low-molecular-weight bioactive chemical compounds, termed specialized metabolites (SMs), which are secreted by microorganisms to the environment, where they can act as nutrient sources, antimicrobials, toxins, cues, and signals for microbial chemical communication.6,7 Significantly, many of these chemical compounds possess a remarkable range of “drug-like” properties, and they have been one of the most © 2017 American Chemical Society

Received: January 12, 2017 Accepted: May 1, 2017 Published: May 1, 2017 4631

DOI: 10.1021/acsnano.7b00258 ACS Nano 2017, 11, 4631−4640

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ACS Nano SMs are thought to be important factors in the homeostasis of microbial communities, and, by extension, disease.13 Nevertheless, it remains unclear under which conditions they are expressed and the specific biological roles they play in nature.14,15 In this context, the ability to detect and visualize SMs within live microbial populations may provide relevant insights into their function, yet there are very few methodologies that allow in vivo detection and imaging of cellular metabolites produced by undisturbed microbial populations. Moreover, gaining a deeper understanding of the complex chemical crosstalk between microbial species is important to understand microbial ecosystems and can assist the rational development of novel therapeutic drugs that specifically target pathogenic populations. Surface-enhanced Raman scattering (SERS) spectroscopy is an analytical tool that enables direct identification of analytes in contact or in close proximity with plasmonic nanostructures.16,17 SERS retains the chemical and structural information provided by Raman spectroscopy but overcomes the intrinsic weakness of the Raman scattering process by exploiting the high electromagnetic field enhancement resulting from the excitation of localized surface plasmon resonances (LSPRs) at metal nanoparticles. The Raman scattering intensity is further enhanced in the case of surface-enhanced resonance Raman scattering (SERRS) spectroscopy by employing a laser wavelength whose excitation frequency overlaps an electronic absorption band of the molecule of interest. Additionally, the advantages of SERS/SERRS to obtain an in-depth understanding of biological systems include outstanding multiplexing capacity for simultaneous molecular detection due to the narrow width of the vibrational Raman bands and optimum contrast with near-infrared (NIR) excitation to reduce autofluorescence background from biological cells and tissues.18−20 We recently reported the use of SERRS for the visualization of a quorum-sensing-regulated metabolite termed pyocyanin in live Pseudomonas aeruginosa (P. aeruginosa) biofilms, in the presence of plasmonic substrates. 21 This phenazine is recognized as a virulence factor with antibiotic properties and displays toxic activity against a broad range of microorganisms and mammalian cells, mainly through the generation of reactive oxygen species.22 Our approach involved the use of nanostructured hybrid materials, comprising a plasmonic component embedded in a porous matrix, as platforms for growing bacterial cultures in droplets and simultaneously detecting pyocyanin by SERRS, upon diffusion into the underlying optical sensor. The hybrid plasmonic substrates were shown to facilitate in situ SERRS detection of pyocyanin produced by small cellular aggregates of P. aeruginosa and biofilms developed in liquid culture and yielded spatially resolved 2D maps of the quorumsensing molecule.21 In the present study we investigate the production of microbial SMs resulting from inter- and intraspecies quorumsensing communication between bacterial colonies of P. aeruginosa and Chromobacterium violaceum (C. violaceum), grown as a coculture on agar-based hybrid plasmonic substrates (Au@ agar) (Figure 1) by surface-enhanced Raman scattering spectroscopy. Agar is a gelling agent used as a standard support matrix for growing microbes on solid culture medium, and its macroporous structure allows the diffusion and exchange of nutrients and metabolites within microbial populations.23 The rationale behind the use of a solid (e.g., agar-based) culture medium for growing bacteria, as opposed to liquid culture used

Figure 1. Schematic illustration of in vivo SERS/SERRS multiplexed detection of violacein and pyocyanin in C. violaceum CV026 and P. aeruginosa PA14 bacterial cocultures grown on Au@agar. The colony of P. aeruginosa PA14 secretes C12- and C4-AHLs, leading to intraspecies activation of quorum sensing and the production of pyocyanin (PYO). C4-AHLs diffuse through agar into neighboring CV026 cells, activating their quorum-sensing system and in turn violacein (VIO) production (interspecies quorum sensing). The dashed lines represent diffusion of the indicated molecules and quorum-sensing signals. Visualization of violacein and pyocyanin was carried out upon illuminating the bacterial colonies on the plasmonic sensor with a NIR laser line (785 nm). The red spheres represent gold nanospheres within the multilayered film of the Au@agar substrate (yellow box).

in our previous study, is that it allows coculturing microbial colonies at predefined locations with controlled separation. By conducting microbial confrontations on agar-based medium, one can readily identify the microorganisms as discrete colonies, as well as the region of “chemical interaction” (i.e., confrontation zone) between them.24,25 We selected P. aeruginosa PA14 and C. violaceum CV026 as a dual species model because their quorum-sensing systems have been largely characterized and they are known to regulate the biosynthesis of pyocyanin and violacein, respectively, which are molecules amenable to Raman spectroscopy detection.21,26,27 P. aeruginosa PA14 produces two types of AHL quorum-sensing signaling molecules termed N-(3-oxododecanoyl)homoserine lactones (C12-AHL) and N-butyryl-L-homoserine lactones (C4-AHL), which are involved in the expression of pyocyanin.28 CV026 is a C. violaceum strain that cannot generate its own AHL signals, but can respond to compatible AHLs bearing short C4 to C8 acyl chains, such as P. aeruginosa C4-AHLs, thereby resulting in the expression of quorumsensing-regulated phenotypes, including the synthesis of violacein.29 The precise biological function of violacein remains to be elucidated, but it has been shown to display diverse pharmacological activities against bacterial competitors and predator organisms.30,31 We demonstrate the suitability of surface-enhanced Raman scattering to visualize pyocyanin and violacein expressed by P. aeruginosa PA14 and C. violaceum CV026 colonies grown on Au@agar. By illuminating the microbial colonies on the plasmonic substrate with a single NIR laser line (785 nm) we were able to simultaneously monitor the expression of pyocyanin and violacein by SERRS and SERS, respectively, in a spatially resolved fashion. Remarkably, the capacity to control the spatial localization of the bacterial colonies on Au@agar allowed us to determine the reduced expression of pyocyanin in coculture, as well as in the presence of added violacein. Moreover, quantitative PCR (qPCR) analysis of gene expression indicated that the decreased levels of pyocyanin are, at least in part, due to the down-regulation of the P. aeruginosa phzS gene responsible for the last step of 4632


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Figure 2. Vis−NIR and SERS/SERRS spectra of violacein and pyocyanin on Au@agar. (a and b) Chemical structures of violacein and pyocyanin, respectively. (c) Normalized vis−NIR spectra of commercial pyocyanin (PYOc) and violacein (VIOc). (d) SERRS spectrum of PYOc (5 μL, 1 mM), SERS spectrum of VIOc (5 μL, 1 mM), and SERS spectrum of Au@agar. All spectra were measured with a 50× objective, a maximum power of 0.64 kWcm−2, and an acquisition time of 10 s. Excitation laser line was 785 nm.

Figure 3. Detection and imaging of violacein expression by CV026 upon induction with commercial C4-AHL on Au@agar. (a) SERS spectra of (1) VIOc (1 mM); (2) colony treated with C4-AHL (100 μM); (3) colony treated with C4-AHL (1 μM); (4) untreated colony; (5) C4-AHL (100 μM); (6) Au@agar. The purple bars of the spectra indicate violacein signals. (b−d) SERS mapping of violacein (727 cm−1). The insets show digital photographs of the bacterial colonies. Measurements on colony were acquired with an excitation laser wavelength of 785 nm, 5× objective, and a laser power of 12.21 kW cm−2 for 10 s.

pyocyanin biosynthesis. Our findings suggest a potential defensive mechanism of C. violaceum in the chemical interplay between the bacterial species. The plasmonic imaging approach described in this work, enabling the visualization of cellular metabolites produced by microbial populations on agar, can help elucidate their functional roles, and thus it may provide researchers with a powerful tool to investigate a broad variety of questions related to microbiota research.

This substrate presents a broad plasmon band centered at 780 nm due to extensive plasmon coupling, as well as a band at around 550 nm from single particles (Figure S1, Supporting Information). Additional details concerning substrate fabrication and characterization are provided in Methods and in the Supporting Information section S1 (Figures S1 and S2, Supporting Information). Commercial violacein (Figure 2a) and pyocyanin (Figure 2b) show broad absorption bands centered at 580 and 695 nm, respectively (Figure 2c). Therefore, illumination of an aqueous solution of either molecule deposited on an Au@agar substrate with a 785 nm laser line leads to SERS (violacein) or SERRS (pyocyanin),21 respectively (Figure 2d). Computational conformation analysis,

RESULTS AND DISCUSSION The Au@agar plasmonic substrate comprised a homogeneous multilayered film of gold nanospheres (60 nm) on glass, covered by a thin layer of nutrient lysogeny-broth (LB) agar. 4633


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Figure 4. Detection of violacein and pyocyanin expressed by CV026 and PA14 colonies cocultured on Au@agar. (a) Illustration of SERS detection of violacein upon illuminating the C. violaceum CV026 colony. SERS spectra recorded in the CV026 colony in coculture with either wild-type PA14 (CV026+PA14) or PA14 Δphz1/2 (CV026+PA14 Δphz1/2) bacteria. (b) Illustration of SERRS detection of pyocyanin upon illuminating the P. aeruginosa PA14 colony in the coculture. SERRS spectrum recorded in the PA14 colony in coculture (CV026+PA14) and in monoculture (PA14), as a control. SERS spectrum of commercial violacein (VIOc) and SERRS spectrum of pyocyanin (PYOc) are included as references. The purple and red bars of the spectra indicate violacein and pyocyanin, respectively. The measurements were performed illuminating the bacterial colonies with an excitation laser wavelength of 785 nm, 5× objective, and a laser power of 12.21 kW cm−2 for 10 s.

(cf. mappings in Figure 3b and c and spectra 3, 4 in Figure 3a). These results show that the expression of violacein can be detected and imaged in situ by SERS in nonpigmented, but AHL-stimulated, CV026 colonies, thereby demonstrating the high sensitivity of this plasmonic detection modality. It is worth noting that the agar matrix enabled the diffusion of violacein, which could be then detected by SERS up to 3 mm away from the bacterial colony (Figure S7, Supporting Information). Once we demonstrated that violacein produced by CV026 colonies upon induction with commercial C4-AHLs can be detected by SERS, we next applied SERS to the detection of violacein expressed as a result of coculture with P. aeruginosa PA14 on Au@agar. PA14 and CV026 bacteria were thus seeded on the Au@agar surface in a 1:100 ratio, with a separation of ca. 2−3 mm and then allowed to grow at 30 °C. Photographs of the coculture taken at 20 and 40 h illustrate the growth of the dual species model on the substrate (Figure S8, Supporting Information). The production of violacein was assessed by SERS at 20 h, upon illuminating the CV026 colony with a 785 nm laser (Figure 4a). The recorded SERS spectrum (CV026+PA14) revealed the violacein fingerprint (Figure 4a, purple bars).26 These specific signals were detected at higher intensities upon coculture of CV026 with a P. aeruginosa Δphz1/2 strain (CV026+PA14Δphz1/2), which is unable to produce phenazines, including pyocyanin.32 The coculture with the P. aeruginosa Δphz1/2 strain consistently resulted in higher amounts of violacein produced by CV026 (not shown). The expression of pyocyanin was next assessed upon illuminating the PA14 colony in coculture (Figure 4b). The spectrum (CV026+PA14) indicated the presence of pyocyanin, as evidenced by the characteristic signals

UV−vis−NIR, and Raman spectroscopic characterization of violacein is provided in the Supporting Information, section S2 (Figures S3−S6). The biosynthesis of violacein by CV026 can be triggered upon incubating bacterial cells with exogenous AHLs bearing acyl chains no longer than eight carbons, including C4-AHLs.29 Thus, individual colonies of CV026 were grown on Au@agar and treated with commercial C4-AHLs to induce violacein expression, or left untreated as a control, and analyzed by SERS (Figure 3). The spectra recorded from CV026 bacterial colonies treated with either 1 or 100 μM AHLs (Figure 3a, spectra 2, 3) present characteristic bands at 727, 807, 870, 950, 1125, and 1535 cm−1 (purple bars), which match those of commercial violacein (Figure 3a, spectrum 1). As expected, these signals were triggered upon AHL induction, and their intensities augment with increasing concentration of C4-AHLs (Figure 3a, cf. spectra 2, 3, 4). SERS interrogation of the uninduced CV026 colony (Figure 3a, spectrum 4), the Au@ agar incubated with 100 μM C4-AHL (Figure 3a, spectrum 5), and the Au@agar substrate (Figure 3a, spectrum 6) did not reveal any meaningful signals, further confirming that they are violacein-specific. Next, the expression of violacein in CV026 colonies was imaged by SERS, on the basis of the 727 cm−1 band (Figure 3b−d). Induction with 100 μM C4-AHL led to the production of large amounts of violacein, which was evidenced by the strong pigmentation developed in the colony (inset of Figure 3d). The superior sensitivity of SERS as compared to colorimetry is demonstrated by analysis of CV026 colonies upon treatment with 1 μM C4-AHL. Whereas no significant pigmentation could be observed (Figure 3c, inset), the production of violacein could be clearly detected by SERS 4634


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Figure 5. Imaging of violacein and pyocyanin expression in coculture on Au@agar. (a, b) SERS mapping of violacein (727 cm−1) (a) and SERRS mapping of pyocyanin (544 cm−1) (b) in a coculture of C. violaceum (CV026) and P. aeruginosa (PA14) grown for 20 h. The dashed squares indicate the confrontation zone. (c) Overlay of the images in a and b. (d) SERS intensities of violacein (727 cm−1) and SERRS intensities of pyocyanin (544 cm−1) measured in c at the spots indicated with white asterisks and plotted as a function of distance. The error bars show the standard deviation from three different measurements. All measurements were acquired with an excitation laser wavelength of 785 nm, 5× objective, and a laser power of 12.21 kW cm−2 for 10 s.

Figure 6. Imaging of bacterial metabolites and RT-qPCR analysis of phenazine gene expression in coculture versus monoculture. (a) Optical image of the CV026 and PA14 coculture, acquired with the Raman microscope, and photograph of the bacterial colonies (inset). (b, c) SERRS mapping of pyocyanin (544 cm−1) (b) and SERS mapping of violacein (727 cm−1) (c) in coculture. The dashed squares indicate the confrontation zone. (d) Optical image of the PA14 colony acquired with the Raman microscope and photograph of the colony (inset). (e, f) SERRS mapping of pyocyanin (544 cm−1) (e) and SERS mapping of violacein (727 cm−1) (f) in monoculture. All measurements were performed with an excitation laser wavelength of 785 nm, 5× objective, and a laser power of 12.21 kW cm−2 for 10 s. (g) Scheme of pyocyanin (PYO) biosynthesis from chorismic acid. (h) Changes in gene expression in coculture relative to monoculture observed by RT-qPCR. Error bars represent the standard error of two independent experiments with three biological replicates each (n = 6). The dotted line represents the value for no change in gene expression.

at 407, 544, 518, 592, 1356, and 1619 cm−1 (Figure 4b, red bars).21 At this point we should mention that among the four

different phenazines produced by P. aeruginosa33,34 only pyocyanin presents an absorption band in the vis−NIR 4635


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Figure 7. Imaging of pyocyanin and RT-qPCR analysis of gene expression in P. aeruginosa colonies grown on violacein. (a, d) Optical images of PA14 colonies grown over VIOc (50 μM in DMSO 1%) or in DMSO (1%) acquired with the Raman microscope and photographs of the bacterial colonies (insets). (b, e) SERRS mappings of pyocyanin (544 cm−1) produced by PA14 bacteria grown over VIOc (50 μM in DMSO 1%) (b) or in DMSO (1%) (e). (c, f) SERS mappings of VIOc (727 cm−1). All measurements were performed with an excitation laser wavelength of 785 nm, 5× objective, and a laser power of 12.21 kW cm−2 for 10 s. (g) Changes in gene expression of PA14 grown on VIOc (50 μM in DMSO 1%) versus control bacteria grown in DMSO (1%) quantified by RT-qPCR. Error bars represent the standard error of two independent experiments with three biological replicates each (n = 6). Dotted line indicates no change in gene expression. (h) Quantification of PA14 growth on VIOc (50 μM in DMSO 1%) and in DMSO (1%).

region,21 leading to an additional enhancement due to the resonant Raman contribution at 785 nm.21 Interestingly, we could also detect the violacein-specific fingerprint when analyzing the PA14 colony in the coculture (Figure 4b, purple bars), which indicated that the molecule diffused through the agar matrix from CV026 cells. We then imaged the spatial distribution of both metabolites in coculture by SERS/SERRS mappings, using the 727 cm−1 (violacein) and 544 cm−1 (pyocyanin) peaks, respectively (Figure 5). This analysis revealed the secretion of violacein and diffusion toward the PA14 colony, into the confrontation zone, indicated with a dashed rectangle (Figure 5a). Interestingly, evaluation of pyocyanin biodistribution showed extremely low levels in this region, as compared to other regions of the substrate colonized by PA14 bacteria (Figure 5b). As shown in Figure 5c,d, and in the SERS/SERRS spectra shown in the Supporting Information Figure S9, the recorded intensities of the metabolite signals were inversely correlated. The low levels of pyocyanin detected in the confrontation zone could not be attributed to an impaired diffusivity in the agar, as we have seen high diffusion in the plasmonic substrate (up to 10 mm) upon expression from PA14 bacteria grown in monoculture (Figure S10, Supporting Information). These results indicate that the expression of pyocyanin by P. aeruginosa is likely affected by the coculture with C. violaceum. To further investigate this phenomenon, we grew PA14 with CV026 in coculture and as a single species in monoculture, and we assessed the expression of pyocyanin. As shown in the SERS/SERRS maps of Figure 6 and the Supporting Information Figure S11, the detected levels of pyocyanin in

coculture were significantly lower than those detected in monoculture (compare Figure 6b and e). Again, in coculture we found reduced levels of pyocyanin in the confrontation zone (Figure 6b). As expected, no violacein signals were detected in the PA14 monoculture (Figure 6f). Similar results can be seen in the assay provided in the Supporting Information (Figure S12a−f). To confirm the above data, we measured the phenazine concentration by UV−vis spectroscopy at 691 nm (λmax of pyocyanin) following chloroform extraction from the agar on which the PA14 colonies were grown. Significantly, whereas the amount of pyocyanin released by PA14 cells in monoculture averaged 2.3 μM, its concentration could not be determined in coculture, as it was below the detection limits of this method (Figure S12g, Supporting Information). Since the growth of PA14 bacteria in monoculture and coculture was very similar (Figure S12h, Supporting Information), the differential expression of pyocyanin could not be attributed to growth defects. In addition, control experiments mixing commercial violacein and pyocyanin showed that both molecules could be simultaneously detected by SERS and SERRS, respectively, on Au@agar, thereby ruling out any potential interaction/screening between both molecules (Figure S13, Supporting Information). Pyocyanin production in P. aeruginosa involves two highly homologous operons constituted by seven genes each, phzA1− G1 (abbreviated as phz1) and phzA2−G2 (abbreviated as phz2), encoding the enzymes required to convert chorismic acid into phenazine-1-carboxylic acid (PCA). Two additional genes, termed phzM and phzS, flanking the phz1 operon, encode the enzymes that catalyze the conversion of PCA into 4636


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developed a dual-species coculture model using C. violaceum and Burkholderia thailandensis to investigate AHL-dependent microbial competition, showing that the capacity of CviR to eavesdrop on B. thailandensis AHLs provided a competitive advantage to C. violaceum.44 It seems plausible that the downregulation of pyocyanin observed in our binary model may provide a fitness benefit for CV026 bacteria. It should be mentioned that although P. aeruginosa PA14 and CV026 can initially coexist, P. aeruginosa eventually overgrows CV026, reducing the viability of its partner in extended cocultures. The advantage of using quorum sensing to control the production of metabolites with antimicrobial properties (i.e., pyocyanin and violacein) is still under debate. The classical view is that quorum-sensing mechanisms may allow a microbial population to coordinate the delivery of a sudden high dose that kills or inhibits the growth of competitors.4 Interestingly, it has been reported that at low subinhibitory concentrations antimicrobials may also function as signaling molecules and cues to modulate gene expression, playing important roles in the regulation of the homeostasis of microbial communities.14,45,46 Microbial SMs have been a source of many pharmaceuticals and therapeutic drugs including antibiotics, antifungals, antitumorals, and immunosuppresants.8 Moreover, diverse metabolites of microbial origin have been pointed out as mediators of human disease. Significantly, these molecules are often characterized by the presence of heterocyclic structures with high Raman cross sections that render them amenable to SERS/SERRS detection.47 Recent examples of Raman-active microbial metabolites include p-coumaric acid,48 quinolones,49 rhabduscin,50 amphotericin B,51 and aflatoxins.52 We envision that our Au@agar substrates could be applied to detect the expression of SERS/SERRS-active SMs with potential medical and/or pharmacological interest, produced by natural and synthetic microbial communities as exemplified in the present study. In addition, this plasmonic platform can be incorporated in existing agar-based devices already in use for growing “uncultivated microorganisms” in their natural environment, such as the iChip.53 Au@agar can also be used as a sensing platform for detecting the expression of SMs produced by the OSMAC (one strain many compounds) approach,54 in which a strain is cultured in a variety of media and under different cultivation parameters in order to activate “silent” metabolic pathways and maximize the diversity of bioactive metabolites produced. Alternatively, this agar-based hybrid plasmonic platform could also be applied for screening chemical inhibitors/activators of SERS/SERRS-active SMs, for monitoring SERS/SERRS-active metabolites produced by genetically modified microbial hosts,55 and to detect SMs produced by metagenomics56 and synthetic biology efforts.57 Owing to its simplicity, Au@agar can be readily implemented in highthroughput array formats by standard microfabrication technology and as a fast analytical method for chemical fingerprinting that can be adopted in drug discovery workflows.

methylphenazine-1-carboxylic acid (5-MCA) and pyocyanin, respectively (Figure 6g).33,35 We assessed whether the observed reduced levels of pyocyanin might be due to inhibition of its biosynthesis, by analyzing the mRNA transcript levels of phzA, phzB, phzM, and phzS genes by reverse transcription (RT)qPCR in PA14 bacteria, grown both in coculture and in monoculture (see Supporting Information Section 3 for details on the RT-qPCR procedure). Remarkably, whereas the levels of expression of phzA, phzB, and phzM genes were slightly upregulated, the expression of the phzS gene was reduced by approximately 30% (Figure 6h). The above data indicate that the down-regulation of pyocyanin is related to factors released by CV026 that interfere with the biosynthesis of the phenazine. In this framework, it has been reported that indole, a metabolite produced by certain Gram-negative bacterial species, as well as native and synthetic indole derivatives36 including 5- and 7-hydroxyindoles, isatin, 3indolylacetonitrile, or 7-fluoroindole, can repress quorumsensing-regulated phenotypes in P. aeruginosa, including pyocyanin production.37−40 Chromobacterium is considered as a genus that does not usually produce indole,41 and CV026 was indole-negative in the Kovacs test (data not shown). Interestingly, violacein is a bis-indole compound formed by a 5-hydroxyindole and an oxindole connected by a 2-pyrrolidone core.31 We therefore decided to investigate whether the bisindolic violacein might be involved in the down-regulation of pyocyanin. We first monitored by SERRS (Figure 7 and Figure S14, Supporting Information) the production of pyocyanin by PA14 colonies grown on commercial violacein (50 μM in 1% DMSO) (see Methods section). We found that pyocyanin levels were significantly reduced in violacein-treated colonies, as compared to colonies grown in the diluent (1% DMSO) without violacein, as a control (compare Figure 7b and e). RTqPCR analysis showed that the mRNA transcript levels of the phzS gene were indeed reduced by approximately 20% in PA14 bacteria grown on commercial violacein, as compared to control bacterial cells grown in 1% DMSO (Figure 7g). Violacein was not found to affect the growth of P. aeruginosa neither on agar (Figure 7h) nor in liquid culture (not shown), whereas it did modulate the expression of pyocyanin, as revealed by SERS/SERRS imaging (Figure 7 and Figure S14, Supporting Information). In this scenario, violacein is thought to act as a cue/coercion molecule42 for chemical manipulation of P. aeruginosa. Although the precise mechanisms involved in pyocyanin inhibition remain to be elucidated, our study indicates that repression of phzS gene expression might be responsible, at least in part, for this process. Finally, since the growth of CV026 colonies was impaired when cocultured with pyocyanin-producing PA14 strains (i.e., wild type and ΔphzH), but not with strains deficient in pyocyanin biosynthesis (i.e., Δphz1/2, ΔphzM, and ΔphzS), it appears that pyocyanin expression might be toxic to C. violaceum (Figure S15, Supporting Information). Our study supports the idea that the promiscuous CviR transcriptional receptor of CV026 can sense C4-AHLs produced by PA1443 and that by detecting P. aeruginosa signals C. violaceum can express violacein, which in turn contributes to pyocyanin down-regulation. Since pyocyanin-expressing PA14 strains compromised the growth of CV026, as opposed to pyocyanin-deficient strains (Figure S15, Supporting Information), the down-regulation of this phenazine suggests a potential defensive mechanism of C. violaceum CV026 in its interplay with P. aeruginosa PA14. Chandler and collaborators

CONCLUSIONS The present study demonstrates the application of surfaceenhanced Raman scattering for in situ detection of violacein and pyocyanin produced by bacterial colonies of C. violaceum CV026 and P. aeruginosa PA14 as a result of quorum-sensing communication. As reported in this study, the coculture induces the expression of violacein by CV026 and reduces the expression of pyocyanin in PA14, as compared to monocultures of this P. aeruginosa strain. Significantly, as 4637


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sodium citrate solution. The final solution was used as seed, and the process was repeated again six times to yield 60 nm Au NPs. To remove the excess of reactants, the colloidal dispersions were centrifuged at 1520g for 20 min and redispersed in the same volume of water. Fabrication of Au Nanoparticle Multilayered Thin Films. Glass slides were washed in piranha solution for 30 min and then copiously rinsed with water and stored in water until use. First, glass slides were immersed in an aqueous PDDA solution (1 mg/mL, 0.05 M NaCl) for 15 min, rinsed with water, and then dried. The process was repeated twice but alternating the polymer solution: PAA solution (1 mg/mL, 0.05 M NaCl) first and then PDDA solution (1 mg/mL, 0.05 M NaCl). For the Au NP film formation, the glass slides were immersed in the 0.93 mM Au NP solution for 3 h followed by rinsing with water. Then, the substrate was immersed in a PDDA solution (1 mg/mL, 0.05 M NaCl) for 15 min followed by rinsing with water. The process was repeated up to two more times to obtain the final material. Note that for each addition a new Au NP solution was used. Moreover, the rinsing step with water was performed 3-fold by dipping the glass slides in a different Eppendorf tube for 1 min. Preparation of the Au@agar Substrate. The glass bearing the gold nanoparticle multilayered thin film was laid on top of a Corning Micro Slide (75 × 25 mm, thickness 0.96 to 1.06) in a Petri dish (90 × 14 mm) and covered with 16.5 mL of molten LB agar (LB agar: 10 g of Bacto-tryptone, 5 g of yeast extract, 10 g of NaCl, and 1.5% agar per liter of water). Once solidified, the agar medium surrounding the Micro Slide was cut with a sterile surgical blade and the Au@agar substrate (e.g., glass bearing the gold nanoparticle multilayered thin film on the Micro Slide layered with solidified LB agar) was transferred to a humidity-controlled chamber to minimize dehydration of the thin agar layer. Bacterial Culture. Bacterial strains used are indicated in Table S4, Supporting Information. P. aeruginosa PA14 and C. violaceum CV026 bacteria were streaked onto LB agar plates and incubated overnight at 30 °C. A single colony was used to inoculate 10 mL of LB medium (LB: 10 g of Bacto-tryptone, 5 g of yeast extract, and 10 g of NaCl per liter of water) and grown at 30 °C with agitation (210 rpm) for 18 h. The bacterial cultures (1 mL) were washed three times by centrifugation (8000g for 3 min) with 1 mL of LB medium, and the cell pellet was resuspended to an optical density (OD) at 600 nm (OD600) of 1.0 in LB medium. The CV026 and PA14 bacterial suspensions were diluted 1/10 and 1/1000 in LB medium, respectively. Next, 2 μL of the bacterial suspensions was spotted on Au@agar, and the bacterial cultures were grown in a humidified chamber at 30 °C. For pyocyanin quantification, PA14 colonies (n = 6) grown in monoculture or in coculture for 18 h were scraped from the agar, and the solid growth substrate was transferred to a glass test tube containing 1 mL of chloroform. The phenazine was recovered by air-drying the solvent and redispersion in 0.5 mL of water. The concentration of pyocyanin was determined spectrophotometrically on the basis of its absorbance at 691 nm (εPYO = 4310 M−1 cm−1).21 The PA14 colonies removed from the agar were pooled together and redispersed in 1 mL of LB to measure the OD600 for determining bacterial growth. For growing P. aeruginosa in the presence of commercial violacein, the molecule was solubilized in LB-containing DMSO 1% (v/v), and 10 μL of this solution was spotted on the substrate. The drop was allowed to dry for 10 min. Next, 2 μL of a bacterial suspension (OD600 of 2.0), previously rinsed three times with 1 mL of LB, was added over the violacein spot. As a control, the bacterial suspension (2 μL) was grown on 10 μL of LB−DMSO 1% (v/v), previously dried on the substrate. Bacteria were cultured in a humidified chamber at 30 °C. To determine bacterial growth, the colonies were scraped off the substrate and resuspended in 500 μL of LB to measure the OD600. Total RNA Isolation and cDNA Synthesis. Total RNA obtained from three colonies of P. aeruginosa per sample was harvested using the NucleoSpin RNA isolation kit (Macherey-Nagel) and subjected to on-column DNase treatment according to the manufacturer’s protocol, followed by an extra DNase treatment with an Ambion DNA-free kit.59 The amount of total RNA was assessed by a NanoDrop ND-

evidenced by SERS/SERRS imaging, the levels of pyocyanin and violacein are inversely proportional in the confrontation zone of the coculture, suggesting a role of violacein in the down-regulation of the phenazine. This was confirmed by surface-enhanced Raman scattering upon growing PA14 bacteria on commercial violacein, which diminished pyocyanin expression. Our experimental evidence indicates that this phenomenon is, at least in part, due to the repression of the P. aeruginosa phzS gene. Since the expression of pyocyanin exerts a detrimental effect in the growth of C. violaceum, its down-regulation seems to be a potential defensive action against P. aeruginosa in the chemical interplay between the bacterial species. This study of interspecies interactions by surface-enhanced Raman scattering illustrates the complexity of the chemical interactions underpinning microbial communities. The reported approach provides an additional tool for the spatiotemporal analysis of interspecies interactions between microbes grown on agar, which is the standard support matrix for culturing microbial cells. Thus, this study can be extended to investigate cellular chemotypes and metabolite exchange processes linked to the development and organization of microbial populations. This sensitive and cost-effective plasmonic substrate can be not only applied in metabolomic studies but also implemented toward the screening and identification of SERS/SERRS-active bioactive metabolites (e.g., SMs) with potential clinical, pharmacological, or biotechnological interest produced by microbes grown in monoculture, in defined synthetic consortia, and in mixed species obtained from environmental samples. Understanding microbial chemical communication processes is important to provide fundamental insights into microbial ecology and the necessary knowledge to manipulate these systems for potential applications in medicine, drug discovery, biotechnology, and synthetic biology.

METHODS Materials. Tetrachloroauric(III) acid trihydrate (HAuCl4·3H2O), trisodium citrate dihydrate, poly(diallyldimethylammonium chloride) (PDDA, average Mw 100 000−200 000), poly(acrylic acid sodium salt) (PAA, Mw 15 000), hydrogen peroxide (H2O2, 28%), sulfuric acid (H2SO4, 98%), and sodium chloride were supplied by Aldrich. Milli-Q grade water was used in all the preparations. Instrumentation. UV−vis−NIR absorption spectra were recorded using an Agilent 8453 spectrophotometer. Transmission electron microscopy (TEM) analysis was performed in a JEOL JEM 1010 microscope operating at an acceleration voltage of 100 kV. Scanning electron microscopy (SEM) images were obtained using a JEOL JSM6700F FEG scanning electron microscope operating at an acceleration voltage of 10.0 kV. The thickness of the agar layer on the gold nanoparticle multilayer thin film was measured by profilometry employing a Dektak XT profilometer (Bruker) equipped with Vision64 analysis software. Raman and SERS measurements were conducted with a Renishaw InVia Reflex system. The spectrograph used a high-resolution grating (1200 grooves cm−1) with additional band-pass filter optics, a confocal microscope, and a 2D-CCD camera. Laser excitation was carried out at the 785 nm laser line. Synthesis of Au Nanoparticles. Citrate-stabilized Au NPs (60 nm in diameter) were prepared following a seeded growth method previously reported.58 Briefly, 150 mL of 2.2 mM trisodium citrate water was heated to boiling under vigorous stirring. After 15 min, 1 mL of 25 mM HAuCl4 was injected into the boiling reaction mixture and incubated for 10 min, and then the reaction mixture was cooled to 90 °C. Subsequently, 1 mL of a 25 mM HAuCl4 aqueous solution was injected into the reaction mixture. After 30 min this addition was repeated. After 30 min, the sample was diluted by extracting 55 mL of sample and adding 53 mL of water and 2 mL of aqueous 60 mM 4638


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ACS Nano 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The integrity of RNA was confirmed by Tris-borate-EDTA agarose (1%) gel electrophoresis. The absence of contaminating genomic DNA was confirmed by PCR and gel electrophoresis. Reverse transcription was performed using a BioRad iScript cDNA synthesis kit according to the manufacturer’s instructions, employing 200 ng of intact, DNA-free total RNA per sample. The resulting cDNA was diluted with nuclease-free water and stored at −20 °C. qPCR Procedure. All qPCR experiments were performed on an Applied Biosystems StepOne Real-Time PCR system using iTaq Universal SYBR Green Supermix (BioRad) according to the manufacturer’s protocol, in MicroAmp Fast Optical 48-well reaction plates (Applied Biosystems). PCR primers (Table S5, Supporting Information) were designed using Primer3 software. Primers were evaluated for their ability to amplify single products from P. aeruginosa genomic DNA before using them in qPCR experiments. It must be noted that the primers amplifying the phzA amplicon do not discriminate between the phzA1 or phzA2 genes. However, the primers for phzB specifically amplify the phzB2 gene (Table S5, Supporting Information). Calibration curves were established for each primer pair (Figure S16, Supporting Information). Data were normalized with rpoD as reference gene.59,60 Statistical analyses were performed on qPCR measurements performed in triplicate from two independent studies. Relative quantification of gene expression was determined by the comparative CT method (ΔΔCT).61

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00258. Characterization of the plasmonic substrates; theoretical study of violacein; SERS/SERRS detection of microbial metabolites, bacterial culture on Au@agar, and qPCR procedure (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: (G. Bodelón): [email protected]. *E-mail: (L. M. Liz-Marzán): [email protected]. ORCID

Isabel Pastoriza-Santos: 0000-0002-1091-1364 Luis M. Liz-Marzán: 0000-0002-6647-1353 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS L.M.L.-M. acknowledges support by the European Research Council through an ERC Advanced Grant (267867 Plasmaquo). Funding from Spanish Ministerio de Economiá y Competitividad (Grant MAT2013-45168-R) is gratefully acknowledged. V.M.-G. acknowledges the financial support of an FPU scholarship from the Spanish MECD. REFERENCES (1) Blaser, M. J.; Cardon, Z. G.; Cho, M. K.; Dangl, J. L.; Donohue, T. J.; Green, J. L.; Knight, R.; Maxon, M. E.; Northen, T. R.; Pollard, K. S.; Brodie, E. L. Toward a Predictive Understanding of Earth’s Microbiomes to Address 21st Century Challenges. mBio 2016, 7, e00714−00716. (2) Stubbendieck, R. M.; Vargas-Bautista, C.; Straight, P. D. Bacterial Communities: Interactions to Scale. Front. Microbiol. 2016, 7, 1234. (3) Nadell, C. D.; Drescher, K.; Foster, K. R. Spatial Structure, Cooperation and Competition in Biofilms. Nat. Rev. Microbiol. 2016, 14, 589−600. 4639


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Imaging Bacterial Interspecies Chemical Interactions by Surface

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