Yeast two-hybrid (Y2H) assays

Yeast two-hybrid experiments were conducted following procedures described before^20,21,39. Saccharomyces cerevisiae Mav 203 strain (MATα, leu2-3,112, trp1-901, his3-Δ200, ade2-101, gal4Δ, gal80Δ, SPAL10::URA3, GAL1::lacZ, HIS3UAS GAL1::HIS3, LYS2, can1R, and cyh2R) was transformed with the different expression vectors. First, BlsA and AbaR were analyzed for self-activation. For this purpose, MaV203 yeast strain containing the pGAD-T7 empty vector was transformed with the DNA DB-fusion protein expressing vectors (pGBK-X) (X = BlsA or AbaR). Conversely, MaV203 yeast strain containing the pGBK-T7 empty vector was then transformed with the AD-fusion protein expressing vectors (pGAD-Y) (Y = BlsA or AbaR). In addition, these strains were used for determination of the optimal 3-amino-1,2,4-triazole (3AT) concentration required to titrate basal HIS3 expression. MaV203/pGBK-X strains were afterward transformed with each pGAD-Y plasmids. Transformations using one or both Y2H plasmids were performed by the lithium acetate/single-stranded carrier DNA/polyethylene glycol method described in Gietz and Woods⁴⁵, and plated in convenient minimal selective medium [synthetic complete (SC) medium without leucine (-leu) for pGAD-Y transformants, SC without tryptophan (-trp) for pGBK-X transformants, and SC-leu-trp transformants carrying both plasmids]. The plates were then incubated at 30 °C for 72 h to allow growth of transformants. A “Master Plate” was then prepared using SC-leu-trp media, in which we patched: four to six clones of each pGBK-X/pGAD-Y containing yeasts, four to six self-activation control clones pGBK-X/pGAD and pGBK/pGAD-Y (Y DNA-binding negative control), and two isolated colonies of each of the five yeast control strains (A–E). The plates were incubated for 48–72 h at 23 °C in the dark. This Master Plate was then replica plated to SC–leu–trp–hisC 3AT and to SC–leu–trp–ura to test for growth in the absence of histidine (his) and uracil (ura), respectively (his3 and ura3 reporter activation), under the different conditions analyzed, i.e., dark/light; 23/30 °C, for at least 72 h. For development of blue color as a result of β -galactosidase (β-Gal) expression, transformed yeasts were replica plated on a nitrocellulose filter on top of a YPAD medium plate and grown at the different conditions (dark/light; 23/30 °C). Then, the cells on the nitrocellulose filter were permeabilized with liquid nitrogen and soaked in X-Gal solution (5-bromo- 4-chloro-3-indolyl-b-d-galactopyranoside in Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, pH 7.0), for 24 h at 37 °C following the manufacturer's recommendations (Invitrogen).

Retrotranscription and qRT-PCR analysis were done as described in Tuttobene et al.²¹, using primers listed in Table ​Table3.3. Data are presented as NRQ (Normalized relative quantities) calculated by the qBASE method⁴¹, using recA and rpoB genes as normalizers.

Cell motility was tested on swimming agarose media: 1% tryptone, 0.5% NaCl and 0.3% agarose plates inoculated on the surface by depositing 3 µl of LB cultures grown to an optical density at 660 nm (OD₆₆₀) of 0.3. The plates were incubated 24 or 48 h in the presence or absence of blue light at 37 °C or 23 °C, respectively. For AHL detection assays, the aqueous media containing the cells on the surface of motility plates was recovered, homogenized, and the optical density at 660 nm was determined. Cultures were then centrifuged, filtered-sterilized and amounts normalized to OD₆₆₀ = 1.5 were loaded on biosensor plates, as is indicated in section AHLs detection using biosensors. Triplicate assays were done using fresh samples each time.

For biofilm assays, two milliliters of fresh swimming agarose broth medium contained into glass tubes were inoculated with 0.01 ml of an overnight shaking culture grown at 37 °C. The cultures were then incubated stagnantly at 23 °C or 37 °C either in darkness or under blue light for 48 or 24 h, respectively. Biofilms and pellicles that formed on the walls of the glass tubes were detected by visual inspection¹⁴. Replicate tubes were homogenized by vortexing and the cell density was determined by measurement of OD_660. In general, no significant differences were registered between the different strains incubated under blue light vs. dark conditions, at each temperature. Nonetheless, whether any difference in optical density appeared, the amount of supernatants was normalized to OD₆₆₀ = 0.5. Each culture was then centrifuged, and the supernatants were filter-sterilized and used for AHL detection, as indicated in the following item. Triplicate assays were done using fresh samples each time.

Agrobacterium tumefaciens NT1 (pZLR4), C. subtsugae (formerly C. violaceum) ATCC 31532 CV026⁴², and C. violaceum ATCC 12472 VIR07²⁹, were used to detect the presence of AHLs in A. baumannii cultures recovered from motility plates or biofilm tubes. The A. tumefaciens NT1 (pZLR4) AHL biosensor, which contains a plasmid-localized traG-lacZ fusion (pZLR4)^25,26, responds to AHLs of chain lengths ranging from C6 to C12²⁷. C. subtsugae cviI mutant CV026²⁸ does not produce AHLs, but induces the CviR upon exposure to exogenous short-chain AHLs, which results in rapid synthesis of a visually clear purple pigmentation, violacein. Particularly, violacein is inducible by compounds with N-acyl side chains from C₄, to C₈, in length, with varying degrees of sensitivity²⁸. Violacein production in C. violaceum ATCC 12472 cviI mutant VIR07 is induced in response to the long-chain AHLs (C10–C16)²⁹. Supernatants recovered from motility plates or biofilm tubes (approximately 500 µl), were loaded in a central well of LB plates previously inoculated with C. subtsugae CV026 or C. violaceum VIR07. Chromobacterium inoculation was performed by addition of 500 µL of overnight cultures grown to an OD₆₆₀ = 2.5–5 ml of 0.7% agar media, which was then incorporated on top of the 1.5% agar LB plates. The plates were then incubated at 30 °C in the dark for 24 h.

Cells recovered from motility plates of the indicated strains incubated under blue light or in the dark at 23 °C or 37 °C for 48 or 24 h respectively, were normalized to OD₆₆₀ = 1.5. The cells were recovered by centrifugation at 3000 g for 10 min and resuspended in 500 μl in swimming media (1% tryptone, 0.5% NaCl). The cells were sonicated and then centrifuged. Post-sonication supernatants were incubated with 2 µg of commercial standards, C8-AHL or C10-AHL, for 6 h in a shaker at 37 °C. Aliquots (500 μl) of the resulting supernatant were used to detect AHL degradation in a well diffusion assay in double agar plates, in which C. subtsugae CV026 or C. violaceum VIR07 were added to soft agar to detect inhibition of violacein³⁰. Quantification of violacein production in the different strains was determined by measuring the area and integrated density of each complete plate and subtracting the corresponding values measured in the negative control, using ImageJ software (NIH). The values were normalized to the positive control, which received the arbitrary value of 100. The data shown are the means of three independent experiments, and error bars represent the standard deviation of the mean.

Extracts for analytical TLC were prepared from 10-ml cultures (2 tubes of 5 ml each) grown in swimming media pH 7 under blue light or in the dark at 23 °C for 48 h or 37 °C for 24 h. The tubes were then vortexed to disrupt biofilms and the OD₆₆₀ was measured. No significant differences in OD₆₆₀ were detected between the samples. Bacteria were then removed by centrifugation for 5 min at 5000 rpm and the pH was checked, and confirmed to be to be 7 ± 0.1 for all the samples analyzed. Then, the supernatants were filtered-sterilized and extracted twice with equal volumes of ethyl acetate acidified with 0.1% acetic acid. The organic phase was evaporated under N₂ flow.

Procedures followed those described in Shaw et al.²⁷. Extracted AHL samples were dissolved in 20 µl HPLC-grade acetonitrile and 2 µl were applied to C₁₈ TLC plates (RP-18 F254 S, Merck) and developed with 60% methanol. After development, the solvent was dried, and plates were overlaid with a 0.75% soft agar layer seeded with an overnight culture of C. subtsugae CV026 or C. violaceum VIR07. After the agar solidified, the coated plates were incubated at 28 °C for 18 h.

AHL extracts were dissolved in 100 µl HPLC-grade acetonitrile. For silylation, 100 μl of a mixture of 99% bis(trimethylsilyl)-trifluoro-acetamide (BSTFA) and 1% trimethylchlorosilane (TMCS) (Catalog Number 15238, Sigma-Aldrich, USA) was added. After purged with nitrogen, the mixture was allowed to react for 1 h at 80 °C. One μl of extracted sample was injected (Split 1:10) into the gas chromatograph-mass spectrometer Agilent 7890B gas chromatograph coupled to an Agilent 5977 A mass spectrometer (Agilent Technologies Inc., Palo Alto, CA, USA) and a 30 m HP-5 ms Ultra Inert with a 0.25 mm inner diameter and 0.25 μm film thicknesses (Agilent Technologies Inc., Palo Alto, CA, USA). The sample was injected at 250 °C with a gas flow rate of 1 ml/min. The temperature program was isothermal for 5 min at 70 °C, followed by a 5 °C/min ramp to 240 °C and a hold at 240 °C for 2 min. The total running time was 41 min. Ions were generated by a ionization voltage of 70 eV with a scan range of 50–600 Da.

Components of each of the GC peaks were identified by comparison of retention indexes and mass spectra data in the NIST 2011 Mass Spectral Library and also with an in-house database created with standards. Standard mixtures of C8, C10 and C12-AHL as well as 3OHC8, 3OHC10 and 3OHC12-AHL were also analyzed by GC–MS demonstrating that mass fragmentation patterns are unique to each AHL. Areas of ion chromatograms were obtained taking into account that the quantitation ion and the confirming ion are unique to that acyl homoserine lactones retention times.

Assays were performed as described before^14,22, with the modification of incubating 1-ml of the co-cultures without shaking at 37 °C from 24 to 72 h under dark or blue light conditions. Fungal CFU counts per ml were determined at each time point studied by plating convenient dilutions of the co-cultures on yeast extract-peptone-dextrose (YPD) agar containing 60 mg/ml tetracycline, 30 mg/ml chloramphenicol, and 30 mg/ml gentamicin, following incubation at 28 °C for 48 h.