L. monocytogenes EGD-e cells were grown overnight in BHI media at 37°C and constant shaking. The stationary phase cultures were diluted 1:100 in the Activation media: 1x LB broth buffered with 50 mM MOPS pH = 7.3 and supplemented with 25mM glucose-1-phosphate and 1% Amberlite XAD4 resin (Sigma-Aldrich) (Ermolaeva et al., 2004 (link); Ripio et al., 1997 (link)). The culture was grown at constant shaking at 37°C for 5 hours until early stationary growth phase. RNA was isolated from cells. 1 μg of RNA was treated with DNase I (Roche) and purified on RNeasy MinElute columns (QIAGEN). cDNA synthesis was performed with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The levels of hly and prsA2 mRNAs were measured by quantitative RT-PCR with respective primers (Table S6 ) and normalized to the level of 16S rRNA. Quantitative RT-PCR was performed with Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific).
Amberlite xad4 resin
Manufactured by Merck Group
Amberlite XAD4 is a polymeric adsorbent resin commonly used in laboratory applications. It is a non-ionic, macroporous resin with a high surface area and porous structure. The resin is designed for the adsorption and separation of organic compounds from aqueous solutions.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rneasy minelute column, by Qiagen (2 mentions)
Maxima sybr green qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Roche (2 mentions)
Bhi medium, by BD (2 mentions)
1220 infinity lc system, by Agilent Technologies (2 mentions)
1 step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Hydrogen peroxide, by Thermo Fisher Scientific (1 mentions)
Styrene, by Merck Group (1 mentions)
Sulphuric acid, by Merck Group (1 mentions)
N hexane, by Thermo Fisher Scientific (1 mentions)
Ammonium sulphate, by Merck Group (1 mentions)
Pfbha, by Merck Group (1 mentions)
Nitric oxide, by Linde (1 mentions)
Luna omega 5 μm polar c18 lc prep column, by Phenomenex (1 mentions)
8 protocols using amberlite xad4 resin
1
Listeria monocytogenes Virulence Regulation
2
Listeria monocytogenes Growth and Induction
3
Listeria monocytogenes Virulence Regulation
4
Listeria monocytogenes Growth and Induction
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For cloning purposes and reporter gene assay we used E. coli strain DH5a, which was grown in LB medium at constant shaking. For RNA structure profiling and other experiments L. monocytogenes EGDe strain and its mutants (Table S8 ) were used. Before each experiment L. monocytogenes cells were grown overnight in BHI medium (BD Biosciences) at 37°C and constant shaking. For structure probing, the bacteria were diluted 1:100 in 25 mL fresh BHI and grown to OD600 = 1.0. Growth was followed using a spectrophotometer (Amersham Biosciences).
For the experiments requiring induction of PrfA virulence regulator, L. monocytogenes cells were diluted 1:100 in 25 mL of the Activation medium: 1x LB broth buffered with 50 mM MOPS pH = 7.3 and supplemented with 25mM glucose-1-phosphate and 1% Amberlite XAD4 resin (Sigma-Aldrich) (Ermolaeva et al., 2004 (link); Ripio et al., 1997 (link)). As a negative control of the Activation medium, the bacteria were grown in 1x LB broth. The cultures were grown at constant shaking at 37°C for 5 hours until early stationary growth phase.
For the experiments requiring induction of PrfA virulence regulator, L. monocytogenes cells were diluted 1:100 in 25 mL of the Activation medium: 1x LB broth buffered with 50 mM MOPS pH = 7.3 and supplemented with 25mM glucose-1-phosphate and 1% Amberlite XAD4 resin (Sigma-Aldrich) (Ermolaeva et al., 2004 (link); Ripio et al., 1997 (link)). As a negative control of the Activation medium, the bacteria were grown in 1x LB broth. The cultures were grown at constant shaking at 37°C for 5 hours until early stationary growth phase.
5
Direct ELISA for Bacterial Detection
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For direct ELISA, bacteria were grown in the BHI supplemented with 1% (wt/vol) Amberlite XAD4 resin (Sigma-Aldrich) overnight. Bacteria were harvested, washed, and resuspended in PBS at the concentration of 10 10 cfu/mL. Decimal dilutions of the initial bacterial suspension were prepared in PBS. Corner Notch Pierce 96-Well Polystyrene Plates (Thermo Scientific) were coated with 100 μL each of bacterial suspensions and incubated at +4°C overnight. After 3 times washing with the TTBS solution, a-InlB-IgG-HRP conjugate diluted 1:10,000 was added. Plates were incubated at room temperature for 1 h, washed 3 times with TTBS, and the HRP substrate 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added. Results of the colorimetric reaction was read with the iMark (Bio-Rad) plate reader at 450 nm.
6
Characterization of Volatile Organic Compounds
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Reagents were obtained from commercial sources at the following purities: styrene (!99%, Sigma-Aldrich), hydrogen peroxide (>60%, Fisher Chemical), nitric oxide (!99.5%, Linde), sulphuric acid (99.99%, Sigma-Aldrich), ammonium sulphate (99%, Sigma-Aldrich), methanol (99.9%, Sigma-Aldrich), n-hexane (>97%, Acros), derivatization agent: PFBHA (>98%, Sigma-Aldrich), amberlite XAD-4 resin (20e60 mesh, Sigma-Aldrich) and glacial acetic acid (99.7%, Sigma-Aldrich). Synthetic air (99.995%, Praxair) and N 2 (99.999%, Praxair) were employed as bath gases for the experiments, and He (99.999%, Praxair) was used as GC carrier gas. Ultrapure water (18.2 MU cm) was obtained from an ELGA Purelab ® Ultra water purification system (Veolia Water Technologies).
7
Purification of Pyoverdine from P. aeruginosa
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An LB overnight culture of P. aeruginosa PAO1 was diluted 100-fold into 300 mL of M9 medium [1% wt/vol 5X M9 Salts (BD Difco, Franklin Lakes, NJ, USA), 1.5% wt/vol Bacto Casamino Acids with low iron and salt content (BD Difco), 1 mM MgSO4, and 1 mM CaCl2] in a 2-L flask and grown aerobically for 24 h at 37°C. Bacteria were then removed by centrifugation and filtration through a 0.22 µm membrane. The filtrate was incubated with 10% wt/vol amberlite XAD-4 resin (MilliporeSigma) at room temperature for 4 h with constant agitation. After rinsing the resin with copious amounts of water, pyoverdine was eluted in 50% methanol. This eluent was diluted in water to 15% methanol and loaded onto a Luna Omega 5 µm Polar C18 LC prep column (Phenomenex, Torrance, CA, USA) for high-performance liquid chromatography on a 1220 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA). Pyoverdine was eluted from the column by a 0%–100% methanol gradient across 4 h at a flow rate of 5 mL/min. Fractions were collected every other minute for pyoverdine content analysis (Fig. 5B ). The fractions with the highest pyoverdine content were pooled. Methanol was evaporated using a SpeedVac vacuum concentrator. The final purified product was analyzed by HPLC on an analytical column to verify sample purity (Fig. 5C ).
8
Purification of Pyoverdine from P. aeruginosa
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A LB overnight culture of P. aeruginosa PAO1 was diluted 100-fold into 300 mL of M9 medium (1% w/v 5X M9 Salts (BD Difco, Franklin Lakes, NJ), 1.5% w/v Bacto Casamino Acids with low iron and salt content (BD Difco), 1 mM MgSO4, 1 mM CaCl2) in a 2 L flask and grown aerobically for 24 h at 37 °C. Bacteria were then removed by centrifugation and filtration through a 0.22 μm membrane. The filtrate was incubated with 10% w/v amberlite XAD-4 resin (MilliporeSigma) at room temperature for 4 h with constant agitation. After rinsing the resin with copious amounts of water, pyoverdine was eluted in 50% methanol. This eluent was diluted in water to 15% methanol and loaded onto a Luna Omega 5 μm Polar C18 LC prep column (Phenomenex, Torrance, CA) for high-performance liquid chromatography on a 1220 Infinity LC system (Agilent Technologies, Santa Clara, CA). Pyoverdine was eluted from the column by a 0–100% methanol gradient across 4 h at a flowrate of 5 mL/min. Fractions were collected every other minute for pyoverdine content analysis (Fig. 5B ). The fractions with the highest pyoverdine content were pooled. Methanol was evaporated using a SpeedVac vacuum concentrator. The final purified product was analyzed by HPLC on an analytical column to verify sample purity (Fig. 5C ).
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