Rnawiz

Monolayers of infected SHK-1 cells were rinsed with PBS and resuspended in 1.0 ml of RNAWIZ (Ambion), and total RNA was purified using the RiboPure-Bacteria kit (Ambion) following manufacturer's instructions. The fraction of intracellular P. salmonis RNA was enriched by subtracting eukaryotic ribosomal and messenger RNA using the MICROBEnrich™ Kit (Life Technologies). Briefly, 25 μg of total RNA was mixed with 10 μl of oligocapture in 300 μl of binding buffer. The mixes were incubated at 70°C by 10 min, and at 37°C by 1 h. Then, magnetic oligo-MagBeads were added and incubated at 37°C by 15 min, the beads were captured by using a magnetic stand, and the supernatant containing bacterial enriched RNA was recovered. The recovered RNA was precipitated with sodium acetate, ethanol 100%, and glycogen and incubated at −20°C overnight. The pellets were centrifuged at 4°C, washed twice with cold 70% ethanol, and resuspended in 40 μl of TE buffer. The quantity and quality of RNA were determined by NanoQuant Spectrophotometer (Tecan Technologies) and 2200 TapeStation Bioanalyzer (Agilent Technologies).

To isolate total RNA from exponential or stationary phase P. salmonis cultures, bacteria were collected from the broth by centrifugation, resuspended in 0.5 ml of RNAWIZ (Ambion), mixed with 200 μl of zirconia beads, and disrupted using the Fast-prep 24 (MP) in three cycles of 40 s at 6.0 M/s with intervals of 5 min on ice. The bacterial lysate was recovered by centrifugation and zirconia beads were discarded. The bacterial lysate was mixed with 0.2 volumes of chloroform and then centrifuged. The aqueous phase was mixed with 0.5 volumes of 100% ethanol and bacterial RNA was purified by passing through a filter cartridge (Qiagen); successive washes were carried out according to manufacturer's instructions. RNA was eluted with 0.1 ml of TE buffer in DEPC-treated water and incubated for 30 min at 37°C with RNase-Free DNase I (Ambion) to remove residual gDNA. The quantity and quality of RNA were determined by measuring the absorbance at 260/280 nm using a NanoQuant Spectrophotometer (Tecan Technologies) and integrity was confirmed using 2200 TapeStation Bioanalyzer (Agilent Technologies).

The organization of piscibactin genes into operon(s) was tested by reverse transcription PCR. V. anguillarum RV22 was grown until exponential phase (ca. OD₆₀₀ = 1) in 10 mL CM9 medium containing 5 μM EDDA or 10 μM Fe₂(SO₄)₃. Cells were pelleted by centrifugation at 10,000 ×g for 10 min and total RNA was isolated with RNAwiz (Ambion) following the manufacturer’s recommendations. RT-PCR was performed with 1 μg RNA pre-treated with RQ1 RNase-Free DNase (Promega) by using the M-MLV reverse transcriptase (Invitrogen). An appropriate primer (Table 2), located at the 3′-end of irp5 gene, was used to obtain a cDNA that was then used as template for three PCR reactions targeted into araC1 (PCR1), frpA (PCR2) and between irp2 3′-end and irp1 5′-end (PCR3) (Figure 1). All primers used are listed in Table 2. A negative control reaction was performed with total RNA without M-MLV reverse transcriptase to confirm the lack of genomic DNA contamination in each reaction mixture. The PCR positive control reaction was done using 100 ng of genomic DNA as template.

Probes for in situ hybridization were generated by RT-PCR from poly(A)‘ RNA purified with RNAwiz (Ambion, Austin, TX) from four lab-raised stage 31 larvae [63 (link)] from the San Joaquin River in Friant, CA, USA.
The following primers were used to amplify a 765-bp fragment from exon 2, 305 bp of which are in the 3 ^′ untranslated region:

5 ^′-CCAGGCCAAAAGGTCATTCTC-3 ^′

5 ^′-GAGGAAGTTTCACCAGAAGC-3 ^′

This fragment was cloned into the pCR4-TOPO vector (Thermo Scientific), cut with NotI enzyme, and transcribed with T3 polymerase (Promega, Madison, WI) as described [9 (link)]. Stage 30/31 larvae (20 days post hatching) from a lab-raised in vitro fertilized cross of Matadero Creek fish (Palo Alto, CA, USA) were used for in situ hybridization. RNA in situ hybridization was performed as described [64 ] with the following modifications: larvae were bleached in a 4:1 mix of 30 % H₂O₂ and phosphate-buffered saline with 0.1 % Triton X-100 for 1 hour under bright light. The proteinase K treatment lasted 5 minutes, the hybridization temperature was 65 °C, and the coloration reaction used BM Purple (Roche Diagnostics, Indianapolis, IN).

Total RNA samples were prepared from H. austinensis embryos at selected stages [44 (link)] with RNA Wiz (Ambion, Austin, TX, USA) according to the manufacturer's instructions, using 40 embryos for each stage. The total RNA obtained (3 µg) was reverse transcribed using a first-strand cDNA synthesis kit (BD Biosciences, Palo Alto, CA, USA). The PCR mixture (50 μl) contained 10 × Taq buffer, 0.3 U Taq polymerase (PerkinElmer, Wellesley, MA, USA), 2.5 mM of dNTPs, 5 pmol of each set of primers (for the details of primer information, see Additional file 1: Table S1), and 50 ng of cDNA from the selected stages as template. The PCR reactions were performed under the following cycling conditions: an initial denaturation at 94 °C for 5 min, followed by 25–35 cycles of denaturation at 94 °C for 30 s, and elongation at 72 °C for 10 min. A 6 μl aliquot of each PCR reaction was removed after 25 cycles, while the remaining material underwent five additional cycles of amplification. The 18 rRNA sequence was used as an internal standard (QuantumRNA, Ambion). The extent of amplification was chosen empirically to avoid saturation of the amplified bands. To quantify PCR products, each sample was run in a 1.5% agarose gel, and then stained with ethidium bromide. Band intensity was measured with an Alphalmager (Alpha Innotech Corp., San Leandro, CA, USA) using the Alphaease (v3.3b) program.

C. perfringens strain SM101, grown under identical conditions in BHI alone or BHI containing nisin and bile acids, was used to isolate RNA. The RNA was extracted from the cells according to a method previously described [25 (link)]. Cells harvested at various intervals after incubation were used for RNA extraction. The cells were centrifuged (15,000 ×g, 10 min, 4°C), washed with 10 mM Tris and 1 mM EDTA (pH 8.0), and suspended in lysozyme buffer [25 (link)] containing 10 mg/ml of lysozyme (Sigma). Following incubation for 10 min at room temperature, the cells were centrifuged (15,000 ×g, 10 min, 4°C). The cell pellets were suspended in 50 μl of TE (10 mM Tris, 1 mM EDTA) and mixed with 350 μl of RNAWIZ from Ambion (Grand Island, NY). 75 μl of chloroform was added to the samples, which were incubated for 30 min on ice. The samples were then centrifuged (15,000 ×g, 30 min, 4°C) and the clear phases containing RNA were removed to different tubes. The RNA samples were applied to an RNeasy Mini Kit from Qiagen, Inc. (Valencia, CA), to further purify RNA. Contaminating DNA was removed using a Turbo DNA-free kit (Ambion). A Nanodrop ND-1000 spectrophotometer (NanoDrop Technology, Wilmington, DE) was used to determine the quantity and quality of total RNA. To avoid RNA degradation, the RNA was stored at −80°C and used for qRT-PCR within a week of extraction.