To induce abrupt iron starvation, triplicate cultures were harvested by centrifugation, washed twice with iron-free media, divided and re-suspended in iron-replete (1µM) or iron-free media. RNA (collected as in Lindell et al, 2007) and cell number samples were collected at 0, 16, 28, 53, and 72 hours with iron addition to the iron-deprived cultures at 54 hours to match replete (1µM FeCl3) iron concentrations.
Growth protocol
MIT9313 was grown at 20µE m-2s-1 (continuous) at 25ºC, in PRO99 media (Moore et al, 2007) modified for trace metal clean work through microwave sterilization of seawater, increase in EDTA to 11.7µM, treatment of major nutrients with Chelex-100 resin (Biorad, Hercules, CA), and treatment of polycarbonate culture vessels sequentially with 0.1% Citranox (Alconox, White Plains, NY), 10% Baker Instra-analyzed HCl (Mallinckrodt Baker, Phillpsburg, NJ), and pH2 HCl for 24 hours each (Keller et al, 1988; Price et al, 1988; Saito et al, 2002).
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using the mirVana miRNA kit (Ambion, Austin, TX, USA) as in Lindell et al. (2007) with lysozyme added (Tolonen et al, 2006). DNA was removed from RNA using Turbo DNase (Ambion) and SuperaseIN (Ambion) to protect RNA (as in Lindell et al, 2007). Due to low RNA yield for MIT9313, DNase-treated MIT9313 mRNA samples were concentrated using Micron Y-30 columns (Millipore, Billerica, MA) then amplified using the Message Amp TM II-Bacteria Prokaryotic RNA Amplification Kit (Ambion) following the manufacturer’s protocol with a 10-hour linear amplification step.
Label
biotin
Label protocol
Synthesis of complementary DNA (cDNA), labeling, hybridization, staining and scanning was carried out according to Affymetrix protocols for E.coli with minor changes. Total RNA (2 µg) was denatured at 70 °C and annealed to random hexamer primers (25 ng/µl) at 25 °C for 10 min. The RNA was reverse transcribed to produce cDNA with Superscript II (25 U/µl – Invitrogen Life Technologies) and 0.5 mM dNTPs in the presence of 1 U/µl RNase Out RNase Inhibitor (Invitrogen). The mix was incubated at 25 °C for 10 min followed by 60 min incubations at 37 °C and 42 °C respectively. Superscript II was inactivated with a 10 min incubation at 70 °C. Sodium hydroxide (0.25 N) was used to remove RNA during a 30 min incubation at 65 °C, followed by neutralization with HCl. The cDNA was purified with MinElute PCR purification columns (Qiagen). Fragments of cDNA, 50-200 nt long, were produced from a 10 min incubation at 37 °C with DNase I (0.6 U per µg cDNA), followed by heat inactivation of the DNase I enzyme (10 min at 98 °C). The cDNA fragments were end-labeled with biotin using the BioArray Terminal Labeling Kit (Enzo) during a 60 min incubation at 37 °C. The reaction was stopped by freezing at –20 °C. The quality of biotin end-labeling was verified by gel-shift assays with NeutrAvidin (Pierce Chemicals) on 1% TBE agarose gels.
Hybridization protocol
The cDNA was hybridized to the MD4-9313 custom Affymetrix array in aqueous hybridization solution (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween-20, 0.1 mg/mL Herring Sperm DNA, 0.5 mg/mL BSA, 7.8 % DMSO and 3 nM prelabeled Affymetrix hybridization B2 oligo control probe mix) during a 16 h incubation at 45 °C in a GeneChip Hybridization Oven 320 rotating at 60 rpm. Washes and stains were carried out on a GeneChip Fluidics Station 450 (Affymetrix) following the ProkGE_WS2v3 Affymetrix protocol. Briefly, following two stringency washes the array was sequentially incubated with 10 µg/mL streptavidin (Pierce Chemical), 5 µg/mL biotinylated anti-streptavidin goat antibody (Vector Laboratories) and 0.1 mg/mL goat IgG (Sigma) and 10 µg/mL streptavidin-phycoerythrin conjugate (Mol. Probes) each for 10 min at 25 °C and given a final wash.
Scan protocol
The arrays were scanned with the GeneChip Scanner (Affymetrix) at a 2.5 µm resolution with excitation set for 570 nm.
Description
Gene expression of MIT9313 after resuspension in iron-replete media, control treatment
Data processing
Robust multi-chip average (RMA) to normalize background signal between arrays (performed on strain-specific probesets) and lowess normalization at the probe-set level to correct influences of expression-signal intensity on fold change, both implemented in Matlab (The Mathworks, Inc., Natick, MA), and using an Affymetrix library file that was updated in September 2008 to include recently discovered ORFs and non-coding RNAs (MD4-9313a520062.Sep08.cdf). rRNA probesets were disregarded due to amplification bias (Frias-Lopez, pers. comm.).
