Bacteria were recovered from frozen stock by culturing on Mueller-Hinton (MH) agar overnight at 37 oC. Bacteria removed from agar were suspended in DNase-, RNase-free water, and genomic DNA was isolated using the Puregene system (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions with the following modifications. Proteinase K was added to a final concentration of 200 µg/ml prior to incubating cell lysates at 80oC for 5 min, the final concentration of RNAse was increased to 25 µg/ml, and precipitated proteins were removed by course filter through a 1 ml syringe packed to the 0.2 ml demarcation with glass wool prior to the clarifying centrifugation. Pelleted DNA was resuspended in DNase-, RNase-free water, and DNA quality was assessed by A260/A280 ratios and agarose gel electrophoresis.
Label
Alexa Dye 647
Label protocol
Four micrograms of bacterial genomic DNA were labeled with Alexa Fluor 555 or Alexa Fluor 647 ester dyes using the BioPrime Plus Array CGH Indirect Genomic Labeling System (Invitrogen Corp., Carlsbad, CA) following the manufacturer’s specifications with two modifications. In place of ethanol precipitation, DNA was dried for 2 h under medium heat after Klenow amplification using the Savant DNA 120 vacuum centrifuge (Thermo Scientific, Asheville, NC), and DNA was completely dried after dye incorporation for 2 h by vacuum centrifugation with no heat.
Hybridization protocol
Dried DNAs from two isolates labeled with each fluorophore were resuspended together in hybridization buffer (6x SSPE, 23% formamide, and 0.05% Tween 20), heated for 5 min at 95°C, and cooled on ice for 5 min before loading onto the arrays. Coverslips were rinsed with 2% SDS, followed by water and then 100% ethanol. After two more washes with water, coverslips were overlaid on the slide to cover both arrays. Covered slides were deposited in hybridization chambers and incubated in a 48°C water bath for 16-20 h. Arrays were then sequentially washed in 6X SSPE for 2 min at 48°C and 1X SSPE for 2 min at 48°C, and then quickly dipped ten times in 0.25X SSPE at room temperature before drying by centrifugation at 2000 xg for 30 s.
Scan protocol
Arrays were scanned in an Axon 4000B scanner (Molecular Devices, Inc., Sunnyvale, CA) using the Genepix v5.1 software and a 5 μm pixel size. PMT values were customized for each channel to obtain the complete range of intensities. Images were saved in TIFF format. Before extracting data, spots were reviewed and flagged as unreadable if scratches, dust or salts precipitates were present. Data were extracted from both channels, but each channel was treated independently in the final analysis
Global background, calculated as 5% median of the intensity of all target probes, was subtracted from each spot intensity. Arrays were then normalized using a set of 159 probes from 78 genes whose sequences were exact matches in all 5 of the genomes used for the array design. The median spot intensity for the group of conserved probes was arbitrarily set to 10,000 and applied as a correction factor to each array. Probes with adjusted signal intensities greater than 1594 units. Data from independent hybridization reactions were consolidated to a single present/absent call for each probe, with present being defined as at least 50% of all replicate spots being called present. Finally, genes were called present if at least 50% of the probes targeted to that element were called present.
