Microarray slides

WT and mutantcultures were harvested by rapid cooling, followed by centrifugation at 4,230 × g for 10 min; samples were taken at mid-exponential phase (10h, ~5 × 10⁷ cells/mL), the bifurcation point in the growth curve of the two strains (14h, 9.7 × 10⁷ cells/mL for WT and 1.2 × 10⁸ cells/mL for the mutant), and stationary phase (24h, 2.7 × 10⁸ cells/mL for WT and 1.6 × 10⁹ cells/mL for mutant). After centrifugation, cells were washed in ice-cold TE buffer and stored at −80°C until further processing. Total RNA was isolated by re-suspending the cells in TRIzol reagent (Invitrogen, San Diego, CA). RNA was purified using an RNEasy kit (Qiagen, Valencia, CA). RNA was then reverse transcribed to cDNA using Superscript III (Invitrogen), random primers (Invitrogen), and the incorporation ofaminoallyl-DUTP, 5-(3-aminoallyl)-2’-deoxyuridine 5’-triphosphate (Ambion, Austin, TX), as described previously (Chou et al. 2007 (link)). cDNA was labeled with either Cy3 or Cy5 dye (GE Healthcare, Little Chalfont, United Kingdom), and hybridized to one of six microarray slides (Corning, Acton, MA) in the loop design (see Figure S1), following protocols previously described(Chou et al. 2007 (link)).

A spotted, whole-genome hybrid oligonucleotide microarray, with probes for 2,032 P. furiosus COM1 genes and 19 additional probes for heterologous genes from M. sedula 3HP/4HB cycle enzymes, was used as described previously for other microarrays, with 5 replicates of each probe spotted on the slide (Hawkins et al., 2014 (link)). Total RNA from two separate biological experiments was pooled and reverse-transcribed (Superscript III, Invitrogen), re-purified, labeled with either Cy3 or Cy5 dye (GE Healthcare), and hybridized to the microarray slides (Corning). Slides were scanned on a GenePix 4000B Microarray Scanner (Molecular Devices, Sunnyvale, CA), and raw intensities were quantitated using GenePix Pro version 6.0. Data normalization and statistical analysis were performed using JMP Genomics 5 (SAS). In general, significant differential transcription was defined to be relative changes in expression of ≥ 2-fold (where a log₂ value of ±1 means a 2-fold change) having −log₁₀p-values of ≥ 4.9 (Bonferroni correction equivalent to a p-value of 1.2 × 10⁻⁷ for these experiments).