Supplementary Material for: Global Transcriptional Analysis of <i>Bacillus licheniformis</i> Reveals an Overlap between Heat Shock and Iron Limitation Stimulon

In this study, we characterized the heat shock stimulon of the important industrial microorganism <i>Bacillus licheniformis</i> using DNA microarrays. While sharing a high degree of homology with the closely related model organism <i>Bacillus subtilis</i>, the heat shock stimulon of <i>B. licheniformis</i> exhibited several novel and unexpected features. Most notably, heat shock in <i>B. licheniformis</i> resulted in decreased amounts of mRNA from the <i>ytrABCEF</i> operon, encoding a putative acetoin uptake system, and stimulated the transcription of purine biosynthesis and iron uptake genes. Unexpectedly, deletion of the <i>ytrEF</i> genes did not affect acetoin uptake, but increased heat sensitivity. To investigate the connection between heat stress and iron uptake further, we analyzed the iron limitation response of <i>B. licheniformis</i> by DNA microarrays and concluded that the response mostly involves the genes related to iron uptake and metabolism, while the only heat shock gene affected by iron limitation was <i>clpE</i>. We also attempted to delete the <i>fur </i>gene (encoding the ferric uptake repressor), but unexpectedly found it to be essential in <i>B. licheniformis</i>. Using the fluorescent protein-encoding reporter gene under control of the <i>dhb </i>promoter, which responded to both heat shock and iron-starvation, we confirmed the overlap between these responses.