| Development and application of versatile high density microarrays for genome-wide analysis of Streptomyces coelicolor: characterization of the HspR regulon (2009) | |||||||||||
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| Abstract Background DNA microarrays are a key resource for global analysis of genome content, gene expression and the distribution of transcription factor binding sites. We describe the development and application of versatile high density ink-jet in situ -synthesized DNA arrays for the G+C rich bacterium Streptomyces coelicolor . High G+C content DNA probes often perform poorly on arrays, yielding either weak hybridization or non-specific signals. Thus, more than one million 60-mer oligonucleotide probes were experimentally tested for sensitivity and specificity to enable selection of optimal probe sets for the genome microarrays. The heat-shock HspR regulatory system of S. coelicolor , a well-characterized repressor with a small number of known targets, was exploited to test and validate the arrays for use in global chromatin immunoprecipitation-on-chip (ChIP-chip) and gene expression analysis. Results In addition to confirming dnaK , clpB and lon as in vivo targets of HspR, it was revealed, using a novel ChIP-chip data clustering method, that HspR also apparently interacts with ribosomal RNA ( rrnD operon) and specific transfer RNA genes (the tRNA Gln /tRNA Glu cluster). It is suggested that enhanced synthesis of Glu-tRNA Glu may reflect increased demand for tetrapyrrole biosynthesis following heat-shock. Moreover, it was found that heat-shock-induced genes are significantly enriched for Gln/Glu codons relative to the whole genome, a finding that would be consistent with HspR-mediated control of the tRNA species. Conclusions This study suggests that HspR fulfils a broader, unprecedented role in adaptation to stresses than previously recognized - influencing expression of key components of the translational apparatus in addition to molecular chaperone and protease-encoding genes. It is envisaged that these experimentally optimized arrays will provide a key resource for systems level studies of Streptomyces biology. | |||||||||||
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