Development and application of versatile high density microarrays for genome-wide analysis of Streptomyces coelicolor: characterization of the HspR regulon

• Published in: Genome biology Vol. 10; no. 1; p. R5
• Main Authors: Bucca, Giselda, Laing, Emma, Mersinias, Vassilis, Allenby, Nicholas, Hurd, Douglas, Holdstock, Jolyon, Brenner, Volker, Harrison, Marcus, Smith, Colin P
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 01.01.2009; BioMed Central
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Chromatin - metabolism; Cluster Analysis; Gene Expression Profiling; Genes, Bacterial; Genomics - instrumentation; Genomics - methods; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; Immunoprecipitation; Oligonucleotide Array Sequence Analysis - methods; Protein Binding; Regulon - genetics; Repressor Proteins - genetics; Repressor Proteins - metabolism; RNA, Ribosomal - metabolism; RNA, Transfer - genetics; Streptomyces coelicolor - genetics
• ISSN: 1474-760X; 1465-6906; 1474-760X; 1465-6914
• DOI: 10.1186/gb-2009-10-1-r5

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. 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 tRNAGln/tRNAGlu cluster). It is suggested that enhanced synthesis of Glu-tRNAGlu 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. 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.