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


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.
Bucca
Giselda
G
Microbial Sciences Division, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK. g.bucca@surrey.ac.uk
Laing
Emma
E
Mersinias
Vassilis
V
Allenby
Nicholas
N
Hurd
Douglas
D
Holdstock
Jolyon
J
Brenner
Volker
V
Harrison
Marcus
M
Smith
Colin P
CP
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D005798:Genes, Bacterial; D023281:Genomics / Q000295:instrumentation / Q000379:methods*; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D047468:Immunoprecipitation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics; D018085:Regulon / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000235:genetics*
D001426:Bacterial Proteins; D002843:Chromatin; D006360:Heat-Shock Proteins; C108444:HspR protein, bacteria; D012335:RNA, Ribosomal; D012097:Repressor Proteins; D012343:RNA, Transfer