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

ABSTRACT: 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.

SUBMITTER: Bucca G

PROVIDER: S-EPMC2687793 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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

Bucca Giselda G 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

Genome biology 20090116 1

<h4>Background</h4>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 ...[more]

PMID: 19146703

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Project description:Most currently used antibiotics originate from Streptomycetes and phosphate limitation is an important trigger of their biosynthesis. Understanding the molecular processes underpinning such regulation is of crucial importance to exploit the great metabolic diversity of these bacteria and get a better understanding of the role of these molecules in the physiology of the producing bacteria. To contribute to this field, a comparative proteomic analysis of two closely related model strains, Streptomyces lividans and Streptomyces coelicolor was carried out. These strains possess identical biosynthetic pathways directing the synthesis of three well-characterized antibiotics (CDA, RED and ACT) but only S. coelicolor expresses them at a high level. Previous studies established that the antibiotic producer, S. coelicolor, is characterized by an oxidative metabolism and a reduced triacylglycerol content compared to the none producer, S. lividans, characterized by a glycolytic metabolism. Our proteomic data support these findings and reveal that these drastically different metabolic features could, at least in part, due to the weaker abundance of proteins of the two component system PhoR/PhoP in S. coelicolor compared to S. lividans. In condition of phosphate limitation, PhoR/PhoP is known to control positively and negatively, respectively, phosphate and nitrogen assimilation and our study revealed that it might also control the expression of some genes of central carbon metabolism. The tuning down of the regulatory role of PhoR/PhoP in S. coelicolor is thus expected to be correlated with low and high phosphate and nitrogen availability, respectively and with changes in central carbon metabolic features. These changes are likely to be responsible for the observed differences between S. coelicolor and S. lividans concerning energetic metabolism, triacylglycerol biosynthesis and antibiotic production. Furthermore, a novel view of the contribution of the bio-active molecules produced in this context, to the regulation of the energetic metabolism of the producing bacteria, is proposed and discussed.

Project description:Many microorganisms, including bacteria of the class Streptomycetes, produce various secondary metabolites including antibiotics to gain a competitive advantage in their natural habitat. The production of these compounds is highly coordinated in a population to expedite accumulation to an effective concentration. Furthermore, as antibiotics are often toxic even to their producers, a coordinated production allows microbes to first arm themselves with a defense mechanism to resist their own antibiotics before production commences. One possible mechanism of coordination among individuals is through the production of signaling molecules. The gamma-butyrolactone system in Streptomyces coelicolor is a model of such a signaling system for secondary metabolite production. The accumulation of these signaling molecules triggers antibiotic production in the population. A pair of repressor-amplifier proteins encoded by scbA and scbR mediates the production and action of one particular gamma-butyrolactone, SCB1. Based on the proposed interactions of scbA and scbR, a mathematical model was constructed and used to explore the ability of this system to act as a robust genetic switch. Stability analysis shows that the butyrolactone system exhibits bistability and, in response to a threshold SCB1 concentration, can switch from an OFF state to an ON state corresponding to the activation of genes in the cryptic type I polyketide synthase gene cluster, which are responsible for production of the hypothetical polyketide. The switching time is inversely related to the inducer concentration above the threshold, such that short pulses of low inducer concentration cannot switch on the system, suggesting its possible role in noise filtering. In contrast, secondary metabolite production can be triggered rapidly in a population of cells producing the butyrolactone signal due to the presence of an amplification loop in the system. S. coelicolor was perturbed experimentally by varying concentrations of SCB1, and the model simulations match the experimental data well. Deciphering the complexity of this butyrolactone switch will provide valuable insights into how robust and efficient systems can be designed using "simple" two-protein networks.

Dataset Information

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

Publications

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

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