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LysM is an Lrp-like transcriptional regulator from S. solfataricus that was previously shown to bind to the control region of the lysWXJK operon, the sole known target of LysM (Brinkman et al J. Biol. Chem.: 277: 29537-29549). To identify all LysM binding sites in the genome of S. solfataricus we have performed a LysM-specific nanobody-based chromatin immunoprecipitation assay coupled to array hybridization (ChIP-chip). The utilization of a LyM-specific nanobody avoided the need for overexpressing or epitope-tagging the transcription factor. Consequenttly, the genomic association profile was determined at homogenous expression levels of the regulator. In total 72 novel ChIP enriched regions (chers) with an enrichment of at least four-fold were identified. In each cher the best LysM binding site was predicted using an energy-based position weight matrix determined from a saturation mutagenesis of the LysM consensus binding site. These results were validated for a subset of selected target sites with in vitrobinding assays, electrophoretic mobility shift assays (EMSas) and in gel cupper-phenanthroline footprinting. High affinity targets were found in both intergenic control regions and in ORF's, and both classes comprise sites that are efficiently bound in vitro and in vivo, whereas others are only bound efficiently in vivo. Various LysM binding sites are positioned in close proximity to promoter sequences. Transcription units, of which the expression is potentially influenced by LysM binding include: amino acid metabolism, central metabolism, transport, CRISPR immunity system, translation and hypothetical proteins. In vitro binding assays demonstrated high affinity binding of LysM to the control region of leuA-2 (2-isopropylmalate synthase), gltB (glutamate synthase) and Sso1906 and Sso2043, both encoding amino acid transporter related proteins. Binding of LysM to all these targets was decreased in the presence of lysine, and lysine supplementation to the growth medium resulted in a reduction of the corresponding gene expression levels, as demonstrated with qRT-PCR. Therefore, it appears that LysM is a much more versatile regulator than previously thought and that LysM stimulates the expression of all target genes when the intracellular lysine concentration is low. E.P. is a post-doctoral fellow of the Research Foundation Flanders (FWO-Vlaanderen). L.v.O. is a predoctoral fellow of the FWO-Vlaanderen. |
