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| Home > Publications database > Regulation durch verzweigtkettige Aminosäuren in Corynebacterium glutamicum |
| Dissertation / PhD Thesis/Book | PreJuSER-34993 |
2004
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/100
Report No.: Juel-4124
Abstract: In this work, global gene expression changes of Corynebacterium glutamicum in the presence of L-valine, L-leucine or L-isoleucine and regulation due to the transcriptional regulator Lrp were characterised. The following results were obtained: 1. C. glutamicum is not able to utilize branched chain amino acids as carbon or nitrogen sources. For that reason, export via the recently identified exporter BrnFE is thought to reduce exceedingly high intracellular valine, leucine or isoleucine concentrations. The presence of 100 mM leucine or isoleucine in the culture medium caused a slight growth inhibition of the wild type while the same concentration of valine had no effect. Expression of three genes of branched chain amino acid biosynthesis (ilvN, leuC, leuD) was repressed in the presence of these amino acids. 2. In contrast to the wild type, the valine production strain C. glutamicum VAL1 (13032ΔilvAΔpanBC(pJC1ilvBNCD)) showed severe growth inhibition in the presence of valine or leucine. Transcriptome analyses with DNA microarrays, proteome analyses and enzyme assays revealed unexpectedly increased mRNA, protein and activity ratios of the acetohydroxy acid synthase (ilvBN gene product) due to valine in VAL1, but not in the wild type. As the inhibitory effect of valine and leucine was relieved through an increased isoleucine concentration, it could be ascribed to an isoleucine limitation in the isoleucine auxotrophic strain VAL1. This limitation is caused by the fact that all three branched chain amino acids are taken up into the cell via the same transporter, BrnQ. Thus, high valine or leucine concentrations competitively inhibit the uptake of the supplement isoleucine. This mechanism was proven by relieve of the valine or leucine inhibition if isoleucine was supplied in the form of the dipeptide isoleucyl-isoleucine which is not imported into the cell via BrnQ. 3. In C. glutamicum, the gene encoding the transcriptional regulator Lrp (“Leucine responsive regulatory protein”) is arranged divergently to the adjacent genes brnFE of the branched chain amino acid exporter. A comparison of the transcriptomes of the wild type and an lrp deletion mutant showed that expression of 60 genes was directly or indirectly regulated by Lrp. By means of the “ChIP-to-Chip” method, which allowed identification of DNA sequences directly bound to Lrp in vivo, three DNA fragments were identified. This indicated a local role for the regulator Lrp. Among these three DNA fragments was the promoter region between lrp and brnFE. The exact binding position of Lrp in this region was determined in vitro by DNase I Footprinting with purified Lrp and indicated activation of brnFE transcription and a negative autoregulation of lrp transcription. In accordance with this model, a strongly reduced export of branched chain amino acids in the lrp deletion mutant was shown previously. The multitude of gene expression changes in the lrp deletion mutant and the growth inhibition in the presence of branched chain amino acids are a likely consequence of the lack of brnFE induction that caused increased intracellular concentrations of these amino acids.
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