Transcriptional regulation of central amino acid metabolism in Lactococcus lactis

This thesis describes the functional characterisation of the transcriptional regulators GlnR, ArgR and AhrC of Lactococcus lactis, which are responsible for the control of genes involved in the metabolism of the amino acids glutamine, glutamate and arginine. A chromosomal glnR deletion mutant was made and compared to the wild-type strain, by transcriptome analysis, during growth in either rich or poor nitrogen media. L. lactis GlnR was shown to repress the expression of glnRA (encoding the regulator itself and glutamine synthetase), amtBglnK (encoding putative ammonium transport and PII signal transduction proteins), and glnPQ (encoding putative glutamine ABC transport and substrate binding proteins), in nitrogen excess. Promoter-deletion analysis and electrophoretic mobility shift assays showed that the L. lactis GlnR operator strongly resembles the TnrA/GlnR operator of Bacillus subtilis. Finally, glutamine and ammonium were shown to be the main nitrogen-effector molecules for GlnR-mediated regulation in L. lactis. A random integration knockout screening identified both ArgR and AhrC as essential for the control of arginine metabolic genes in L. lactis. DNA microarray analyses determined ArgR and AhrC to be dedicated to the regulation of arginine metabolism, and demonstrated the effect of disrupted arginine regulation on related metabolic pathways. Using purified, His-tagged derivatives of ArgR and AhrC it was shown that AhrC does not bind to DNA, but that ArgR binds to DNA in an arginine-independent manner. Arginine-dependent DNA-binding was only obtained when the two regulators were mixed. In the presence of arginine, AhrC increased the binding of ArgR/AhrC to operators in the promoters of the arginine biosynthetic operons, but inhibited binding of ArgR to the promoter of the arginine catabolic operon. Consequently, the arginine-dependent repression of arginine biosynthesis and activation of catabolism is proposed to be mediated through direct protein-protein interaction between ArgR and AhrC in L. lactis. The work described in this thesis contributes to the understanding of transcriptional gene regulation in L. lactis, where relatively few regulators have been characterised so far. The study illustrates the strength of combining a global transcriptome approach with classical molecular techniques, and the care that must be taken when attempting to predict global regulatory circuits.