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ThepyrGgene fromLactococcus lactisencodes CTP syn-thase (EC 6.4.3.2), an enzyme converting UTP to CTP. A series of strains were constructed with different levels of pyrGexpression by insertion of synthetic constitutive pro-moters with different strengths in front ofpyrG.These strains expressed pyrGlevels in a range from 3 to 665% relative to thewild-type expression level.Decreasing the level of CTP synthase to 43% had no effect on the growth rate, showing that the capacity of CTP synthase in the cell is in excess in a wild-type strain. . | Eur. J. Biochem. 271 2438-2445 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04168.x Expression of the pyrG gene determines the pool sizes of CTP and dCTP in Lactococcus lactis Casper M. Jorgensen Karin Hammer Peter R. Jensen and Jan Martinussen Bacterial Physiology and Genetics BioCentrum-DTU Technical University of Denmark Kgs. Lyngby Denmark The pyrG gene from Lactococcus lactis encodes CTP synthase EC 6.4.3.2 an enzyme converting UTP to CTP. A series of strains were constructed with different levels of pyrG expression by insertion of synthetic constitutive promoters with different strengths in front of pyrG. These strains expressed pyrG levels in a range from 3 to 665 relative to the wild-type expression level. Decreasing the level of CTP synthase to 43 had no effect on the growth rate showing that the capacity of CTP synthase in the cell is in excess in a wild-type strain. We then studied how pyrG expression affected the intracellular pool sizes of nucleotides and the correlation between pyrG expression and nucleotide pool sizes was quantified using metabolic control analysis in terms of inherent control coefficients. At the wild-type expression level CTP synthase had full control of the CTP concentration with a concentration control coefficient close to one and a negative concentration control coefficient of -0.28 for the UTP concentration. Additionally a concentration control coefficient of 0.49 was calculated for the dCTP concentration. Implications for the homeostasis of nucleotide pools are discussed. Keywords pyrG CTP synthase metabolic control analysis metabolism EC 6.4.3.2. Synthesis of ribonucleotides and deoxyribonucleotides is an essential part of cellular metabolism as synthesis of RNA I ui iees bonnueleolides add INNA I i dicaff n is dependent on deoxyribonucleotides. The involvement of nucleotides in these central cellular pathways suggests that it is important for the cell to control the synthesis of nucleotides and to be able to maintain a steady
