DNA of prokaryotes is in a nonequilibrium structural state, characterized as ÔactiveÕ DNA supercoiling. Alterations in this state a ect many life processes and a homeostatic control of DNA supercoiling has been suggested [Menzel, R. & Gellert, M. (1983) Cell 34, 105±113]. We here report on a new method for quantifying homeostatic control of the high-energy state of in vivo DNA. The method involves making small perturbation in the expression of topoisomerase I, and measuring the e ect on DNA supercoiling of a reporter plasmid and on the expression of DNA gyrase. In a separate set of experiments the expression of DNA gyrase was manipulated and the control on DNA supercoiling and topoisomerase I expression was measured [part of these latter experiments has been published in Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerho , H.V. & Snoep, J.L. (1999) Eur. J. Biochem. 266, 865±877]. Of the two regulatory mechanisms via which homeostasis is conferred, regulation of enzyme activity or regulation of enzyme expression, we quanti®ed the ®rst to be responsible for 72% and the latter for 28%. The gene expression regulation could be dissected to DNA gyrase (21%) and to topoisomerase I (7%). On a scale from 0 (no homeostatic control) to 1 (full homeostatic control) we quanti®ed the homeostatic control of DNA supercoiling at 0.87. A 10% manipulation of either topoisomerase I or DNA gyrase activity results in a 1.3% change of DNA supercoiling only. We conclude that the homeostatic regulation of the nonequilibrium DNA structure in wild-type Escherichia coli is almost complete and subtle (i.e. involving at least three regulatory mechanisms).Keywords: metabolic control analysis (MCA); hierarchical control analysis (HCA); homeostasis coe cient.DNA in the bacterial nucleoid is negatively supercoiled and it has been estimated that roughly 50% of the supercoiling is constrained by proteins binding to the DNA [1]. This constraint does not depend on the continuous expenditure of ATP. The remaining supercoils are maintained actively at the cost of ATP hydrolysis, via topoisomerase activities. Four topoisomerases have been identi®ed in Escherichia coli (reviewed in [2]). Topoisomerase I [3,4] and DNA gyrase (topoisomerase II) are mostly held responsible for maintaining the supercoiled state of the DNA while topoisomerase III and IV manage the decatenation reactions. A recent publication suggested that topoisomerase IV may also be important for the relaxation of DNA supercoiling [5].The importance of DNA gyrase and topoisomerase I for supercoiling has been shown in studies involving mutants with activities differing greatly from the wild-type activity. Such studies cannot be used to assess the homeostasis of supercoiling in the physiological situation, where the response to smaller challenges is important. When challenged suf®ciently, all systems will respond in drastic manners, or fail. It may well be that a system is robust with respect to small challenges, whilst it fails to deal with the same but larger challenges, or vice v...

Two mutant strains of Lactococcus lactis in which the promoter of the las operon, harboring pfk, pyk, and ldh, were replaced by synthetic promoters were constructed. These las mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions. One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth rate observed in glucosesaturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6-phosphate and fructose-6-phosphate were subsequently found to be increased two-to fourfold in the las mutants, which indicates that phosphofructokinase exerts strong control over the concentration of these metabolites.Lactococcus lactis plays an important role in dairy fermentations, mainly in the production of cheeses. During such fermentation processes, lactose is present at very high concentrations (50 g/liter) and is converted through glycolysis to primarily form lactic acid as well as minor amounts of other compounds (homolactic fermentation). The resulting low pH contributes to the texture and flavor of cheeses and inhibits the growth of other bacterial species. During homolactic fermentation more than 90% of the lactose consumed is recovered as by-products (42), which shows that the glycolytic pathway functions almost exclusively as a catabolic pathway to supply ATP to the cells. When sugar becomes limiting for growth, or in the presence of a less readily metabolized carbon source, the pattern of product formation switches from homolactic to mixedacid fermentation, i.e., to the formation of formate, ethanol, and acetate with smaller amounts of lactate (43).The mechanisms responsible for regulation of glycolytic flux and the shift between different fermentation modes in L. lactis have been studied intensively. The concentrations of intermediary metabolites and cofactors are affected by the external sugar concentration (8,10,37,46). In the presence of excess sugar, the concentrations of fructose-1,6-bisphosphate, the triose-phosphates, and pyruvate and the NADH/NAD ϩ ratio are high, whereas the concentrations of phosphoenolpyruvate and inorganic phosphate are relatively low. The glycolytic flux was proposed to be regulated through the level of fructose-1,6-bisphosphate (43), which is known to activate both pyruvate kinase ...

A series of mutant strains of Lactococcus lactis were constructed with lactate dehydrogenase (LDH) activities ranging from below 1% to 133% of the wild-type activity level. The mutants with 59% to 133% of lactate dehydrogenase activity had growth rates similar to the wild-type and showed a homolactic pattern of fermentation. Only after lactate dehydrogenase activity was reduced ninefold compared to the wild-type was the growth rate significantly affected, and the ldh mutants started to produce mixed-acid products (formate, acetate, and ethanol in addition to lactate). Flux control coefficients were determined and it was found that lactate dehydrogenase exerted virtually no control on the glycolytic flux at the wild-type enzyme level and also not on the flux catalyzed by the enzyme itself, i.e. on the lactate production. As expected, the flux towards the mixed-acid products was strongly enhanced in the strain deleted for lactate dehydrogenase. What is more surprising is that the enzyme had a strong negative control (C J F1 LDH ¼ 2 1.3) on the flux to formate at the wild-type level of lactate dehydrogenase. Furthermore, we showed that L. lactis has limited excess of capacity of lactate dehydrogenase, only 70% more than needed to catalyze the lactate flux in the wild-type cells.Keywords: lactic acid bacteria; metabolic control analysis; gene expression; fermentation.Lactococcus lactis plays an important role in dairy fermentations, mainly in the production of cheeses. In these fermentation processes, lactose is present at high concentrations (50 g·L 21 ) and is converted through glycolysis to lactic acid, with minor amounts of other compounds being produced in addition (homolactic fermentation). The resulting low pH contributes to the texture and flavor of cheeses and inhibits the growth of other bacterial species. Under conditions where sugar becomes limiting for growth of Lactococcus lactis, the metabolism shifts to mixed-acid products, i.e. formate, acetate and ethanol along with smaller amounts of lactate [1,2].Work has been performed in the past to study the mechanisms involved in the shift between the two different fermentation modes in L. lactis. In the presence of excess sugar, the concentration of fructose 1,6-bisphosphate, the triose-phosphates, pyruvate, and the NADH/NAD þ ratio are high, whereas the concentration of phosphoenolpyruvate and inorganic phosphate are relative low [3 -6]. In contrast, when sugar is limiting the concentration of these metabolites and cofactors are reversed to the opposite, high or low level. Particularly, the level of fructose-1,6-bisphosphate, which is known to activate both pyruvate kinase and lactate dehydrogenase, has been suggested to play a key role in the regulation of the fermentation mode [1].Work has also been performed to determine the factors that control the flux through glycolysis by applying metabolic control analysis [7,8]. Based on inhibitor titration, Poolman et al. [9] suggested that glyceraldehyde 3-phosphate dehydrogenase had a large amount of control ove...

No abstract