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- The intricate side of systems biology


It is usually assumed that the accumulation of intermediates in a linear pathway is disadvantageous, because their storage is unnecessary yet chemically costly (68). The observation of high 3-PGA and PEP pools in Lactococcus therefore appears to point to a suboptimal pathway design. However, closer scrutiny corrects this conclusion. Many homofermentative lactic acid bacteria, including Lactococcus, live in environments where glucose availability may fluctuate widely between high concentrations and extended periods of starvation. As long as glucose is plentiful, the bacteria employ efficient transporters that feed the substrate into metabolic pathways that, in turn, use it for energy production and population growth. In situations without sugars in the medium, the organisms cannot grow. During these periods of starvation, it becomes crucial to be well prepared for future availability of glucose, which the organisms must use quickly for energy generation and for the excretion of lactate, whose acidity helps them create and maintain an advantage over potential competitors. To achieve this readiness, the organisms must enter a holding pattern that is characterized by high concentrations of PEP, which is needed as phosphate donor for glucose consumption.

Without an effective control design, a holding pattern of this type would not be possible. All glucose would be converted into lactate and other end products, such as acetate, acetoin, and ethanol. In particular, without PEP, Lactococcus would be less competitive against organisms by using ATP for glucose phosphorylation and any sudden availability of glucose would be of little benefit, because of the organism’s sluggishness in taking up the substrate. Lactococcus and other homofermentative lactic acid bacteria therefore are faced with the design task of maintaining PEP at relatively high concentration levels that last long enough to bridge normal periods of starvation. It appears that these concentration levels are fine tuned so that the amount of phosphate donors necessary for rapid glucose utilization after starvation are just sufficient to hold over until de novo trioses are provided through glycolysis.

Our comparative studies with lacking FBP and/or Pi regulation demonstrate the power of detailed systems biological analyses. Experimentally, such studies are very difficult, if not impossible (12), but their computational equivalents are easily capable of deciphering the advantages of one regulatory design over another. In the present case, they show that accomplishing the goal or retaining PEP not only requires a clever structural and regulatory design but also critically depends on precise timing. If the outlet of the pathway, catalyzed by pyruvate kinase, is closed too rapidly, unnecessary amounts of material are stored in the form of trioses. Otherwise, if pyruvate kinase is deactivated too slowly, most glycolytic material is converted into lactate, thereby causing PEP depletion that is detrimental for future glucose utilization.

Lactococcus operates the glycolytic pathway and its needed PEP holding pattern with a feedforward activation mechanism that is rare in metabolic systems. This mechanism is fortified with a second mechanism of feedforward inhibition by Pi that, by itself, appears to be inferior. The observed design, when elucidated in this fashion, proves to be very effective. The strong transient peak of FBP (Fig. 2) facilitates a very quick conversion of PEP into pyruvate and lactate while glucose is available but is also an effective stop of PK activity when glucose is no longer available. The source and position of activation appear to be optimal. In contrast to G6P, which is a major metabolic branch and control point, and to F6P, which is in very fast equilibrium with G6P, FBP is the first intermediate dedicated to glycolysis but not much else. By virtue of the fact that FBP activates PK, and not some other intermediate step, glycolysis stops and holds at the perfect position, namely PEP.

The regulation by FBP is accompanied by a secondary regulatory mechanism involving Pi. Without FBP, this regulation would be sensitive to Pi fluctuations anywhere in the cell, thereby disqualifying Pi as sole regulator. Such Pi fluctuations are frequent occurrences, because they are connected with many changes in the energy status of the cell that, in turn, affects the activity of glycolysis, because PFK is activated by ADP. Under normal conditions, Pi inhibition and FBP activation have complementary roles, and Pi therefore solidifies the start-and-hold mechanism controlling glucose utilization. An interesting detail is the surprisingly large pool of FBP that accumulates transiently during glucose consumption. Although there is no experimental proof, one may surmise its role as a protectant against situations where glucose is available, but Pi is high for extraneous reasons, for instance, because large quantities of ATP had been used somewhere in the cell. The high Pi level would inhibit PK, thereby leading to an accumulation of trioses at an inopportune time that would be controlled by factors outside glycolysis. Only a strong peak in FBP would overcome this incidentally inappropriate control.

One may speculate why Lactococcus uses the PTS system rather than ATP for glucose phosphorylation. Despite the clear disadvantage of a strong dependence on a well timed, reliable PEP dynamics, this design has the notable advantage for Lactococcus that most of the glycolytic process is short-circuited through the PTS system. Thus, the organism uses the first available glucose directly to produce pyruvate and then lactate, thereby souring the surrounding medium at a critical time when potential competitors attempt to take up glucose. Of note is that this process is independent of FBP, which at that point is still in its depleted state.

The mechanism of feedforward activation is unusual. Feedback inhibition is widely recognized as a ubiquitous mechanism of controlling the sizes of metabolite pools. Feedforward inhibition has been observed in a number of pathway systems and, under the right conditions, can have a stabilizing effect on the pathway (1314). Feedback activation usually is dreaded, because it often leads to instability. Feedforward activation has been reported in neuronal systems, but hardly in a metabolic context, and activation of PK by FBP is indeed the best-known example. We have shown here that feedforward activation, properly embedded in a regulatory system, provides a potent tool of pathway control.

Stepping back from these pathway-specific features, our analysis demonstrates how important it is to investigate the timing and the regulatory features within a system in intricate detail. Such investigations are possible only if they are based on data that are obtained under physiological conditions and that are crisp enough to permit differentiating analyses. Combining such data with nonlinear dynamic analyses seems to be the most promising path toward discovering natural design principles and developing a true understanding of complex systems in biology.

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