**2. The regulation of the energetic metabolism in lactic acid bacteria**

Despite the wide use of LAB in food production and the role of some species for their health benefits for the human gastro intestinal tract, the regulatory mechanisms that govern the main energetic metabolism of these bacteria have still not been completely disclosed. Most of the studies have been carried out on the 'domesticated' *L. lactis* species, a member of the LAB widely used in the industrial manufacture of milk-fermented products. The most important industrial application of *L. lactis* is based on its energetic metabolism, which leads mainly to the production of high amounts of lactic acid. Anaerobic glycolysis is the principal energy-generating process of *L. lactis*, it is thus considered exclusively as a fermenting microorganism. Nevertheless, in aerobic conditions and in presence of an exogenous source of heme, *L. lactis* may be able to carry out oxidative phosphorylation (Duwat *et al.*, 2001). This cofactor-dependent respiration capacity has also been discovered in other LAB species (Lechardeur *et al.*, 2011). Although named and used for their capacity to produced lactic

Optimisation of Cell Bioenergetics in Food-Associated Microorganisms 223

While it is usually assumed that the accumulation of intermediates in a linear pathway is disadvantageous because their storage is chemically costly, in *L. lactis* it a strongly persistent accumulation of trioses (3-phosphoglycerate and phosphoenolpyruvate) at relatively high concentrations (6-20 mM) after glucose consumption was observed (Voit *et al.*, 2006). The reason for the accumulation of trioses in the glycolytic pathway was identified when the overall primary metabolism was considered (Figure 2) together with the nutritional characteristics of the environments where many homofermentative bacteria, including *L. lactis*, live. These environments are characterised by the availability of glucose, which fluctuates widely between high concentrations and extended periods of starvation. As long as glucose is available, the glycolytic pathway is efficiently fed so as to obtain energy production and population growth. During glucose starvation, it becomes crucial to be wellprepared for future carbohydrate availability, when the cell must use them as fast as possible in order to restart the flux of the glycolytic pathway and grow. The maintenance of the high concentration of trioses is, therefore, necessary because glucose transport across the membrane depends upon phosphoenolpyruvate (PEP) as phosphate donor through a PTS system (Figure 2) (Voit *et al.*, 2006). As such, it can be speculated that *L. lactis* and other homofermentative LAB have evolved regulatory mechanisms to be able to control the level

The maintenance of the 'necessary' concentration of PEP (PEP holding pattern) during starvation periods requires a fine tuning of downstream reactions in the pathway. If pyruvate kinase is closed too rapidly, unnecessary amounts of materials are stored in the form of trioses. Otherwise, if pyruvate kinase is deactivated too slowly, the glycolytic flux is accelerated towards the production of lactate. In other words, this regulatory mechanism has evolved to use the phosphotransferase system rather than ATP for glucose phosphorylation, thereby having most of the glycolytic process short-circuited through the PTS system. The main ecological advantage of such metabolic control is that cells use the first available glucose directly in order to produce pyruvate and than lactate, thereby acidifying the local environment when potential competitors attempt to take up glucose

Beside the PEP holding pattern, a further interesting metabolic control mechanism developed by *L. lactis* is represented by the 'feed-forward activation', which is quite rare in metabolic systems. The observation of a transient high concentration of fructose 1,6 biphosphate (FBP) during glucose consumption led to the hypothesis of a regulatory role for this glycolytic intermediate. It was suggested that FBP represents a strong activator of the pyruvate kinase (PK), thereby facilitating the very quick conversion of PEP into pyruvate and lactate while glucose is available. On the other hand, the reduction of glucose availability and, therefore a drop in FBP concentration, allows the decrease of PK activity until an effective stop when glucose is no longer available. The specific activation of PK by FBP has also affected the tuning of PEP concentration. This complex regulation of the energetic metabolism was strictly driven by environmental and ecological constraints. In a more general view, the 'PEP holding' strategy and the FBP 'feed-forward activation' represent an adaptive prediction of environmental changes (in this case related to the availability of carbohydrates). The increasing concentration of PEP during glucose starvation represents a metabolic anticipation of the next environmental stimulus (i.e. new

of PEP in order to bridge normal periods of starvation.

(Voit *et al.*, 2006).

acid, numerous LAB can be induced through a respiratory metabolism, thereby improving the population size and its survival. It follows that *L. lactis* is currently industrially produced as biomass using a heme-dependent respiration, while in the manufacture of fermented milk and cheeses the homolactic fermentation has a key role in the food matrix's transformation or preservation as a consequence of the sizable production lactic acid. Due to the relatively recent timing of studies on the respiratory behaviour of *L. Lactis*, most of the work carried out in order to elucidate the intricate regulation of the energetic metabolism in this species has focused on lactic fermentation. A detailed description of the dynamics of metabolic pools were obtained through *in vivo* measurements, and kinetic analysis by using cell extracts and the techniques of nonlinear systems modelling (Voit *et al.*, 2006). The monitoring of the glycolytic intermediates made, at first glance, intuitive sense. During homolactic fermentation, glucose was taken-up by lactococcal cells and converted into glucose 6-phosphate and then fructose 1,6-biphosphate. The latter is converted into trioses, which ultimately form lactate (Figure 2).

Fig. 2. Simplified representation of glycolysis, homolactic fermentation and heme-dependent respiration in *L. lactis*. The Black arrows show the metabolic fluxes. The red arrow shows the regeneration of NAD+ occurring during the heme-dependent respiration. Glucose-6 phosphate (G6P), dihydroxyacetone phosphate (DHA-P), glyceraldehyde-3-phosphate (GA3P), 1,3-biphosphoglycerate (1-3PGA), 3-phosphoglycerate (3PGA), 2-phosphoglycerate (2PGA) phosphoenolpyruvate (PEP), pyruvate (PYR). The green arrows show the role of PEP as a phosphate donor in the glucose uptake, PTS-dependent phosphoenolpyruvate phosphotransferase system (PTS). EIIC, EIIB, EIIA, HPr, HPr-His-P, EI, and EI-P are components of the PTS system.

acid, numerous LAB can be induced through a respiratory metabolism, thereby improving the population size and its survival. It follows that *L. lactis* is currently industrially produced as biomass using a heme-dependent respiration, while in the manufacture of fermented milk and cheeses the homolactic fermentation has a key role in the food matrix's transformation or preservation as a consequence of the sizable production lactic acid. Due to the relatively recent timing of studies on the respiratory behaviour of *L. Lactis*, most of the work carried out in order to elucidate the intricate regulation of the energetic metabolism in this species has focused on lactic fermentation. A detailed description of the dynamics of metabolic pools were obtained through *in vivo* measurements, and kinetic analysis by using cell extracts and the techniques of nonlinear systems modelling (Voit *et al.*, 2006). The monitoring of the glycolytic intermediates made, at first glance, intuitive sense. During homolactic fermentation, glucose was taken-up by lactococcal cells and converted into glucose 6-phosphate and then fructose 1,6-biphosphate. The latter is converted into trioses,

Fig. 2. Simplified representation of glycolysis, homolactic fermentation and heme-dependent respiration in *L. lactis*. The Black arrows show the metabolic fluxes. The red arrow shows the

regeneration of NAD+ occurring during the heme-dependent respiration. Glucose-6 phosphate (G6P), dihydroxyacetone phosphate (DHA-P), glyceraldehyde-3-phosphate (GA3P), 1,3-biphosphoglycerate (1-3PGA), 3-phosphoglycerate (3PGA), 2-phosphoglycerate (2PGA) phosphoenolpyruvate (PEP), pyruvate (PYR). The green arrows show the role of PEP as a phosphate donor in the glucose uptake, PTS-dependent phosphoenolpyruvate phosphotransferase system (PTS). EIIC, EIIB, EIIA, HPr, HPr-His-P, EI, and EI-P are

which ultimately form lactate (Figure 2).

components of the PTS system.

While it is usually assumed that the accumulation of intermediates in a linear pathway is disadvantageous because their storage is chemically costly, in *L. lactis* it a strongly persistent accumulation of trioses (3-phosphoglycerate and phosphoenolpyruvate) at relatively high concentrations (6-20 mM) after glucose consumption was observed (Voit *et al.*, 2006). The reason for the accumulation of trioses in the glycolytic pathway was identified when the overall primary metabolism was considered (Figure 2) together with the nutritional characteristics of the environments where many homofermentative bacteria, including *L. lactis*, live. These environments are characterised by the availability of glucose, which fluctuates widely between high concentrations and extended periods of starvation. As long as glucose is available, the glycolytic pathway is efficiently fed so as to obtain energy production and population growth. During glucose starvation, it becomes crucial to be wellprepared for future carbohydrate availability, when the cell must use them as fast as possible in order to restart the flux of the glycolytic pathway and grow. The maintenance of the high concentration of trioses is, therefore, necessary because glucose transport across the membrane depends upon phosphoenolpyruvate (PEP) as phosphate donor through a PTS system (Figure 2) (Voit *et al.*, 2006). As such, it can be speculated that *L. lactis* and other homofermentative LAB have evolved regulatory mechanisms to be able to control the level of PEP in order to bridge normal periods of starvation.

The maintenance of the 'necessary' concentration of PEP (PEP holding pattern) during starvation periods requires a fine tuning of downstream reactions in the pathway. If pyruvate kinase is closed too rapidly, unnecessary amounts of materials are stored in the form of trioses. Otherwise, if pyruvate kinase is deactivated too slowly, the glycolytic flux is accelerated towards the production of lactate. In other words, this regulatory mechanism has evolved to use the phosphotransferase system rather than ATP for glucose phosphorylation, thereby having most of the glycolytic process short-circuited through the PTS system. The main ecological advantage of such metabolic control is that cells use the first available glucose directly in order to produce pyruvate and than lactate, thereby acidifying the local environment when potential competitors attempt to take up glucose (Voit *et al.*, 2006).

Beside the PEP holding pattern, a further interesting metabolic control mechanism developed by *L. lactis* is represented by the 'feed-forward activation', which is quite rare in metabolic systems. The observation of a transient high concentration of fructose 1,6 biphosphate (FBP) during glucose consumption led to the hypothesis of a regulatory role for this glycolytic intermediate. It was suggested that FBP represents a strong activator of the pyruvate kinase (PK), thereby facilitating the very quick conversion of PEP into pyruvate and lactate while glucose is available. On the other hand, the reduction of glucose availability and, therefore a drop in FBP concentration, allows the decrease of PK activity until an effective stop when glucose is no longer available. The specific activation of PK by FBP has also affected the tuning of PEP concentration. This complex regulation of the energetic metabolism was strictly driven by environmental and ecological constraints. In a more general view, the 'PEP holding' strategy and the FBP 'feed-forward activation' represent an adaptive prediction of environmental changes (in this case related to the availability of carbohydrates). The increasing concentration of PEP during glucose starvation represents a metabolic anticipation of the next environmental stimulus (i.e. new

1998).

Optimisation of Cell Bioenergetics in Food-Associated Microorganisms 225

other Gram-positive bacteria. CcpA belongs to the LacI/GalR family of bacterial regulator proteins, and the disruption of the *ccpA* gene reduces the carbon catabolite repression (CR) of several genes involved in carbohydrate metabolisms. CcpA-mediated regulation depends basically on three elements: i) a specific *cis*-acting DNA sequence, termed cataboliteresponsive element (*cre*) which is present near the promoter region of genes affected by CR, ii) the HPr protein, a phosphotransferase protein of the PTS system, and iii) the concentration of glycolytic intermediates (such as FBP). A metabolite-activated kinase has been shown to phosphorylate HPr on residue serine 46. This phosphorylated form of HPr [HPr(Ser-P)] interacts with CcpA, and this interaction enhances the binding of CcpA to *cre* on the promoter region of genes, so affecting the level of their expression. Indirectly, the phosphorylation of HPr on serine residue, enhanced by high level of glycolytic intermediates (*e.g.* FBP), reduces the number of HPr molecules that can be phosphorylated on histidine residue so as to ensure the functionality of the PTS system in sugar uptake across the membrane (Figure 1). Besides the role of CcpA in the control of sugar metabolism (mainly the sugar uptake), it was demonstrated that the role of this protein in the transcriptional activation of the glycolytic *las* operon, encoding the enzymes phosphofructokinase, pyruvate kinase and L-lactate dehydrogenase (Luesink *et al.*, 1998). Specifically, the disruption of the *ccpA* gene lowered the activity of pyruvate kinase and Llactate dehydrogenase, resulting in the production of metabolites that are characteristic of a mixed-acid fermentation. It was, therefore, speculated that homolactic fermentation in *L. lactis* is maintained by CcpA-mediated repression of mixed-acid fermentation (Luesink *et al.*,

The regulatory function of CcpA on the energetic metabolism was further confirmed when its primary role in the regulation of aerobic and respirational growth of *L. lactis* was described (Gaudu *et al.*, 2003). CcpA was found to repress NADH oxidase activity, thus maintaining a correct NADH/NAD+ ratio that directed the metabolism in favour of respiration. Moreover, it was proposed that a CcpA-mediated repression of the heme transportation system thereby prevented the oxidative damage provoked by precocious heme uptake at the start exponential growth. CcpA thus appears to govern a regulatory

**3. The regulation of the glycolytic pathway in** *Saccharomyces cerevisiae* 

The *S. cerevisiae* metabolism has been exploited by humans for several millennia through a variety of food processes in order to produce alcoholic beverages and leavened bread. Alcoholic fermentation began due to the presence of indigenous yeast in grapes, must, wort and dough, and with total ignorance regarding the existence of microorganisms and their fermentative role. In practice, humans started to apply microbiology before the role of yeast in beer, wine and bread production was formally proven by Pasteur in 1860 (Pasteur, 1860). Starting with the work of Emil Christian Hansen at the Carlsberg Laboratory in Copenhagen, in the early 1880s, the control of the *S. cerevisiae* metabolism became of crucial importance to enhance the efficiency of fermentation processes as well as the quality of the various products. Alcoholic fermentation is not the unique energetic metabolism in *S. cerevisiae* since it can use the more energetically favourable respiration, which sees a

network that coordinates oxygen, iron and the energetic metabolism.

significant increase of ATP being produced per mole of glucose (Figure 3).

glucose availability). The anticipation of environmental change is considered an adaptive trait because pre-exposure to the stimulus – which typically appears early in the ecology – improves the organism's fitness when it encounters a second stimulus (Mitchell *et al.*, 2009). In the regulatory mechanisms described above, carbohydrates-availability and carbohydrates-starvation represent two consecutive and predictable environmental stimuli for fermentative domesticated LAB. In *L. lactis* the FBP 'feed-forward activation' represents a clear example of the relevance of allosteric regulatory mechanisms (Figure 1) on the modulation of the energetic metabolism.

A study done to explain the ability of *L. lactis* to grow, retain an active metabolism and survive at low pH highlights the complexity and the interplay of the overlapping regulatory mechanisms that operate in the regulation of the energetic metabolism. Culturing the microorganisms at low environmental pH sees the biomass yield diminished and the energy dedicated to maintenance increased as a response to the organic acid inhibition and cytoplasmic acidification (Even *at al.*, 2003). The request for energy for maintenance in acid conditions resulted in an increase in glucose consumption and the glycolytic rate with a significant reduction of biomass yield relative to ATP production. The adjustment of the metabolic flux in response to a low environmental pH was determined by an increase in the enzymes' capacity and by a specific modulation of the enzyme activities of the glycolytic pathway. A transcription profile and regulation analysis were effective in evaluating the contribution of each layer of regulatory mechanisms in the observed phenomena, highlighting the primary contribution of translational regulation to the increased concentration of glycolytic enzymes in acidic conditions, and confirming that the translation apparatus of *L. lactis* was optimised under acid stress conditions (Even *et al.*, 2003). In this case, the decrease of intracellular pH due to the acidity of the extracellular environment determines an important decrease in enzyme activity that was compensated for by an increase in the enzyme capacity through the efficiency of the translation machinery. In this context, it should be underlined that the enzyme concentration results from the rate of protein synthesis, corrected by dilution coefficient, which is affected by protein turnover (normally negligible except under conditions of stress) and the rate of cell division (at each cell division, the enzymes cellular content will be halved). It follows that cells growing at different rates will have substantially different rates of protein synthesis, even though the specific activities may remain similar (Even *et al.*, 2001). More recently, the primary role of allosteric regulatory mechanisms in controlling the glycolytic flux of *L. lactis* has been questioned, underlining the predominant regulatory role of the enzymes' concentration. This statement was supported by a new methodology whereby experimental measurements of fluxes and enzyme concentrations can be integrated into flux functions capable of predicting the 'fulsome' from the proteome (Rossel *et al.*, 2011). Nevertheless, by such an approach the understanding of the role of each layer of regulation can only be partially addressed. In this case, the approach of regulation analysis is more informative in delineating which regulatory layer is responsible for establishing fluxes through a given enzyme (Gerosa & Sauer, 2011).

Concerning the hierarchical (i.e. expression and post-translational modification) regulation of the energetic metabolism of LAB, the little information available are related to the catabolite control protein CcpA, the major regulator of the carbon metabolism in *L. lactis* and

glucose availability). The anticipation of environmental change is considered an adaptive trait because pre-exposure to the stimulus – which typically appears early in the ecology – improves the organism's fitness when it encounters a second stimulus (Mitchell *et al.*, 2009). In the regulatory mechanisms described above, carbohydrates-availability and carbohydrates-starvation represent two consecutive and predictable environmental stimuli for fermentative domesticated LAB. In *L. lactis* the FBP 'feed-forward activation' represents a clear example of the relevance of allosteric regulatory mechanisms (Figure 1) on the

A study done to explain the ability of *L. lactis* to grow, retain an active metabolism and survive at low pH highlights the complexity and the interplay of the overlapping regulatory mechanisms that operate in the regulation of the energetic metabolism. Culturing the microorganisms at low environmental pH sees the biomass yield diminished and the energy dedicated to maintenance increased as a response to the organic acid inhibition and cytoplasmic acidification (Even *at al.*, 2003). The request for energy for maintenance in acid conditions resulted in an increase in glucose consumption and the glycolytic rate with a significant reduction of biomass yield relative to ATP production. The adjustment of the metabolic flux in response to a low environmental pH was determined by an increase in the enzymes' capacity and by a specific modulation of the enzyme activities of the glycolytic pathway. A transcription profile and regulation analysis were effective in evaluating the contribution of each layer of regulatory mechanisms in the observed phenomena, highlighting the primary contribution of translational regulation to the increased concentration of glycolytic enzymes in acidic conditions, and confirming that the translation apparatus of *L. lactis* was optimised under acid stress conditions (Even *et al.*, 2003). In this case, the decrease of intracellular pH due to the acidity of the extracellular environment determines an important decrease in enzyme activity that was compensated for by an increase in the enzyme capacity through the efficiency of the translation machinery. In this context, it should be underlined that the enzyme concentration results from the rate of protein synthesis, corrected by dilution coefficient, which is affected by protein turnover (normally negligible except under conditions of stress) and the rate of cell division (at each cell division, the enzymes cellular content will be halved). It follows that cells growing at different rates will have substantially different rates of protein synthesis, even though the specific activities may remain similar (Even *et al.*, 2001). More recently, the primary role of allosteric regulatory mechanisms in controlling the glycolytic flux of *L. lactis* has been questioned, underlining the predominant regulatory role of the enzymes' concentration. This statement was supported by a new methodology whereby experimental measurements of fluxes and enzyme concentrations can be integrated into flux functions capable of predicting the 'fulsome' from the proteome (Rossel *et al.*, 2011). Nevertheless, by such an approach the understanding of the role of each layer of regulation can only be partially addressed. In this case, the approach of regulation analysis is more informative in delineating which regulatory layer is responsible for establishing fluxes through a given

Concerning the hierarchical (i.e. expression and post-translational modification) regulation of the energetic metabolism of LAB, the little information available are related to the catabolite control protein CcpA, the major regulator of the carbon metabolism in *L. lactis* and

modulation of the energetic metabolism.

enzyme (Gerosa & Sauer, 2011).

other Gram-positive bacteria. CcpA belongs to the LacI/GalR family of bacterial regulator proteins, and the disruption of the *ccpA* gene reduces the carbon catabolite repression (CR) of several genes involved in carbohydrate metabolisms. CcpA-mediated regulation depends basically on three elements: i) a specific *cis*-acting DNA sequence, termed cataboliteresponsive element (*cre*) which is present near the promoter region of genes affected by CR, ii) the HPr protein, a phosphotransferase protein of the PTS system, and iii) the concentration of glycolytic intermediates (such as FBP). A metabolite-activated kinase has been shown to phosphorylate HPr on residue serine 46. This phosphorylated form of HPr [HPr(Ser-P)] interacts with CcpA, and this interaction enhances the binding of CcpA to *cre* on the promoter region of genes, so affecting the level of their expression. Indirectly, the phosphorylation of HPr on serine residue, enhanced by high level of glycolytic intermediates (*e.g.* FBP), reduces the number of HPr molecules that can be phosphorylated on histidine residue so as to ensure the functionality of the PTS system in sugar uptake across the membrane (Figure 1). Besides the role of CcpA in the control of sugar metabolism (mainly the sugar uptake), it was demonstrated that the role of this protein in the transcriptional activation of the glycolytic *las* operon, encoding the enzymes phosphofructokinase, pyruvate kinase and L-lactate dehydrogenase (Luesink *et al.*, 1998). Specifically, the disruption of the *ccpA* gene lowered the activity of pyruvate kinase and Llactate dehydrogenase, resulting in the production of metabolites that are characteristic of a mixed-acid fermentation. It was, therefore, speculated that homolactic fermentation in *L. lactis* is maintained by CcpA-mediated repression of mixed-acid fermentation (Luesink *et al.*, 1998).

The regulatory function of CcpA on the energetic metabolism was further confirmed when its primary role in the regulation of aerobic and respirational growth of *L. lactis* was described (Gaudu *et al.*, 2003). CcpA was found to repress NADH oxidase activity, thus maintaining a correct NADH/NAD+ ratio that directed the metabolism in favour of respiration. Moreover, it was proposed that a CcpA-mediated repression of the heme transportation system thereby prevented the oxidative damage provoked by precocious heme uptake at the start exponential growth. CcpA thus appears to govern a regulatory network that coordinates oxygen, iron and the energetic metabolism.
