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

Optimisation of Cell Bioenergetics in Food-Associated Microorganisms 227

ecological factors that drive the evolution of organisms, the presence in *S. cerevisiae* of two different metabolic strategies for exploiting resources (fermentation and respiration) represents an ecological advantage that has allowed this species to survive under different environmental conditions. The duality of the *S. cerevisiae* metabolism increases the complexity of the regulatory mechanisms interacting with each other to control the energetic

Despite *S. cerevisiae* has been extensively studied with regard to several of its characteristics, little information is available concerning the complexity of the regulatory mechanisms acting on the glycolytic pathway, i.e. the common pathway for fermentative and respiratory metabolism (Figure 3). Glycolysis is a highly conserved pathway from bacteria to yeast and humans, and presumably it has been under intense evolutionary pressure for its robust efficiency. It therefore represents an interesting model for investigating the correlation between the different levels of gene expression. As stated by the central dogma of molecular biology (DNA encodes mRNA and mRNA encodes proteins), a strong correlation was anticipated amongst mRNA concentrations, protein concentrations and metabolic fluxes. However, all attempts to verify these correlations – starting from the data on mRNA and protein levels, enzyme activities and *in vivo* fluxes – were far from perfect. A recent study developed a method to dissect the hierarchical regulation of *S. cerevisiae* glycolysis into contributions by transcription, translation, protein degradation and post-translational modification (Daran-Lapujade *et al.*, 2007). The authors propose the calculation of two coefficients, the hierarchical regulation coefficient *ρ<sup>h</sup>* and the metabolic regulation coefficient *ρm*. *ρ<sup>h</sup>* quantifies to what extent the local flux through the enzyme is regulated by a change in enzyme capacity which is affected by a cascade of gene expression, from transcription to post-translational modification. *ρm* quantifies the relative contribution of changes in the interaction of the enzyme with the rest of the metabolism to the regulation of the enzyme's local flux. While *ρh* can be measurable, *ρm* is calculated assuming that *ρh* + *ρ<sup>m</sup>* = 1. It follows that a reaction that is purely regulated by a cascade of gene expression would have a *ρh* of 1, whereas a reaction that is solely metabolically regulated would have *ρh* of 0 and *ρm* of 1. A study by Daran-Lapujade compared different cultivation conditions in order to compare a fully respiratory metabolism with a fully anaerobic fermentative metabolism. Moreover, the anaerobic fermentative metabolism was studied by increasing the carbon fluxes in glycolysis by adding to the culture the non-metabolisable weak acid benzoic acid. The comparison of the three different cultivation conditions, carried out using a glucose-limited chemostat at the same dilution rate, highlights an increase of carbon fluxes (5- to 11-fold) in anaerobic rather than in aerobic cultures, with a further increase in the presence of benzoic acid. The dissection analysis revealed that in most cases the fluxes resulted from both hierarchical and metabolic regulatory mechanisms (*ρh* between 0.2 and 0.5). Surprisingly, the increase of glycolytic fluxes stimulated by benzoic acid revealed a dominant contribution of metabolic regulation because most of the reactions showed small *ρh* values and *ρm* values which were close to 1 (with the exception of the reactions governed by phosphofructokinase, fructosebisphosphate aldolase, triose-phosphate isomerase and pyruvate kinase) (Daran-Lapujade, 2007). A further dissection approach was useful for analysing the contribution of transcription, mRNA degradation, translation, protein degradation or post-translational modification, to the hierarchical regulation of enzymes' capacities. The main conclusion was that fluxes through glycolytic enzymes were only marginally regulated by mRNA levels,

metabolism under different environmental stimuli.

Fig. 3. Simplified representation of the glycolysis, alcoholic fermentation and respiration metabolism in *S. cerevisiae*. Black arrows show the metabolic fluxes. 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).

A fundamental characteristic of *S. cerevisiae* is the ability to ferment sugars, even in the presence of oxygen in aerobic conditions. This phenomenon is called the Crabtree effect, in honour of Herbert Grace Crabtree who first described the reversible switch between the glycolytic and oxidative metabolism in some cancer cells (Crabtree 1929). In more general terms, the duality of the *S. cerevisiae* metabolism allows this microorganism to use two different strategies for exploiting resources: the 'selfish' strategy and the 'cooperative' strategy. According to the 'selfish' strategy the individuals quickly consume resources and increase their own reproduction rate, whilst according to the 'cooperative' strategy the individuals exploit resources slowly but efficiently. A high rate of ATP production per unit of time is associated with a high reproduction rate and is considered to be a 'selfish' strategy (e.g., fermentation), whereas a high yield of ATP production (the number of units of ATP per unit of resource consumed) is associated with a low reproduction rate but with high biomass production, and is therefore considered to be a 'cooperative' strategy (e.g., respiration) (Pfeiffer, 2001). Given that, resource supply is one of the most important

Fig. 3. Simplified representation of the glycolysis, alcoholic fermentation and respiration metabolism in *S. cerevisiae*. Black arrows show the metabolic fluxes. Glucose-6-phosphate (G6P), dihydroxyacetone phosphate (DHA-P), glyceraldehyde-3-phosphate (GA3P), 1,3 biphosphoglycerate (1-3PGA), 3-phosphoglycerate (3PGA), 2-phosphoglycerate (2PGA)

A fundamental characteristic of *S. cerevisiae* is the ability to ferment sugars, even in the presence of oxygen in aerobic conditions. This phenomenon is called the Crabtree effect, in honour of Herbert Grace Crabtree who first described the reversible switch between the glycolytic and oxidative metabolism in some cancer cells (Crabtree 1929). In more general terms, the duality of the *S. cerevisiae* metabolism allows this microorganism to use two different strategies for exploiting resources: the 'selfish' strategy and the 'cooperative' strategy. According to the 'selfish' strategy the individuals quickly consume resources and increase their own reproduction rate, whilst according to the 'cooperative' strategy the individuals exploit resources slowly but efficiently. A high rate of ATP production per unit of time is associated with a high reproduction rate and is considered to be a 'selfish' strategy (e.g., fermentation), whereas a high yield of ATP production (the number of units of ATP per unit of resource consumed) is associated with a low reproduction rate but with high biomass production, and is therefore considered to be a 'cooperative' strategy (e.g., respiration) (Pfeiffer, 2001). Given that, resource supply is one of the most important

phosphoenolpyruvate (PEP), pyruvate (PYR).

ecological factors that drive the evolution of organisms, the presence in *S. cerevisiae* of two different metabolic strategies for exploiting resources (fermentation and respiration) represents an ecological advantage that has allowed this species to survive under different environmental conditions. The duality of the *S. cerevisiae* metabolism increases the complexity of the regulatory mechanisms interacting with each other to control the energetic metabolism under different environmental stimuli.

Despite *S. cerevisiae* has been extensively studied with regard to several of its characteristics, little information is available concerning the complexity of the regulatory mechanisms acting on the glycolytic pathway, i.e. the common pathway for fermentative and respiratory metabolism (Figure 3). Glycolysis is a highly conserved pathway from bacteria to yeast and humans, and presumably it has been under intense evolutionary pressure for its robust efficiency. It therefore represents an interesting model for investigating the correlation between the different levels of gene expression. As stated by the central dogma of molecular biology (DNA encodes mRNA and mRNA encodes proteins), a strong correlation was anticipated amongst mRNA concentrations, protein concentrations and metabolic fluxes. However, all attempts to verify these correlations – starting from the data on mRNA and protein levels, enzyme activities and *in vivo* fluxes – were far from perfect. A recent study developed a method to dissect the hierarchical regulation of *S. cerevisiae* glycolysis into contributions by transcription, translation, protein degradation and post-translational modification (Daran-Lapujade *et al.*, 2007). The authors propose the calculation of two coefficients, the hierarchical regulation coefficient *ρ<sup>h</sup>* and the metabolic regulation coefficient *ρm*. *ρ<sup>h</sup>* quantifies to what extent the local flux through the enzyme is regulated by a change in enzyme capacity which is affected by a cascade of gene expression, from transcription to post-translational modification. *ρm* quantifies the relative contribution of changes in the interaction of the enzyme with the rest of the metabolism to the regulation of the enzyme's local flux. While *ρh* can be measurable, *ρm* is calculated assuming that *ρh* + *ρ<sup>m</sup>* = 1. It follows that a reaction that is purely regulated by a cascade of gene expression would have a *ρh* of 1, whereas a reaction that is solely metabolically regulated would have *ρh* of 0 and *ρm* of 1. A study by Daran-Lapujade compared different cultivation conditions in order to compare a fully respiratory metabolism with a fully anaerobic fermentative metabolism. Moreover, the anaerobic fermentative metabolism was studied by increasing the carbon fluxes in glycolysis by adding to the culture the non-metabolisable weak acid benzoic acid. The comparison of the three different cultivation conditions, carried out using a glucose-limited chemostat at the same dilution rate, highlights an increase of carbon fluxes (5- to 11-fold) in anaerobic rather than in aerobic cultures, with a further increase in the presence of benzoic acid. The dissection analysis revealed that in most cases the fluxes resulted from both hierarchical and metabolic regulatory mechanisms (*ρh* between 0.2 and 0.5). Surprisingly, the increase of glycolytic fluxes stimulated by benzoic acid revealed a dominant contribution of metabolic regulation because most of the reactions showed small *ρh* values and *ρm* values which were close to 1 (with the exception of the reactions governed by phosphofructokinase, fructosebisphosphate aldolase, triose-phosphate isomerase and pyruvate kinase) (Daran-Lapujade, 2007). A further dissection approach was useful for analysing the contribution of transcription, mRNA degradation, translation, protein degradation or post-translational modification, to the hierarchical regulation of enzymes' capacities. The main conclusion was that fluxes through glycolytic enzymes were only marginally regulated by mRNA levels,

Optimisation of Cell Bioenergetics in Food-Associated Microorganisms 229

through lactic acid production as the final product of their energetic metabolism. It follows that the growth of LAB determines a significant change in the environmental chemical composition, with a progressive decrease in sugar concentration and a simultaneous increase in lactic acid concentration thereby determining a decrease in environmental pH. Consequently, LAB during each fermentation process are faced with 'predictable' environmental changes, ending with the cessation of growth due to carbon source starvation and, mostly, with an environmental pH which is incompatible with the metabolic processes of the microorganism. The exposure to low pH for long period times determines the arresting of growth and a dramatic decrease of glycolytic fluxes, structural damage to the cell membrane and macromolecules (such as DNA and proteins), and a progressive loss in viability. Weak acids, such as lactic acid, have potent antimicrobial activity because the undissociated forms of weak acids pass freely through the cell membrane. Since the cytoplasmic pH is generally higher than that of the growth medium, inside the cell the weak acid dissociates by releasing a proton and leading to the acidification of the cytoplasm. Due to environmental constraints, LAB have developed through evolution a 'make-accumulateconsume' metabolic strategy (Pfeiffer *et al.*, 2001; Rozpedowska *et al.*, 2011) in order to have a faster sugar consumption, lactic acid production and accumulation. This strategy is aimed at rapidly monopolising sugars and creating an unfavourable environment so as to outcompete other microorganisms by the rapid secretion of fermentation products. In order to survive themselves, LAB have therefore developed a series of mechanisms to counteract low environmental pH and the negative effects of weak organic acids produced by their own metabolism. Several of these mechanisms have been extensively studied (Cotter & Hill, 2003) to understand how LAB protect themselves from the challenge posed by low-pH environments, such as food and gastric juice, and how they develop the strategies by which they can be aided or impeded. Nevertheless, the role of these mechanisms in the regulation of the energetic metabolism was barely investigated even though the loss of the activity of the relatively acid-sensitive glycolytic enzymes (which severely affects the ability to produce ATP) was well known. Indeed, even if LAB species are acid-tolerant bacteria, they cannot be considered to be acidotrophic, and the optimum pH of the highly conserved glycolytic

enzymes is close to neutral-alkaline values (Hutkins & Nannen, 1993).

Fig. 4. Reaction catalysed by urease (EC 3.5.1.5) and the spontaneity of carbamate in

ammonia and carbonic acid.

whereas most of the observed gene-expression regulation was exerted at the level of protein synthesis and/or degradation and the post-translational level. It was, therefore, speculated that in *S. cerevisiae*, the whole glycolytic regulation is an interplay of purely hierarchical regulation (*ρh* close to 1), purely metabolic regulation (*ρm* close to 1), cooperative regulation (*ρm* and *ρm* between 0 and 1) and antagonistic regulation (both *ρh* and *ρm* negative). The nature and the role of post-translational modification, which appeared to be relevant in the control of glycolytic fluxes, has not yet been investigated systematically for all glycolytic enzymes, even though phosphorylation seems to be the predominant mechanism of protein modification.

The ability of *S. cerevisiae* to switch from respiratory to fermentative metabolism is an important characteristic in the evolutionary and ecological context and for many of its industrial applications. In the natural – evolutionary – context, this ability may have helped this organism to quickly recover sugars and create a hostile environment for competing microorganisms. Concerning the industrial application of *S. cerevisiae*, yeast biomass starved of glucose during storage must rapidly adapt to a high sugar concentration when it is added to bread dough or wort. As has been reported, the shift from respiratory to fermentative metabolism resulted in a rapidly increase of the yeast glycolytic flux in order to compensate the differences in the ATP yield of the two metabolisms. The dynamics of glycolytic regulation during the adaptation of *S. cerevisiae* to fermentative metabolism have been investigated with the aim of understanding the time-dependent, multilevel regulation of glycolytic enzymes during the metabolic switch just described (van de Brink *et al.*, 2008). It was reported that within 45 min of the switch from respiratory to fermentative metabolism, the glycolytic flux increases eightfold without any changes in the glycolytic enzymes' capacities, thereby highlighting an increase of the enzymes activities via metabolic regulation (i.e. the regulation of activities by interaction with low-molecular-weight substrates, products and effectors). By prolonging the incubation during the fermentative metabolism under anaerobic, glucose-excessive conditions, a hierarchical regulation of enzymes was also observed. Specifically, the capacity of the kinases of the upper part of the glycolysis remained unaffected, whereas the enzymes' capacities of the lower part of the glycolysis increased, establishing a new homeostasis of glycolytic metabolites. The delay of the transcriptional regulation compared to the metabolic regulation of glycolytic enzymes observed after the metabolic switch was ascribed to the dramatic change in the rate of ATP production. While the glucose consumption rate increased more than 12-fold during the 2 hours after the switch, the rate of ATP decreased during the first 15 minutes as a result of the reduced ATP yield under fermentative conditions. It was, therefore, speculated that cells energy levels influence the induction of the enzymatic capacity in glycolysis. Due to the fact that an increased level of glycolytic enzymes was only observed 45 minutes after the metabolic switch, and given that the majority of the relevant transcripts were induced after 10 minutes, the step was severely affected by the cellular energetic status which was identified in the translation machinery.

#### **4. Alkalising reactions and cell bioenergetics**

Food associated bacteria, and in particular LAB, have been selected and used by humans in several food processes because of their ability to acidify milk or vegetables in order to obtain a more stable and safer food products. Acidification occurs in homofermentative LAB

whereas most of the observed gene-expression regulation was exerted at the level of protein synthesis and/or degradation and the post-translational level. It was, therefore, speculated that in *S. cerevisiae*, the whole glycolytic regulation is an interplay of purely hierarchical regulation (*ρh* close to 1), purely metabolic regulation (*ρm* close to 1), cooperative regulation (*ρm* and *ρm* between 0 and 1) and antagonistic regulation (both *ρh* and *ρm* negative). The nature and the role of post-translational modification, which appeared to be relevant in the control of glycolytic fluxes, has not yet been investigated systematically for all glycolytic enzymes, even though phosphorylation seems to be the predominant mechanism of protein

The ability of *S. cerevisiae* to switch from respiratory to fermentative metabolism is an important characteristic in the evolutionary and ecological context and for many of its industrial applications. In the natural – evolutionary – context, this ability may have helped this organism to quickly recover sugars and create a hostile environment for competing microorganisms. Concerning the industrial application of *S. cerevisiae*, yeast biomass starved of glucose during storage must rapidly adapt to a high sugar concentration when it is added to bread dough or wort. As has been reported, the shift from respiratory to fermentative metabolism resulted in a rapidly increase of the yeast glycolytic flux in order to compensate the differences in the ATP yield of the two metabolisms. The dynamics of glycolytic regulation during the adaptation of *S. cerevisiae* to fermentative metabolism have been investigated with the aim of understanding the time-dependent, multilevel regulation of glycolytic enzymes during the metabolic switch just described (van de Brink *et al.*, 2008). It was reported that within 45 min of the switch from respiratory to fermentative metabolism, the glycolytic flux increases eightfold without any changes in the glycolytic enzymes' capacities, thereby highlighting an increase of the enzymes activities via metabolic regulation (i.e. the regulation of activities by interaction with low-molecular-weight substrates, products and effectors). By prolonging the incubation during the fermentative metabolism under anaerobic, glucose-excessive conditions, a hierarchical regulation of enzymes was also observed. Specifically, the capacity of the kinases of the upper part of the glycolysis remained unaffected, whereas the enzymes' capacities of the lower part of the glycolysis increased, establishing a new homeostasis of glycolytic metabolites. The delay of the transcriptional regulation compared to the metabolic regulation of glycolytic enzymes observed after the metabolic switch was ascribed to the dramatic change in the rate of ATP production. While the glucose consumption rate increased more than 12-fold during the 2 hours after the switch, the rate of ATP decreased during the first 15 minutes as a result of the reduced ATP yield under fermentative conditions. It was, therefore, speculated that cells energy levels influence the induction of the enzymatic capacity in glycolysis. Due to the fact that an increased level of glycolytic enzymes was only observed 45 minutes after the metabolic switch, and given that the majority of the relevant transcripts were induced after 10 minutes, the step was severely affected by the cellular energetic status which was

Food associated bacteria, and in particular LAB, have been selected and used by humans in several food processes because of their ability to acidify milk or vegetables in order to obtain a more stable and safer food products. Acidification occurs in homofermentative LAB

modification.

identified in the translation machinery.

**4. Alkalising reactions and cell bioenergetics** 

through lactic acid production as the final product of their energetic metabolism. It follows that the growth of LAB determines a significant change in the environmental chemical composition, with a progressive decrease in sugar concentration and a simultaneous increase in lactic acid concentration thereby determining a decrease in environmental pH. Consequently, LAB during each fermentation process are faced with 'predictable' environmental changes, ending with the cessation of growth due to carbon source starvation and, mostly, with an environmental pH which is incompatible with the metabolic processes of the microorganism. The exposure to low pH for long period times determines the arresting of growth and a dramatic decrease of glycolytic fluxes, structural damage to the cell membrane and macromolecules (such as DNA and proteins), and a progressive loss in viability. Weak acids, such as lactic acid, have potent antimicrobial activity because the undissociated forms of weak acids pass freely through the cell membrane. Since the cytoplasmic pH is generally higher than that of the growth medium, inside the cell the weak acid dissociates by releasing a proton and leading to the acidification of the cytoplasm. Due to environmental constraints, LAB have developed through evolution a 'make-accumulateconsume' metabolic strategy (Pfeiffer *et al.*, 2001; Rozpedowska *et al.*, 2011) in order to have a faster sugar consumption, lactic acid production and accumulation. This strategy is aimed at rapidly monopolising sugars and creating an unfavourable environment so as to outcompete other microorganisms by the rapid secretion of fermentation products. In order to survive themselves, LAB have therefore developed a series of mechanisms to counteract low environmental pH and the negative effects of weak organic acids produced by their own metabolism. Several of these mechanisms have been extensively studied (Cotter & Hill, 2003) to understand how LAB protect themselves from the challenge posed by low-pH environments, such as food and gastric juice, and how they develop the strategies by which they can be aided or impeded. Nevertheless, the role of these mechanisms in the regulation of the energetic metabolism was barely investigated even though the loss of the activity of the relatively acid-sensitive glycolytic enzymes (which severely affects the ability to produce ATP) was well known. Indeed, even if LAB species are acid-tolerant bacteria, they cannot be considered to be acidotrophic, and the optimum pH of the highly conserved glycolytic enzymes is close to neutral-alkaline values (Hutkins & Nannen, 1993).

Fig. 4. Reaction catalysed by urease (EC 3.5.1.5) and the spontaneity of carbamate in ammonia and carbonic acid.

Optimisation of Cell Bioenergetics in Food-Associated Microorganisms 231

are exposed to an increasingly acidic intracellular environment and must maintain cell energy homeostasis when the pHout and pHin decrease as a result of lactic acid production. Urease biogenesis is only important when the cells are actively growing, since it increases the fermentative capacity of *S. thermophilus* and leads to rapid growth and an increased acidification rate in milk (i.e. urease favour a cytoplasmic background suitable for a 'makeaccumulate-consume' strategy). If we consider that energetic limitation is an important factor for organisms in their natural environment, we then expect that the properties of ATP-production pathways have been under strong selection during evolution (Pfeiffer *et al.*, 2001). Similarly, the regulatory mechanisms which act in optimising the efficiency of the ATP-production pathway should be under the same evolutionary selection. In this context, it is notable that eleven genes are necessary in order for the maintenance of an active urease, which accounts for 0.9% of the estimated core genome of *S. thermophilus*. This enzyme has been found in all the previously characterised *S. thermophilus* strains, and urease-negative mutants are not common in nature. The *S. thermophilus* genome has mainly evolved following divergent evolution from the phylogenetically related pathogenic streptococci bacteria. Loss-of-function mutations, counterbalanced by the acquisition of relevant traits (e.g. lactose utilisation) have resulted in a *S. thermophilus* genome that is well-adapted for dairy colonisation (Bolotin *et al.*, 2004). Because urease is not common in pathogenic streptococci (Mora *et al.*, 2005), its acquisition and maintenance within the *S. thermophilus* genome is likely to be dependent upon its contribution to the environmental fitness of this microorganism when linked to the environmental availability of urea. Urea is the major nitrogenous waste product of most terrestrial animals. Urea is produced in the liver, carried in the bloodstream to the kidneys and excreted in urine. Urea is also present in milk and in the secretions of the major and minor exocrine glands at concentrations approximately equivalent to serum, so a large proportion of circulating urea is translocated onto epithelial surfaces by secretory systems or else in tissue exudates. In this context, it is not surprisingly that urease is present in a high number of human pathogenic bacteria and represents an

important factor in infection and disease (Burne and Chen, 2000; Mora *et al.*, 2005).

benefits from the local transient increase of pH (Arioli *et al*., 2010).

Since the activity of the bioenergetic machinery is modulated by the intracellular pH, the mechanism of metabolism regulation in other urease-positive bacteria, including human pathogens, should be further analysed. All of the metabolic reactions that result in the alkalisation of the cytosol of acidogenic organisms (such as those involved in the arginine deiminase (ADI) pathway, the citrate metabolism or else those involved in malolactic conversion) should be analysed in light of these novel findings. Indeed, and not surprisingly, all previous pathways act by subtracting protons from the cytoplasm and are strongly induced by an acidic environmental pH (Magni *et al.*, 1999; Cotter & Hill, 2003; Broadbent *et al*., 2010). The conserved role of alkalising reactions across acidogenic bacteria is also supported by the data obtained for *L. lactis* IL1403-945 and *S. pneumoniae* SP292-945 in the presence of glucose and cellobiose as a carbon source (Arioli *et al.*, 2010). In both cases, the rate of ATP produced during the sugar catabolism was increased, alkalising with the ammonia the cytoplasm. Besides the selfish utility of urease for cells harbouring this enzymatic activity, the cooperative behaviour of urease in an ecological context in which different microbial species share the same environment was also underlined. Urea hydrolysis results in a rise of both pHin and pHout due to the rapid diffusion of ammonia outside the cell. It follows that in the presence of urea and a urease-positive microorganism, (or a urease-negative microorganism) sharing the same micro-environment, there will be

Fig. 5. Simplified representation of glycolysis, homolactic fermentation and urease activity in *S. thermophilus*. The inset represents the raw isothermal titration calorimetry data (heat flux versus time) of *S. thermophilus* lactose metabolism either alone (blue line) or in the presence of ammonia (green line) or urea (red line) (for the detailed experimental procedure see Arioli *et al.*, 2010).

Quite recently, the urease activity – an enzymatic reaction known as a stress response to counteract environmental acidic pH in several bacteria – has been described as a metabolic regulatory mechanism of the energetic metabolism in the dairy bacterium *Streptococcus thermophilus* (Arioli *et al.*, 2010). Urease is a multi-subunit urea amidohydrolase (EC 3.5.1.5) that catalyses the hydrolysis of urea to yield ammonia and carbamate, which spontaneously decomposes to yield a second molecule of ammonia and carbonic acid (Figure 4). The released carbonic acid and the two molecules of ammonia are in equilibrium with their deprotonated and protonated forms respectively, and the net effect of these reactions is an increase in intracellular (pHin) and extracellular (pHout) pH (Figure 5). Urea hydrolysis increases the catabolic efficiency of *S. thermophilus* by modulating the intracellular pH and thereby increasing the activity of β-galactosidase, glycolytic enzymes, and lactate dehydrogenase. Moreover, urease increases the overall change in enthalpy generated by the microbial metabolism as a consequence of an increased glycolytic flux (Figure 5).

In light of these considerations, urease activity – which is stimulated when environmental pH is weakly acidic (pH 5.8-6) (Mora *et al.,* 2005) – should be considered as a regulatory system that has evolved to optimise the activity of the glycolytic enzymes. These enzymes

Fig. 5. Simplified representation of glycolysis, homolactic fermentation and urease activity in *S. thermophilus*. The inset represents the raw isothermal titration calorimetry data (heat flux versus time) of *S. thermophilus* lactose metabolism either alone (blue line) or in the presence of ammonia (green line) or urea (red line) (for the detailed experimental procedure

Quite recently, the urease activity – an enzymatic reaction known as a stress response to counteract environmental acidic pH in several bacteria – has been described as a metabolic regulatory mechanism of the energetic metabolism in the dairy bacterium *Streptococcus thermophilus* (Arioli *et al.*, 2010). Urease is a multi-subunit urea amidohydrolase (EC 3.5.1.5) that catalyses the hydrolysis of urea to yield ammonia and carbamate, which spontaneously decomposes to yield a second molecule of ammonia and carbonic acid (Figure 4). The released carbonic acid and the two molecules of ammonia are in equilibrium with their deprotonated and protonated forms respectively, and the net effect of these reactions is an increase in intracellular (pHin) and extracellular (pHout) pH (Figure 5). Urea hydrolysis increases the catabolic efficiency of *S. thermophilus* by modulating the intracellular pH and thereby increasing the activity of β-galactosidase, glycolytic enzymes, and lactate dehydrogenase. Moreover, urease increases the overall change in enthalpy generated by the

microbial metabolism as a consequence of an increased glycolytic flux (Figure 5).

In light of these considerations, urease activity – which is stimulated when environmental pH is weakly acidic (pH 5.8-6) (Mora *et al.,* 2005) – should be considered as a regulatory system that has evolved to optimise the activity of the glycolytic enzymes. These enzymes

see Arioli *et al.*, 2010).

are exposed to an increasingly acidic intracellular environment and must maintain cell energy homeostasis when the pHout and pHin decrease as a result of lactic acid production. Urease biogenesis is only important when the cells are actively growing, since it increases the fermentative capacity of *S. thermophilus* and leads to rapid growth and an increased acidification rate in milk (i.e. urease favour a cytoplasmic background suitable for a 'makeaccumulate-consume' strategy). If we consider that energetic limitation is an important factor for organisms in their natural environment, we then expect that the properties of ATP-production pathways have been under strong selection during evolution (Pfeiffer *et al.*, 2001). Similarly, the regulatory mechanisms which act in optimising the efficiency of the ATP-production pathway should be under the same evolutionary selection. In this context, it is notable that eleven genes are necessary in order for the maintenance of an active urease, which accounts for 0.9% of the estimated core genome of *S. thermophilus*. This enzyme has been found in all the previously characterised *S. thermophilus* strains, and urease-negative mutants are not common in nature. The *S. thermophilus* genome has mainly evolved following divergent evolution from the phylogenetically related pathogenic streptococci bacteria. Loss-of-function mutations, counterbalanced by the acquisition of relevant traits (e.g. lactose utilisation) have resulted in a *S. thermophilus* genome that is well-adapted for dairy colonisation (Bolotin *et al.*, 2004). Because urease is not common in pathogenic streptococci (Mora *et al.*, 2005), its acquisition and maintenance within the *S. thermophilus* genome is likely to be dependent upon its contribution to the environmental fitness of this microorganism when linked to the environmental availability of urea. Urea is the major nitrogenous waste product of most terrestrial animals. Urea is produced in the liver, carried in the bloodstream to the kidneys and excreted in urine. Urea is also present in milk and in the secretions of the major and minor exocrine glands at concentrations approximately equivalent to serum, so a large proportion of circulating urea is translocated onto epithelial surfaces by secretory systems or else in tissue exudates. In this context, it is not surprisingly that urease is present in a high number of human pathogenic bacteria and represents an important factor in infection and disease (Burne and Chen, 2000; Mora *et al.*, 2005).

Since the activity of the bioenergetic machinery is modulated by the intracellular pH, the mechanism of metabolism regulation in other urease-positive bacteria, including human pathogens, should be further analysed. All of the metabolic reactions that result in the alkalisation of the cytosol of acidogenic organisms (such as those involved in the arginine deiminase (ADI) pathway, the citrate metabolism or else those involved in malolactic conversion) should be analysed in light of these novel findings. Indeed, and not surprisingly, all previous pathways act by subtracting protons from the cytoplasm and are strongly induced by an acidic environmental pH (Magni *et al.*, 1999; Cotter & Hill, 2003; Broadbent *et al*., 2010). The conserved role of alkalising reactions across acidogenic bacteria is also supported by the data obtained for *L. lactis* IL1403-945 and *S. pneumoniae* SP292-945 in the presence of glucose and cellobiose as a carbon source (Arioli *et al.*, 2010). In both cases, the rate of ATP produced during the sugar catabolism was increased, alkalising with the ammonia the cytoplasm. Besides the selfish utility of urease for cells harbouring this enzymatic activity, the cooperative behaviour of urease in an ecological context in which different microbial species share the same environment was also underlined. Urea hydrolysis results in a rise of both pHin and pHout due to the rapid diffusion of ammonia outside the cell. It follows that in the presence of urea and a urease-positive microorganism, (or a urease-negative microorganism) sharing the same micro-environment, there will be benefits from the local transient increase of pH (Arioli *et al*., 2010).

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#### **5. Conclusions and perspectives**

The regulation and control of metabolic fluxes in microbes is based on our knowledge of regulatory networks topology, on input-output regulatory logics and metabolic feedback, and on the quantitative effect of control exerted by regulation events. No less important is our understanding of how metabolic regulatory circuits have evolved and what the significance of the impact of environmental constraints on the regulatory configuration will be. It has recently been described that microbes can 'learn' form exposure to a series of new environmental changes and rearrange some regulatory networks so as to predict the new environmental stimuli (Mitchell *et al.*, 2009). The ecological forces and the molecular mechanisms that govern this ability are not clear but it is evident that the regulatory networks that link environmental stimuli to microbial responses are complex and can evolve rapidly (Cooper, 2009). The origin of the adaptability of regulatory networks could be ascribed to microbial cell individuality and the underlying sources of heterogeneity. This heterogeneity is related to stochastic fluctuations in transcription or translation, despite a genetically homogeneous background and constant environmental conditions. Heterogeneity at single-cell level is typically masked in conventional studies of microbial populations, which are based on the average behaviour of thousands or millions of cells, but it has the potential to create variant subpopulations better equipped to persist during environmental perturbation (Avery, 2006). In other words, a population might enhance its fitness by allowing individual cells to make a stochastically transition amongst multiple phenotypes, thus ensuring that some cells are always prepared for erratic, unpredictable environmental fluctuations. It can be therefore be concluded that the regulatory mechanisms that act in the optimisation of the bioenergetics of food-associated bacteria should be analyzed by always taking into consideration the 'predictable' succession of environmental stimuli that have driven their domesticated speciation and evolution.

#### **6. References**


The regulation and control of metabolic fluxes in microbes is based on our knowledge of regulatory networks topology, on input-output regulatory logics and metabolic feedback, and on the quantitative effect of control exerted by regulation events. No less important is our understanding of how metabolic regulatory circuits have evolved and what the significance of the impact of environmental constraints on the regulatory configuration will be. It has recently been described that microbes can 'learn' form exposure to a series of new environmental changes and rearrange some regulatory networks so as to predict the new environmental stimuli (Mitchell *et al.*, 2009). The ecological forces and the molecular mechanisms that govern this ability are not clear but it is evident that the regulatory networks that link environmental stimuli to microbial responses are complex and can evolve rapidly (Cooper, 2009). The origin of the adaptability of regulatory networks could be ascribed to microbial cell individuality and the underlying sources of heterogeneity. This heterogeneity is related to stochastic fluctuations in transcription or translation, despite a genetically homogeneous background and constant environmental conditions. Heterogeneity at single-cell level is typically masked in conventional studies of microbial populations, which are based on the average behaviour of thousands or millions of cells, but it has the potential to create variant subpopulations better equipped to persist during environmental perturbation (Avery, 2006). In other words, a population might enhance its fitness by allowing individual cells to make a stochastically transition amongst multiple phenotypes, thus ensuring that some cells are always prepared for erratic, unpredictable environmental fluctuations. It can be therefore be concluded that the regulatory mechanisms that act in the optimisation of the bioenergetics of food-associated bacteria should be analyzed by always taking into consideration the 'predictable' succession of environmental

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**5. Conclusions and perspectives** 

**6. References** 


**Part 3** 

**New Techniques and Findings** 

**in Bioenergetics Research** 

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