The diversion of lactose carbon through the tagatose pathway reduces the intracellular fructose 1,6-bisphosphate and growth rate of Streptococcus bovis

Bond, Daniel R.; Tsai, B. M.; Russell, James B.

doi:10.1007/s002530051220

Cited by 22 publications

(12 citation statements)

References 15 publications

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“…In order to determine the maximum rates of sugar catabolism, monensin (0.5 M), a K ϩ -Na ϩ ionophore previously used by Bond et al (5), was added to exponentially growing cultures. In the case of Streptococcus bovis, this ionophore led to an increase in glycolytic flux when catabolic capacity was not limiting to compensate for ATP wastage (5).…”

Section: Resultsmentioning

confidence: 99%

Molecular Physiology of Sugar Catabolism in Lactococcus lactis IL1403

2001

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The metabolic characteristics of Lactococcus lactis IL1403 were examined on two different growth media with respect to the physiological response to two sugars, glucose and galactose. Analysis of specific metabolic rates indicated that despite significant variations in the rates of both growth and sugar consumption, homolactic fermentation was maintained for all cultures due to the low concentration of either pyruvate-formate lyase or alcohol dehydrogenase. When the ionophore monensin was added to the medium, flux through glycolysis was not increased, suggesting a catabolic flux limitation, which, with the low intracellular concentrations of glycolytic intermediates and high in vivo glycolytic enzyme capacities, may be at the level of sugar transport. To assess transcription, a novel DNA macroarray technology employed RNA labeled in vitro with digoxigenin and detection of hybrids with an alkaline phosphatase-antidigoxigenin conjugate. This method showed that several genes of glycolysis were expressed to higher levels on glucose and that the genes of the mixed-acid pathway were expressed to higher levels on galactose. When rates of enzyme synthesis are compared to transcript concentrations, it can be deduced that some translational regulation occurs with threefold-higher translational efficiency in cells grown on glucose.Lactococcus lactis is generally recognized as the model organism for the study of lactic acid bacteria, and the complete genome sequence of the IL1403 strain (3, 4) will undoubtedly consolidate this position. When growing on rapidly metabolized sugars, this species shows homolactic metabolism in which more than 90% of metabolized sugar is converted to lactic acid. However, under certain conditions, this homolactic metabolism is replaced by a shift in pyruvate metabolism towards alternative fermentation pathways. Under anaerobic conditions, this shift involves an increased flux through the pyruvate-formate lyase reaction and leads to mixed acid fermentation and the accumulation of formate, acetate, and ethanol. These products accumulate in only low quantities during homolactic metabolism but have been shown to account for the majority of carbon flux under circumstances in which the rate of glycolysis of sugars is significantly diminished.This shift in pyruvate metabolism has been correlated with the NADH/NAD ratio (11). When flux through glycolysis is high, the high NADH/NAD ratio favors lactate dehydrogenase activity and provokes the inhibition of glyceraldehyde-3P dehydrogenase activity upstream of pyruvate. Under such conditions, metabolite pools upstream of glyceraldehyde-3P dehydrogenase (notably triose-Ps) increase within the cell, due to the controlling influence of this enzyme on glycolytic flux (10). Triose-Ps have a negative allosteric effect on pyruvate-formate lyase activity, thereby greatly diminishing the in vivo activity of the enzyme. Sugars metabolized at diminished rates lead to the relaxation of this coordinated control and facilitate the shift towards the more energetically fav...

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Section: Resultsmentioning

confidence: 99%

Molecular Physiology of Sugar Catabolism in Lactococcus lactis IL1403

2001

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“…When 12U1 p was grown on lactose, the FBP pool was much smaller than when grown on glucose. This is probably because lactose is more slowly utilized by S. bovis than glucose, and in addition, the galactose moiety of lactose is fermented by the tagatose pathway (Bond et al, 1998 …”

Section: Effect Of Fba Overexpression On the Intracellular Concentratmentioning

confidence: 99%

“…The concentrated samples were analyzed for glycolytic intermediates by the enzymatic methods reported by Garrigues et al (1997). Intracellular concentrations were calculated on the basis of the data reported for S. bovis that 1 mg of cellular protein represents 4.3 ml of cytoplasmic fluid (Bond et al, 1998).…”

Section: Methodsmentioning

confidence: 99%

“…Cellular nitrogen (N) was determined by digesting cells by the Kjeldahl method, followed by the quantitation of ammonia by the indophenol method (Chaney and Marbach, 1962). Cellular protein was determined according to Bond et al (1998). Briefly, cells were boiled in 0.2 M NaOH for 10 min, and then protein was determined by the Lowry method (Lowry et al, 1951).…”

Section: Methodsmentioning

confidence: 99%

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Effects of the overexpression of fructose-1,6-bisphosphate aldolase on fermentation pattern and transcription of the genes encoding lactate dehydrogenase and pyruvate formate-lyase in a ruminal bacterium, Streptococcus bovis

Asanuma

Yoshii

Kikuchi

et al. 2004

J. Gen. Appl. Microbiol.

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“…The concentrated samples were analyzed for DHAP and GAP by the enzymatic method reported by Garrigues et al (9). The concentrations of intracellular DHAP and GAP were calculated by assuming a ratio of 4.3 l of cytoplasmic fluid to 1 mg of cellular protein in S. bovis (5).…”

Section: Methodsmentioning

confidence: 99%

Effects of pH and Energy Supply on Activity and Amount of Pyruvate Formate-Lyase in Streptococcus bovis

Asanuma

Hino

2000

Appl Environ Microbiol

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The enzyme system of pyruvate formate-lyase (PFL) in Streptococcus bovis was investigated by isolating PFL and PFL-activating enzyme (PFL-AE) from S. bovis, flavodoxin from Escherichia coli, and chloroplasts from spinach. In this study, the PFL and PFL-AE in S. bovis were found to be similar to those in E. coli, suggesting that the activating mechanisms are similar. The optimal pH of S. bovis PFL was 7.5, which is in contrast to the optimal pH of S. bovis lactate dehydrogenase, which is 5.5. The apparent K m of S. bovis PFL was 2 mM. The intermediates of glycolysis, dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (GAP), were shown to inhibit PFL activity. The concentrations of intracellular DHAP and GAP in S. bovis ranged from 1.9 mM to less than 0.1 mM and from 0.6 mM to less than 0.05 mM, respectively, depending on the energy supply. The wide variations in DHAP and GAP levels indicated that PFL activity is allosterically regulated by these triose phosphates in vivo. The amount of PFL protein, as determined by Western blot analysis with polyclonal antibody, changed in parallel with the level of pfl-mRNA, responding to the culture conditions. These observations confirm that PFL synthesis is regulated at the transcriptional level and support the hypothesis that S. bovis shifts the fermentation pathway from acetate, formate, and ethanol production to lactate production when the pH is low and when excess energy is supplied.Streptococcus bovis is one of the prevailing microbes in the rumen, especially in animals receiving a high-starch diet. Its primary fermentation product is lactate (16,19). In this organism, pyruvate is either converted to lactate by lactate dehydrogenase (LDH) or converted to acetyl coenzyme A and formate by pyruvate formate-lyase (PFL) (19) and the acetyl coenzyme A is then converted either to acetate or ethanol. The percentages of lactate in fermentation products differ under different growth conditions, and the percentage is determined by the relative activities of LDH and PFL (2,4,19).We previously reported that S. bovis increases the percentage of lactate by increasing the amount of LDH in response to low pH and an excess energy supply (2-4) and that LDH synthesis is regulated at the transcriptional level (3, 4). The increase in lactate production at low pH is explained in part by the fact that the optimal pH of S. bovis LDH is 5.5, which is similar to the intracellular pH when the extracellular pH is 4.7 (2, 19). The enhanced lactate production in the presence of an excess energy supply can be explained in part by the activation of LDH by an increased concentration of intracellular fructose-1,6-bisphosphate (2, 19). Thus, lactate production by S. bovis is regulated by the ultimate activity of LDH, which includes the amount of enzyme protein and the function of the enzyme.When the pH was low and the energy supply was in excess, the level of pfl-mRNA decreased, while the level of ldh-mRNA increased (4). The activity of PFL decreased with a decrease in the level of pfl-mRNA...

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The diversion of lactose carbon through the tagatose pathway reduces the intracellular fructose 1,6-bisphosphate and growth rate of Streptococcus bovis

Cited by 22 publications

References 15 publications

Molecular Physiology of Sugar Catabolism in Lactococcus lactis IL1403

Molecular Physiology of Sugar Catabolism in Lactococcus lactis IL1403

Effects of the overexpression of fructose-1,6-bisphosphate aldolase on fermentation pattern and transcription of the genes encoding lactate dehydrogenase and pyruvate formate-lyase in a ruminal bacterium, Streptococcus bovis

Effects of pH and Energy Supply on Activity and Amount of Pyruvate Formate-Lyase in Streptococcus bovis

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