Identification of an anaerobically induced phosphoenolpyruvate-dependent fructose-specific phosphotransferase system and evidence for the Embden-Meyerhof glycolytic pathway in the heterofermentative bacterium Lactobacillus brevis

Abstract

Heterofermentative gram-positive bacteria are believed to metabolize sugars exclusively via the pentose phosphoketolase pathway following uptake via sugar:cation symport. Here we show that anaerobic growth of one such bacterium, Lactobacillus brevis, in the presence of fructose induces the synthesis of a phosphotransferase system and glycolytic enzymes that allow fructose to be metabolized via the Embden-Meyerhof pathway.Lactic acid bacteria are a group of gram-positive bacteria that characteristically lack cytochromes and are dependent on substrate level phosphorylation during sugar fermentation for energy (1). These bacteria are subdivided on the basis of their pathways and products of sugar fermentation. Homofermentative lactic acid bacteria use the Embden-Meyerhof pathway (EMP) to generate lactate as the sole product of fermentation, whereas heterofermentative lactic acid bacteria use the pentose phosphoketolase pathway (PKP) to produce a mixture of CO 2 , ethanol, acetate, and lactate (3, 11). While two molecules of phosphoenolpyruvate (PEP) are generated per sugar molecule metabolized via the EMP, only one PEP molecule results from sugar metabolism via the PKP. This fact has been used to rationalize the observation that bacteria that use the EMP usually have the PEP-dependent sugar transporting phosphotransferase system (PTS), while those which use the PKP do not. Indeed, the PTS has been reported to be widespread in homofermentative lactic acid bacteria but absent from heterofermentative bacteria (11,12). Lactobacillus brevis is one heterofermentative lactic acid bacterium which has been reported to lack PTSs specific for glucose, fructose, and lactose (10, 12). Glucose and lactose are known to be transported into L. brevis by sugar:H ϩ symport (10,20,21). Phosphofructokinase and fructose-1,6-bisphosphate aldolase have not been identified in this organism.The PTS catalyzes sugar uptake and phosphorylation via the following scheme: Sugar out ( PEP 3 I ϳ P 3 HPr ϳ P 3 IIA ϳ P 3 IIBC ϳ P 3 Sugar-P in n pyruvate I (enzyme I) and HPr (a heat-stable phosphocarrier protein of the PTS) are the general, energy coupling proteins of the system, while the IIA, IIB, and IIC proteins or protein domains are the constituents of the sugar-specific permeases (enzyme II complexes) that catalyze sugar transport and phosphorylation (6,15). While HPr has been detected in L. brevis, a search for additional components of the PTS gave negative results (9). It was therefore postulated that of the PTS proteins, this heterofermentative organism possesses only HPr and uses it exclusively for regulatory purposes. The use of HPr, the target of an ATP-dependent, metabolite-activated HPr(ser) kinase, to regulate both lactose:H ϩ symport and glucose:H ϩ symport in L. brevis has been extensively documented (9,10,13,(20)(21)(22)(23).We have previously reported that Listeria and Streptomyces species possess fructose-specific PTSs (8,17,18). While examining the properties of L. brevis, as described in Bergey's Manual of Systematic Bacteriolog...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…Owing to the presumed absence of a functional PTS, there seemed to be no obvious regulatory role for the ATP-dependent phosphorylation of HPr at Ser-46 in these organisms. Although the report about the absence of EI and EIIs from heterofermentative lactobacilli later turned out to be wrong (an L. brevis ptsHI operon has been cloned and sequenced) (193) and the presence of a fructose-specific PTS induced under anaerobic conditions has been established (772), it initiated the search for additional functions of P-Ser-HPr, one of them being the regulation of CCR.…”

Hypoxic Response ofMycobacterium tuberculosisStudied by Metabolic Labeling and Proteome Analysis of Cellular and Extracellular Proteins

“…Interestingly, Bai et al (2) observed increased activity of this type of aldolase sensitive to EDTA in cell extracts from M. tuberculosis H37Rv under conditions of low-oxygen tension. In Bacillus subtilis and Lactobacillus brevis, increased levels of this enzyme were also observed under anaerobic conditions (17,24). Fba was not identified in the microarray study or in the proteome studies of the M. bovis BCG response to low-oxygen tension that did not include culture filtrates (4,10,25).…”

Regulation of sugar uptake via the phosphoenolpyruvate-dependent phosphotransferase systems in Bacillus subtilis and Lactococcus lactis is mediated by ATP-dependent phosphorylation of seryl residue 46 in HPr

“…However, the physiological significance of this observation was questioned when in vivo studies conducted with Bacillus subtilis and Staphylococcus aureus failed to reveal appreciable HPr(ser-P)-mediated inhibition of PTS and non-PTS sugar uptake under conditions that were thought to generate HPr(ser-P) in vivo (5,6,17,18,26). On the other hand, extensive studies with vesicles of Lactococcus lactis, Lactobacillus brevis, Enterococcus faecalis, Streptococcus bovis, and Streptococcus pyogenes provided evidence that inducer exclusion (inhibition of sugar uptake) and/or inducer expulsion (stimulated efflux of preaccumulated sugar) were mediated by HPr(ser) phosphorylation (1,2,25,(28)(29)(30)(31)(32)(33)(34)(35). Moreover, Lodge and Jacobson (12) showed that intact cells of Streptococcus mutans exhibit elevated sugar uptake following carbon starvation, and this enhanced activity correlates with decreased 32 P labeling of an acid-stable HPr derivative and not with increased levels of the protein (see reference 9).…”